GENERAL PRINCIPLES │ 1. Behavioral Science ►►Confidence Intervals
Confidence intervals (CI) estimate the population value based on the data from a sample. We give up precision, knowing exactly the
population number, in exchange for confidence. Confidence intervals tell us that reality is most likely within the specified range.
Confidence interval of the mean Where: X = sample mean
Z = Z-score*
( )X±ZS S = standard deviation
√N N = sample size
*Z = 1.96 for 95% confidence
Z = 2.58 for 99% confidence
Interpretation of Confidence Intervals
Confidence intervals for the If the CIs for two means overlap, then they could be the same. Therefore, we have no evidence
mean that they are different. If the CIs do not overlap, then we usually assume that they are different
(statistical significance). In general, any overlap in CIs indicates no difference.
Confidence intervals for relative If the CIs contain the number 1.0, then the population parameters compared in the ratio could be
risk (RR) or odds ratios the same. Therefore, we cannot assume that they are different. If 1.0 is not included in the CI,
then we assume that they are different (statistical significance). A 1.0 in the CI means that it is not
significant.
►►Types of Scales in Statistics
Type of Scale Description Key Words Examples
“This” as opposed to “that”
Nominal (categorical) Different groups Gender, comparing among
Comparative quality, rank treatment interventions
Ordinal Groups in sequence order
Olympic medals, class rank in
Interval Exact differences among groups Quantity, mean, and standard medical school
deviation
Ratio Interval + true zero point Height, weight, blood pressure,
Zero means zero drug dosage
Temperature measured in
degrees Kelvin
►►Types of Scales and Basic Statistical Tests
Variables
Name of Statistical Test Interval Nominal Comment
Is there a linear relationship?
Pearson correlation 20 Any number of groups
Chi-square 02 Two groups only
t-test 11 Two or more groups
One-way ANOVA 11
Two groups, linked data pairs, before and after
Matched pairs t-test 11 More than two groups, linked data
Repeated measures ANOVA 11
36
Biochemistry Chapter 2
Glycolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
The Citric Acid Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Oxidative Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Pyruvate Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Hexose Monophosphate Shunt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Glycogenesis and Glycogenolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Gluconeogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Amino Acid Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Amino Acid Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
Amino Acid Synthesis and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47–48
Urea Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Lipid Synthesis and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50–51
Ketone Body Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Cholesterol Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Lipoprotein Transport and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Lysosomal Storage Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Lipid-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
37
Biochemistry
GENERAL PRINCIPLES │ 2. Biochemistry ►►Glycolysis
Glycolysis is a cytoplasmic pathway used by all cells to generate energy from glucose. One glucose molecule is converted into 2
pyruvate molecules, generating a net of 2 ATPs by substrate-level phosphorylation, and 2 NADHs. When oxygen is present, NADH
delivers electrons to the electron transport chain in mitochondria to generate ATP by oxidative phosphorylation. Under anaerobic
conditions, lactate is generated and NADH is reoxidized to NAD+.
Galactose Regulation: Three irreversible steps
Galactokinase
Lactose Lactase 1
Galactose-1-P
Gal-1-P uridyl transferase Hexokinase Glucokinase*
Glucose-1-P
*Controlled Enzymes Catalyzing Irreversible Steps Most tissues Liver, β-islet cells
*1 ADP Low Km High Km
− G-6-P
ATP Induced by
insulin in liver
Glucose Isomerase PFK–2
Mg2 + Glucose-6-P Fructose-6-P Fructose-2,6-bis-P
Hexokinase
Sucrose Sucrase ATP ADP
Glucokinase (liver) * ATP 2
Fructose 2 PFK–1 +
Fructokinase ADP (Phosphofructokinase)
Fructose-1-P PFK-1 PFK-2
Fructose-1-P aldose Fructose-1, 6-bis P → F-1,6-BP → F-2,6-BP
Glycolysis regulator:
Aldolase Rate-limiting
step of ↑ glycolysis
NAD+ Glyceraldehyde-3-P Dihydroxyacetone-P glycolysis ↓ gluconeogenesis
ETC/O2 Pi ⊕ Insulin
Mitochondria (DHAP) Glycerol-3-P ⊕ AMP − Glucagon
⊕ F-2,6-BP†
NADH Isomerase dehydrogenase − ATP
− Citrate
Glyceraldehyde-3-P Glycerol-3-P
dehydrogenase • Triacylglycerol synthesis 3
• Electron Shuttle
1,3-Bisphosphoglycerate (RBC)
ADP
Phosphoglycerate 2,3-Bisphosphoglycerate
ATP kinase
3-Phosphoglycerate
Mutase Pyruvate kinase
2-Phosphoglycerate ⊕ F-1,6-BP − ATP
− Alanine†
Enolase
Phosphoenolpyruvate (PEP) *Glukokinase mutations may lead to a form of MODY.
†Liver specific
ADP 3*
Glucose Transport
Pyruvate kinase
ATP
Pyruvate Lactate GLUT-1 and -3: basal uptake (most cells)
(aerobic) (anaerobic)
GLUT-2: storage (liver); glucose sensor
(β-islet)
GLUT-4: ↑ by insulin (adipose, skeletal
muscle); ↑ by exercise (skeletal muscle)
Disease Association
Galactokinase deficiency Galactosemia/galactosuria, cataracts in childhood (excess galactose is converted to galactitol
via aldose reductase); Tx: no galactose in diet
Gal-1-P uridyl transferase Same as above, but more severe with vomiting/diarrhea after milk ingestion, liver disease,
deficiency lethargy, mental retardation; Tx: no galactose in diet
Fructokinase deficiency Fructosuria; benign
Fructose-1-P aldolase B Fructosuria, liver and proximal renal tubule disorder; Tx: no fructose in diet
deficiency
Pyruvate kinase deficiency Chronic hemolysis, ↑ 2,3-BPG and other glycolytic intermediates in the RBC, no Heinz bodies,
autosomal recessive
Definition of abbreviations: MODY, mature-onset diabetes of the young; PFK, phosphofructokinase; RBC, red blood cell; Tx, treatment.
38
►►The Citric Acid Cycle GENERAL PRINCIPLES │ 2. Biochemistry
The citric acid cycle (tricarboxylic acid cycle) is a mitochondrial pathway that occurs only under aerobic conditions. Each acetyl-CoA
generated from pyruvate is used to produce 3 NADH, 1 FADH2, and 1 GTP. Both the NADH and FADH2 deliver electrons to the electron
transport chain (ETC) to generate ATP by oxidative phosphorylation.
Acetyl- Regulation
CoA
Citrate
a b
Oxaloacetate cis-Aconitate 1 Isocitrate dehydrogenase
NADH b (Rate-Limiting Step)
h
L-Malate Isocitrate ⊕ ADP − ATP
g 1 − NADH
Fumarate c NADH + CO2 2 α-Ketoglutarate dehydrogenase*
α-Ketoglutarate
FADH2 f − Succinyl CoA
d NADH + CO2 − ATP
Succinate 2 − NADH
e Succinyl-CoA *Similar to pyruvate dehydrogenase
GTP Enzymes complex and uses the same cofactors
a. Citrate synthase
b. Aconitase Links to Other Pathways
c. Isocitrate dehydrogenase
d. α-Ketoglutarate dehydrogenase
e. Succinyl-CoA thiokinase
f. Succinate dehydrogenase
g. Fumarase
h. Malate dehydrogenase
Stoichiometry of the Citric Acid Cycle • Gluconeogenesis (malate shuttle)
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi → 2 CO2 + 3 NADH + FADH2 + GTP + CoA • Fatty acid synthesis (citrate shuttle)
The only net fate of acetyl-CoA as it proceeds through the citric acid cycle is conversion
to CO2. The citric acid cycle is NOT a means to convert acetyl groups to glucose. • Amino acid synthesis (oxaloacetate
Humans lack the capacity to form glucose from acetyl-CoA. and α-ketoglutarate)
• Heme synthesis (succinyl CoA)
39
GENERAL PRINCIPLES │ 2. Biochemistry ►►Oxidative Phosphorylation
Electron transport and the coupled synthesis of ATP are known as oxidative phosphorylation. The electron transport chain
(ETC) is a series of carrier enzymes in the inner mitochondrial membrane that pass electrons, in a stepwise
fashion, from NADH and FADH2 to oxygen, the final electron acceptor. These carriers create a proton gradient across
the inner membrane, which drives the F0/F1 ATP synthase, with a net production of 3 ATPs per NADH and 2 ATPs per FADH2.
Electron Transport Chain
Matrix H20 ADP + P ATP
H+ 02 H+
NADH H+ H+
FADH 2 CoQe- e- e-
ll e- lll
lV
l V
e-
H+ H+ cytc
Cytosol Proton Gradient
(Intermembrane space) H+
Complex I: NADH dehydrogenase Complex III: cytochrome b/c1
Complex II: succinate dehydrogenase Cyt C: cytochrome c
CoQ: coenzyme Q Complex IV: cytochrome a/a3
Complex V: FoF1ATP synthase
Clinical Correlation
Cyanide Poisoning
Blocks cytochrome a/a3; cyanide from burning polyurethane (mattress/furniture stuffing); Tx: nitrites (creates methemoglobin, which
binds cyanide)
Inhibitors
Inhibit ETC and O2 consumption
Inhibit ATP synthesis
• Antimycin A, piscicide (Complex I)
• Cyanide (cyt oxidase)
• R otenone, broad-spectrum insecticides, other pesticides (Complex 1)
• Oligomycin (Fo)
Uncouplers
Increase ETC and O2 consumption
Decrease ATP synthesis
Produce heat
• 2,4-dinitrophenol (2,4-DNP)
• Salicylate (metabolite of aspirin)
• U ncoupling proteins (e.g., thermogenin)
40
►►Pyruvate Metabolism GENERAL PRINCIPLES │ 2. Biochemistry
1 L actate dehydrogenase: Anaerobic tissues: converts pyruvate to lactate, reoxidizing cytoplasmic NADH to NAD+.
Liver: converts lactate to pyruvate for gluconeogenesis or for metabolism to acetyl CoA
2 Pyruvate dehydrogenase: generates acetyl-CoA for fatty acid synthesis and the citric acid cycle; complex of 3 enzymes
3 Pyruvate carboxylase: produces oxaloacetate for gluconeogenesis and the citric acid cycle
4 A lanine aminotransferase (ALT, GPT): Muscle: converts pyruvate to alanine to transport amino groups to the liver.
Liver: converts alanine to pyruvate for gluconeogenesis and delivers the amino group for urea synthesis
Glucose Regulation
Pyruvate dehydrogenase (PDH)
Pyruvate 4 ⊕ ADP − Acetyl-CoA
NAD+ CO2 + ATP 3
1 NAD+ CO 2 2 ⊕ CoA − ATP
NADH + H+ Oxaloacetate ⊕ NAD+ − NADH
Lactate NADH + H+ Alanine
− glucagon
Acetyl-CoA Cofactors: TPP (from thiamine), lipoic
acid, CoA (from pantothenate), FAD
(from riboflavin), NAD (from niacin)
Citric acid cycle Fatty acid Citric acid cycle Gluconeogenesis
synthesis
Stoichiometry of Pyruvate Dehydrogenase Disease Association
Pyruvate + NAD+ + CoA → NADH + CO2 + acetyl-CoA Pyruvate Dehydrogenase
Cori Cycle Deficiency
Lactic acidosis, seizures, mental
retardation, ataxia, spasticity
Muscle or RBC glycolysis pyruvate lactate During fasting or exercise, lactate
Bloodstream lactate from RBCs or skeletal muscles is sent
Liver glucose to the liver to make glucose that can
be returned to the RBCs
glucose pyruvate or muscle.
gluconeogenesis
Definition of abbreviations: TCA, tricarboxylic acid; TPP, thiamine pyrophosphate.
41
GENERAL PRINCIPLES │ 2. Biochemistry ►►Hexose Monophosphate Shunt
The hexose monophosphate (HMP) shunt (pentose phosphate pathway) is a cytosolic pathway that uses glucose-6-phosphate to
reduce NADP to NADPH, and synthesize ribose-5-P. NADPH is important for fatty acid and steroid biosynthesis, maintenance of reduced
glutathione to protect against reactive oxygen species (ROS), and for bactericidal activity in polymorphonuclear leukocytes (PMNs).
Ribose-5-P is required for nucleotide synthesis.
Regulation
Glucose Glucose-6-P-dehydrogenase
NADP
NADPH NADP NADPH ⊕ NADP+ − NADPH
Glucose-6-P 6-phosphogluconate Ribulose-5-P
CO2
Glucose 6-P
dehydrogenase
Ribose-5-P
Nucleotide synthesis
Disease Association
Glucose-6-Phosphate Dehydrogenase Deficiency
Episodic self-limiting hemolytic anemia induced by infection and drugs (common) or chronic hemolysis (rare); X-linked recessive; female
heterozygotes have ↑ resistance to malaria
Glucose 6-
Phosphate
HMP G6PDH NADPH +
Shunt Glutathione NADP
Reductase
Oxidized Reduced Oxidant Stress
Glutathione Glutathione • infection
• drugs
• fava beans
Pentose
Phosphates
Glutathione H2O2 spontaneous O2
Peroxidase
O2 + H2O if
(Se) accumulates
• Hemoglobin Denaturation
(Heinz bodies)
• Membrane Damage
(Hemolytic Anemia)
42
►►Glycogenesis and Glycogenolysis
Glycogen is a branched polymer of glucose, stored primarily in liver and skeletal muscles, which can be mobilized during GENERAL PRINCIPLES │ 2. Biochemistry
hypoglycemia (liver) or muscular contraction (muscles). Synthesis of glycogen (glycogenesis) is mediated by glycogen
synthase, while its breakdown (glycogenolysis) is carried out by glycogen phosphorylase. Branching of the glycogen polymer
occurs via a branching enzyme, which breaks an α-1,4-bond and transfers a block of glucosyl residues to create a new
α-1,6-bond. This is reversed by a debranching enzyme.
Glycogen Metabolism Regulation
Glucose Insulin Glycogen Epinephrine 1 Glycogen synthase
(Liver) (Liver UDP Glucagon (Liver and Muscle)
Muscle)
(Liver)
AMP Liver Skeletal Muscle
Muscle
Pi + ++
++ 2 Glycogen ⊕ Insulin ⊕ Insulin
⊕ Glucose − Epinephrine
1 Glycogen Phosphorylase − Glucagon
Synthase − Epinephrine
(and Debranching
(and Branching
Enzyme) Enzyme)
UDP-Glucose PPi 2 Glycogen phosphorylase
UTP
Glucose 1-P Liver Skeletal Muscle
Glucose Glucose 6-P ⊕ glucagon ⊕ epinephrine
6-Phosphatase Glycolysis ⊕ epinephrine ⊕ AMP
(Muscle) − Insulin ⊕ Ca2+
(Liver) − ATP
Glucose Pyruvate
Branching and Debranching Steps
Glucose-6-phosphate
Glucose-1-phosphate 5
5
1
Limit dextran
UDP-glucose 3 4
2
Storage form of glycogen
1 UDP-glucose pyrophosphorylase 4 Glycogen phosphorylase
2 Glycogen synthase 5 Debranching enzyme
3 Branching enzyme
Glycogen Storage Diseases
Type I: von Gierke disease Severe fasting hypoglycemia, lactic acidosis, hepatomegaly, hyperlipidemia,
(↓ glucose-6-phosphatase) hyperuricemia, short stature
Cardiomegaly, muscle weakness, death by 2 years
Type II: Pompe disease
(↓ lysosomal-α-1,4-glucosidase) Mild hypoglycemia; liver enlargement
Type III: Cori disease Infantile hypotonia, cirrhosis, death by 2 years
(↓ glycogen debranching enzyme)
Muscle cramps/weakness during initial phase of exercise, possible rhabdomyolysis
Type IV: Andersen disease and myoglobinuria
(↓ branching enzyme) Mild fasting hypoglycemia, hepatomegaly, cirrhosis
Type V: McArdle disease 43
(↓ muscle glycogen phosphorylase*)
Type VI: Hers disease
(↓ hepatic glycogen phosphorylase)
* Also known as myophosphorylase.
►►Gluconeogenesis
GENERAL PRINCIPLES │ 2. Biochemistry Gluconeogenesis is a pathway for de novo synthesis of glucose from C3 and C4 precursors using both mitochondrial and cytosolic
enzymes. Occurring only in liver, kidney, and intestinal epithelium, this pathway functions to provide glucose for the body, especially the
brain and RBCs, which require glucose for energy (the brain can also use ketone bodies during fasting conditions). Gluconeogenesis occurs
during fasting, as glycogen stores become depleted. Important substrates for gluconeogenesis are gluconeogenic amino acids (protein
from muscle), lactate (from RBCs and muscle during anaerobic exercise), and glycerol-3-P (from triacylglycerol from adipose tissues).
Pi Glucose Glucokinase Regulation
4 Glucose-6-phosphatase Glucose-6-P Four irreversible steps:
1 Pyruvate carboxylase
Mitochondrial; requires biotin
⊕ acetyl CoA
Pi Fructose-6-P PFK-1 2 PEPCK
3 Fructose-1,6-bisphosphatase Fructose-1,6-bis P
Cytosolic; requires GTP
Induced by glucagon
and cortisol
Glyceraldehyde-3-P DHAP Glycerol-3-P
3 reversible 3 Fructose 1,6-bisphosphatase
reactions
Cytosolic
PEP ⊕ ATP − AMP
− F-2,6-BP‡ (from PFK2)
GDP Pyruvate kinase
GTP PEPCK Pyruvate Alanine 4 Glucose-6-phosphatase
2 Lactate
OAA† In endoplasmic reticulum; only in liver
Cytoplasm
Malate Shuttle Mitochondria ‡ Mediates insulin’s inhibition and glucagon’s
stimulation of this enzyme
Pyruvate Disease Association
ATP CO2 PDH – Glucose-6-Phosphatase Deficiency
OAA† ADP 1 (von Gierke disease)
Pyruvate carboxylase + Acetyl CoA Severe hypoglycemia, lactic acidosis,
(biotin) (from ß-oxidation hepatomegaly, hyperlipidemia,
of fatty acids) hyperuricemia, short stature
Alanine Cycle
Liver ATP Pyruvate (2) Urea A pathway by which muscles release alanine to
Bloodstream Glucose the liver, delivering both a gluconeogenic sub-
Muscle ATP strate (pyruvate) and an amino group for urea
NH2 synthesis
Alanine (2)
Glucose Pyruvate (2) Alanine (2)
ATP
α-amino acid α-ketoacid
Definition of abbreviations: PEPCK, phosphoenolpyruvate carboxykinase; PFK2, phosphofructokinase 2; RBC, red blood cell.
† OAA is not transported across the membrane directly. Instead, it is transported as malate in exchange for asparate via the malate shuttle.
44
►►Amino Acid Structures
Hydrophobic Amino Acids GENERAL PRINCIPLES │ 2. Biochemistry
Nonpolar, Aliphatic Side Chains Aromatic Side Chains
+ COO- + COO- + COO- + COO- + COO- + COO-
CH CH CH CH CH CH
H3N H H3N CH3 H3N H3N CH2 H3N CH2 H3N
CH CH 2
CH3 CH3 C CH
Glycine Alanine Valine NH
Gly Ala Val
+ COO- + COO- + COO- OH
CH CH H
H3N H3N C CH3 H2N Tyrosine
H CH2 C Phenylalanine Tyr Tryptophan
CH2 CH3 H2C CH2 Phe Trp
CH2
CH
CH 3 CH3
Leucine Isoleucine Proline
Leu Ile Pro
Positively Charged R Groups Hydrophilic Amino Acids Negatively Charged R Groups
Polar, Uncharged R Groups
COO- COO- COO- COO- + COO- COO- H 3 N+ COO - + COO-
H3 N+ C H + + H3N CH + H3N
H3N C H H3N C H + C OH H3N C H CH CH
CH 2 H CH2 CH2
CH 2 CH2 CH2 H3N C H CH 2 COO- CH2
CH2 C NH SH COO-
CH2 CH2 C N+ CH CH2OH
NH+3 HH
CH2 Serine CH3 Cysteine Aspartate Glutamate
Lysine Histidine Ser Cys Asp Glu
Lys NH His Threonine
C NH2+ Thr
NH2
+ COO- COO- + COO-
Arginine H3N + H3N CH
Arg CH H3N C H
CH 2 CH 2
CH 2 CH2
S CH 2
C
H2N O C
H2N O
CH 3
Asparagine Glutamine
Methionine Asn Gln
Met
45
GENERAL PRINCIPLES │ 2. Biochemistry ►►Amino Acid Derivatives
Besides being the building blocks of proteins, amino acids are also precursors for various chemicals, such as hormones, neurotransmitters,
and other small molecules.
Amino Acid Product Disease Association
Tyrosine Thyroid hormones (T3, T4); melanin; catecholamines (dopa- Albinism
mine, epinephrine)
Tyrosine hydroxylase (type I) or tyrosine
O2 Tyrosine Aromatic acid O2 Dopamine-β- Phenylethanolamine-N- transporter (type II) deficiency;
Tyrosine hydroxylase decarboxylase hydroxylase methyl transferase (PNMT) ↓ pigmentation of skin, eyes, and hair,
↑ risk of skin cancer, visual defects
DOPA Dopamine Cu + Norepinephrine Epinephrine
Ascorbate Carcinoid Syndrome
THB DHB CO2 SAM S-Adenosylhomocysteine
↑ Serotonin excretion from gastrointestinal
Melanin neuroendocrine tumors (carcinoid tumors);
cutaneous flushing, venous telangiectasia,
Tryptophan Serotonin (5-HT); melatonin; NAD; NADP diarrhea, bronchospasm, cardiac valvular
lesions
O2 Tr yptophan Aromatic amino acid
Tr yptophan hydroxylase decarboxylase Acute Intermittent Porphyria
5-OH-Tr yptophan Serotonin Porphobilinogen deaminase* deficiency;
episodic expression, acute abdominal
THB DHB CO2 pain, anxiety, confusion, paranoia, muscle
weakness, no photosensitivity, port-wine
Glycine Heme urine in some patients, urine excretion
of ALA and PBG; autosomal dominant;
ALA synthase ALA dehydratase Porphobilinogen onset at puberty, 15% penetrance, variable
expression; more common in women
Glycine δ -Aminolevulinate Porphobilinogen deaminase* Uroporphyrinogen-III
Succinyl-CoA
† Porphyria Cutanea Tarda
Uroporphyrinogen decarboxylase†
Protoporphyrin IX deficiency; photosensitivity, skin
inflammation, and blistering; cirrhosis often
Heme synthase Fe2+ associated; autosomal dominant; late onset
(ferrochelatase)
Lead Poisoning
UDP-glucuronyl Biliverdin Heme Inhibits ALA dehydratase and
transferase reductase oxygenase ferrochelatase; microcytic sideroblastic
Bilirubin diglucuronide Bilirubin Biliverdin anemia; basophilic stippling of erythrocytes;
Heme headache, nausea, memory loss, abdominal
UDP UDP- NADPH NADPH O2 pain, diarrhea (lead colic), lead lines in
glucuronate gums, neuropathy (claw hand, wrist-drop),
↑ urine excretion of ALA; Tx: dimercaprol
Glutamate γ-aminobutyric acid (GABA) and EDTA
Glutamate decarboxylase
Glutamate γ-Aminobutyric acid (GABA)
Arginine Nitric oxide (NO) Hemolytic Crisis
+ Ca2 + Jaundice due to ↑ bilirubin from severe
Arginine + O2 NO synthase Nitric oxide + citrulline hemolysis; ↓ hemoglobin; ↑ reticulocytes;
may result from:
NADPH NADP+
(1) G6PD deficiency hemolysis
(2) Sickle cell crisis
(3) Rh disease of newborn
Histidine Histamine UDP-Glucuronyl Transferase Deficiency
Histidine Histidine decarboxylase Histamine Jaundice due to low bilirubin conjugation;
may result from:
Methionine S-adenosylmethionine (SAM; methylating agent) (1) Crigler-Najjar syndromes
Arginine, glycine, SAM Creatine (2) Gilbert syndrome
(3) Physiologic jaundice of newborn,
especially premature infants
* Also known as hydroxymethylbilane synthase; † an enzyme in the pathway between Uroporphyrinogen-III and Protoporphyrin IX.
46
►►Amino Acid Synthesis and Metabolism GENERAL PRINCIPLES │ 2. Biochemistry
Amino acids are required for protein synthesis. Although some amino acids can be synthesized de novo (nonessential), others (essential)
must be obtained from the digestion of dietary proteins. Nonessential amino acids are synthesized from intermediates of glycolysis and
the citric acid cycle or from other amino acids. Degradation of amino acids occurs by transamination of the amino group to glutamate,
while the remaining carbon skeletons of the amino acids may be oxidized to CO2 + H2O, or reverted to citric acid cycle intermediates for
conversion to glucose (glucogenic) or ketones (ketogenic).
Genetic Deficiencies of Amino Acid Metabolism
Phenylalanine Leucine Valine
Isoleucine
Phenylalanine Phenylketonuria Branched chain
hydroxylase ketoacid
Tetrahydrobiopterin dehydrogenase
Tyrosine Maple Syrup Urine
Disease
Acetyl CoA
Homogentisic Acid OAA Citrate
Homogentisate Alkaptonuria Malate
oxidase
Maleylacetoacetate Fumarate α-KG
Succinyl CoA
Methylmalonyl CoA Methylmalonyl CoA
mutase deficiency mutase
B12
Methylmalonyl CoA
Propionyl CoA Propionyl CoA
carboxylase carboxylase deficiency
(Biotin)
Odd-Carbon Fatty Acids Propionyl CoA
Threonine α-Ketobutyrate
Homocysteine Cysteine Homocystinuria
methyltransferase
N5-methyl THF Cystathionine
B6 Cystathionine synthase
B12
Homocysteine
Methionine S-Adenosylhomocysteine
ATP Methyl Groups for Biosynthesis
Pi + PPi • Epinephrine
• N-Methylguanine cap on mRNA
S-Adenosylmethionine (SAM)
Figure I-17-3. Genetic Deficiencies of Amino Acid Metabolism (Continued)
47
GENERAL PRINCIPLES │ 2. Biochemistry ►►Amino Acid Synthesis and Metabolism (Cont'd.) Essential Amino Acids*
Precursors for Nonessential Amino Acids
Arginine† Methionine
Glycolysis TCA cycle Histidine Phenylalanine
Glucose Phosphoglycerate Pyruvate α-Ketoglutarate Oxaloacetate Isoleucine Threonine
Serine Alanine
Glutamate Aspartate Leucine Tryptophan
Glycine Cysteine
Proline Glutamine Asparagine Lysine Valine
* Mnemonic: PVT. TIM HALL; †essential during periods
of growth and pregnancy
Transfer of α-Amino Groups to α-Ketoglutarate Glucogenic and Ketogenic Amino Acids
Amino acid Glutamate Ketogenic Ketogenic and Glucogenic
Glucogenic
NH2
R CH COOH NH2 Leucine Phenylalanine All others
CH2 CH2 CH COOH Lysine Tyrosine
Enz-PLP HOOC Tryptophan
Isoleucine
R C COOH Enz-PLP HOOC CH2 CH2 C COOH Threonine
O NH2 O
α-Ketoacid α-Ketoglutarate
Disease Association
Hartnup disease Transport protein defect with ↑ excretion of neutral amino acids; symptoms similar to pellagra; autosomal
recessive
Phenylketonuria Phenylalanine hydroxylase or dihydrobiopterin reductase deficiency → buildup of phenylalanine; tyrosine
becomes essential; musty body odor, mental retardation, microcephaly, autosomal recessive; Tx: ↓
phenylalanine in diet; avoid aspartame (Nutrasweet®)
Alkaptonuria Homogentisate oxidase deficiency (for tyrosine degradation); ↑ homogentisic acid in blood and urine
(darkens when exposed to air), ochronosis (dark pigment in cartilage), arthritis in adulthood
Homocystinuria ↑ homocystine in urine. Classic homocystinuria, caused by a deficiency in cystathionine synthase, is
associated with dislocated lens, deep venous thrombosis, stroke, atherosclerosis, mental retardation, and
Marfan-like features. Deficiency of pyridoxine, folate, or vitamin B12 can produce a mild homocystinemia with
elevated risk of atherosclerosis (previously listed symptoms absent). Methionine synthase (homocysteine
methyltransferase) deficiency is extremely rare and is associated with megaloblastic anemia and mental
retardation.
Cystinuria Transport protein defect with ↑ excretion of lysine, arginine, cystine, and ornithine; excess cystine
precipitates as kidney stones; Tx: acetazolamide
Maple syrup urine Branched-chain ketoacid dehydrogenase deficiency; branched-chain ketoacidosis from infancy; weight loss,
disease lethargy, alternating hypertonia/hypotonia, maple syrup odor of urine; ketosis/coma/death if untreated; Tx: ↓
valine, leucine, isoleucine in diet
Propionyl-CoA Neonatal ketoacidosis from blocked degradation of valine, isoleucine, methionine, threonine, and
carboxylase odd-carbon fatty acids; Tx: ↓ these amino acids in diet
deficiency
Propionyl-CoA carboxylase deficiency: neonatal metabolic acidosis; hyperammonemia; elevated propionic
Methylmalonyl-CoA acid, hydroxypropionic acid, and methylcitrate; poor feeding, vomiting, lethargy, coma
mutase deficiency
Methylmalonyl-CoA mutase deficiency: symptoms similar to propionyl CoA carboxylase deficiency,
but accumulating metabolites differ (↑ methylmalonic acid)
Definition of abbreviation: PLP, pyridoxal-phosphate, formed from vitamin B6.
48
►►Urea Cycle GENERAL PRINCIPLES │ 2. Biochemistry
Amino acids transported to the liver are transaminated to glutamate, which undergoes deamination to produce NH4+ or
transamination to make aspartate. Both of these are used for synthesis of urea in the liver for excretion via the urea cycle.
Mitochondrial Matrix HCO3– + 2 ATP + NH4+ NH4+ Portal blood
(intestine)
Carbamoyl phosphate synthetase I 1 Glucose
Pyruvate
Carbamoyl phosphate Glutamate α-ketoglutarate
dehydrogenase NADH
Ornithine transcarbamoylase Ornithine NAD+ Alanine
(from muscle)
Citrulline
Glutamate
Cytosol
Citrulline
Argininosuccinate synthetase Aspartate Aspartate oxaloacetate
transaminase α-ketoglutarate
AT P
AMP + PP i
Argininosuccinate
Argininosuccinate lyase Aspartate
Fumarate Arginine
TCA cycle Arginase
Urea Ornithine
Regulation
1 Carbamoyl phosphate synthase I
⊕ N-acetylglutamate*
*High protein diet → ↑ glutamate in mitochondria → ↑ N-acetylglutamate
Disease Association
Carbamoyl Phosphate Synthetase Deficiency Ornithine Transcarbamoylase Deficiency
↑ [NH4+]; hyperammonemia ↑ [NH4+]; hyperammonemia
↑ blood glutamine ↑ blood glutamine
↓ BUN ↓ BUN
No increase in uracil or orotic acid Uracil and orotic acid ↑ in blood and urine*
Cerebral edema Cerebral edema
Lethargy, convulsions, coma, death Lethargy, convulsions, coma, death
*OTC deficiency: ↑ carbamoyl-P stimulates pyrimidine synthesis, causing ↑ orotic acid and uracil
49
GENERAL PRINCIPLES │ 2. Biochemistry ►►Lipid Synthesis and Metabolism
Fatty acids are synthesized from excess glucose in the liver and transported to adipose tissues for storage. Fatty acid synthesis occurs
in the cytosol and involves the transport of acetyl-CoA from the mitochondria via the citrate shuttle, carboxylation to malonyl CoA, and
linking together 2 carbons per cycle to form long fatty acid chains. Synthesis stops at C16 palmitoyl-CoA, requiring 7 ATP and 14 NADPH.
Metabolism of fatty acids occurs by β-oxidation, which takes place in mitochondria, and involves transport of fatty acids from the cytosol
via the carnitine shuttle, then oxidative removal of 2 carbons per cycle to yield 1 NADH, 1 FADH2, and 1 acetyl-CoA.
Fatty Acid Synthesis and Oxidation
Inner mitochondria
membrane
Mitochondrial matrix Cytosol
Citrate shuttle
pyruvate acetyl-CoA citrate citrate 1
oxaloacetate
Acetyl CoA
carboxylase
oxaloacetate acetyl-CoA Malonyl CoA
malate Fatty acid Triglycerides
NADPH† synthase and VLDL
(well-fed)
pyruvate Palmitoyl CoA
pyruvate AMP ATP Fatty acids
+PPi from adipose
NADH NAD FADH2 FAD (fasting)
FA-CoA
FA-CoA
Acetyl-CoA Fatty acyl CoA 2 Carnitine CoA
dehydrogenase acyltransferase
Other Liver (LCAD, MCAD) (CAT-1)
Tissues
FA-carnitine
Citric Ketones Carnitine
acyltransferase-2
Acid Cycle (CAT-2)
FA-carnitine
Carnitine
transporter
†Another important source of NADPH is the HMP shunt.
Triacylglycerols (triglycerides), the storage form of fatty acids, are formed primarily in the liver and adipose tissues by attaching 3
fatty acids to a glycerol-3-P. Triacylglycerols are transported from liver to adipose as VLDL. Fatty acids from the diet are transported
as chylomicrons. Both are digested by lipoprotein lipase (induced by insulin) in the capillaries of adipose and muscle. Fatty acids may be
mobilized from triacylglycerols in adipose by hormone-sensitive lipase. Free fatty acids are delivered to tissues for beta oxidation.
Regulation
1 Acetyl-CoA carboxylase 2 Carnitine acyltransferase-1 (CAT-1)
Rate-limiting for fatty acid synthesis; requires biotin Rate-limiting for fatty acid oxidation
⊕ insulin − glucagon − malonyl-CoA
⊕ citrate − palmitoyl-CoA
Disease Association
Myopathic CAT-2/CPT-2 Deficiency Medium Chain Acyl-Dehydrogenase (MCAD) Deficiency
Muscle aches/weakness, myoglobulinuria provoked by prolonged Fasting hypoglycemia, no ketone bodies, dicarboxylic acidemia,
exercise, ↑ muscle triacylglycerols C8–C10 acyl carnitines in blood, vomiting, coma, death;
Tx: give IV glucose, avoid fasting, maintain high carb/low fat diet,
including short chain FAs, which can be metabolized
(Continued)
50
►►Lipid Synthesis and Metabolism (Cont’d.) GENERAL PRINCIPLES │ 2. Biochemistry
Triacylglycerol (Triglyceride) Synthesis
DHAP Glucose Glucose Glucose DHAP
1 1 Glycerol-3-P
dehydrogenase
Glycerol-3-P
dehydrogenase 2 Glycerol kinase
Glycerol Glycerol-3-P TAG
storage
Glycerol-3-P Lipoprotein 3 FA CoA
lipase
3
3 FA CoA VLDL VLDL Triacylglycerol
Triglyceride
(storage)
4 Hormone- Gluconeogenesis
TAG sensitive Glycerol Glycerol Glycerol Glucose
lipase
Fatty Fatty Acids Fatty β-Oxidation
Acids Albumin Acids
Acetyl CoA
Ketogenesis TAG
mobilization
Ketone Ketone Citric
Bodies Bodies Acid
Muscle Cycle
(Brain)
ADIPOSE LIVER
Notes
1 Glycerol-3P-dehydrogenase (adipose, liver) Triacylglycerol synthesis from fatty acids
2 Glycerol kinase (liver only)
3 Lipoprotein lipase Located on luminal membrane of endothelial cells in
adipose tissue
Regulation
3 Lipoprotein Lipase 4 Hormone sensitive lipase
Digests TGL in VLDL and chylomicrons. Mobilizes fatty acids from
Fatty acids enter adipose triacylglycerols
Induced by insulin Repressed by insulin ⊕ Epinephrine – insulin
⊕ ApoC-II Induced by cortisol
Definition of abbreviations: CAT, carnitine acyltransferase (a.k.a. CPT, carnitine palmitoyl transferase); L/MCAD, long/medium chain acyl-
dehydrogenase; TAG, triacylglycerols.
Diabetic ketoacidosis results from overactive hormone-sensitive lipase often in the context of stress, trauma, or infection.
51
►►Ketone Body Metabolism
GENERAL PRINCIPLES │ 2. Biochemistry During fasting, the liver converts excess acetyl-CoA from beta-oxidation of fatty acids into two ketone bodies, acetoacetate and
β-hydroxybutyrate, which can be used by muscle and brain tissues. Ketosis represents a normal and advantageous response to fasting/
starvation, whereas ketoacidosis is a pathologic condition associated with diabetes and other diseases.
β-Oxidation Disease Association
FA CoA Acetyl CoA Diabetic Ketoacidosis
HMG CoA Synthase
Excess ketone bodies in blood asso-
HMG CoA ciated with type 1 diabetes mellitus
HMG CoA Lyase not adequately managed with insulin,
or precipitated by infection or trauma.
Acetoacetate Acetone Liver Characterized by polyuria, dehydration,
NADH CNS depression and coma, sweet fruity
Blood breath (acetone).
NAD Muscle
Renal Cortex With the prevalence of obesity and
Mitochondrial 3-Hydroxybutyrate Brain in Prolonged Fast stressful environments, ketoacidosis is
Matrix (β-Hydroxybutyrate) now becoming more prevalent in type
2 diabetics, e.g., a diabetic in ketoaci-
Cytoplasm 3-Hydroxybutyrate Acetone dosis cannot be assumed to be type 1.
Acetoacetate
Alcoholic Ketoacidosis
Cytoplasm
Excess ketone bodies due to high
Mitochondrial 3-Hydroxybutyrate NADH/NAD ratio in liver; symptoms
Matrix same as above
Acetoacetate Note: In either type of ketoacidosis,
3-hydroxybutyrate (β-hydroxybutyrate)
Activation of NADH NAD is the predominant ketone body
acetoacetate formed (not detected by the urine test).
in extrahepatic Measure 3-hydroxybutyrate to more
accurately evaluate ketoacidosis.
tissues
Acetoacetyl CoA
2 Acetyl CoA Citric Acid Cycle
Figure I-16-4. Ketogenesis (Liver) and Ketogenolysis (Extrahepatic)
►►Cholesterol Synthesis
Cholesterol is obtained from diet (about 20%) or synthesized de novo (about 80%). Synthesis occurs primarily in the liver for storage and
bile acid synthesis, but also in adrenal cortex, ovaries, and testes for steroid hormone synthesis. Cholesterol may also be esterified into
cholesterol esters by acyl-cholesterol acyl-transferase (ACAT) in cells for storage.
Regulation
2 NADPH 1 HMG-CoA reductase
HMG-CoA
3 Acetyl-CoA Mevalonic acid Rate-limiting step
HMG-CoA reductase
⊕ insulin − glucagon
1
⊕ thyroxine − cholesterol
Cell membranes Cholesterol Vitamin D Pharmacology
Steroids (adrenal, Bile acids (liver) HMG-CoA Inhibitors (“Statins”),
ovaries, testes) e.g., lovastatin, pravastatin
↓ LDL; used for hypercholesterolemia; side
effects: myopathy, liver dysfunction
52
►►Lipoprotein Transport and Metabolism GENERAL PRINCIPLES │ 2. Biochemistry
Free fatty acids are transported by serum albumin, whereas neutral lipids (triacylglycerols and cholesterol esters) are transported by
lipoproteins. Lipoproteins consist of a hydrophilic shell and a hydrophobic core and are classified by their density into chylomicrons,
VLDL, LDL, and HDL.
~ 80% Regulation
1 Lipoprotein lipase
Dietary fat HDL Released from
liver and small
~ 20% intestine
Intestine Liver LDL Extra Hepatic Hydrolyzes fatty acids from
Dietary Endogenous (B-100) Tissues triacylglycerols from chylomicrons
Cholesterol Cholesterol and VLDL
Hepatic LCAT
Lipase Induced by insulin
⊕ ApoC-II
Chylomicrons CETP Hyperlipidemias
(E, CII, B-48)
IDL Type I Hypertriglyceridemia
Remnants VLDL (E, B-100) HDL (cholesterol-rich)
(E, B-48) (E, CII, B-100) Lipoprotein lipase deficiency; ↑
triacylglycerols and chylomicrons;
LP Lipase (Fatty acid) LP Lipase (Fatty acid) Deliver cholesterol orange-red eruptive xanthomas, fatty
1 1 to liver and liver, acute pancreatitis, abdominal
steroidogenic pain after fatty meal; autosomal
Adipose Tissue and Muscle Adipose Tissue and Muscle tissues via SR-B1 recessive
LCAT, lecithin cholesterol acyltransferase Type II Hypercholesterolemia
CETP, cholesterol ester transfer protein
SR-B1, scavenger receptor-B1 LDL receptor deficiency; ↑ risk
of atherosclerosis and CAD,
Classes of Lipoproteins and Important Apoproteins xanthomas of Achilles tendon,
tuberous xanthomas on elbows,
Lipoprotein Functions Apoproteins Functions xanthelasma (lipid in eyelid), corneal
arcus, homozygotes die <20 years;
Chylomicrons Transport dietary apoB-48 Secreted by epithelial cells autosomal dominant
triglyceride and cholesterol apoC-II Activates lipoprotein lipase
from intestine to tissues apoE Uptake by liver Pharmacology
VLDL Transports triglyceride apoB-100 Secreted by liver Cholestyramine/Colestipol
from liver to tissues apoC-II Activates lipoprotein lipase
apoE Uptake of remnants ↑ Elimination of bile salts leads to ↑
LDL receptor expression, leading to
by liver ↓ LDL; for hypercholesterolemia; side
effect: GI discomfort
LDL Delivers cholesterol apoB-100 Uptake by liver and other
Gemfibrozil/Clofibrate (“Fibrates”)
into cells tissues via LDL receptor
(apoB‑100 receptor)
IDL Picks up cholesterol from apoE Uptake by liver ↑ elimination of VLDL leads to
(VLDL HDL to become LDL apoA-1 ↓ triacylglycerols and ↑ HDL; for
remnants) Activates LCAT to produce hypertriglyceridemia; side effect:
Picked up by liver cholesterol esters muscle toxicity ; acts via PPAR-α
HDL to induce LP lipase gene
Picks up cholesterol
accumulating in blood Nicotinic Acid
vessels
↓ VLDL synthesis leads to ↓
Delivers cholesterol to LDL; for hypercholesterolemia
liver and steroidogenic and hypertriglyceridemia; side
tissues via scavenger effects: GI irritation; hyperuricemia,
receptor (SR-B1) hyperglycemia, flushing, pruritus
Shuttles apoC-II and apoE
in blood
Definition of abbreviations: CAD, coronary artery disease; CETP, cholesterol ester transfer protein; HDL, high-density lipoprotein; LCAT,
lecithin-cholesterol acyl transferase; LDL, low-density lipoprotein; PPAR, peroxisome proliferator-activated receptor; SR-B1, scavenger
receptor B1; VLDL, very-low-density lipoprotein.
53
►►Lysosomal Storage Diseases
GENERAL PRINCIPLES │ 2. Biochemistry Disease Deficiency and Accumulated Features AR*
Tay-Sachs disease Substrate AR
• P sychomotor retardation AR*
Niemann-Pick disease ↓ Hexosaminidase A • C herry red spots in macula
↑ GM2 ganglioside • O nset in 1st 6 mos.; death <2 years XR
(whorled membranes in lysosomes) AR
• Hepatosplenomegaly AR
↓ Sphingomyelinase • Microcephaly
↑ Sphingomyelin • Mental retardation XR
(zebra bodies in lysosomes) • Foamy macrophages AR
• Neonatal onset
Gaucher disease ↓ β-glucocerebrosidase
↑ Glucocerebroside • T hree clinical subtypes; type 1 is most common
• Hepatosplenomegaly
Fabry disease ↓ α-galactosidase A • Bone involvement, including fractures and bone pain
↑ Ceramide trihexoside • Neurologic defects (rare, types 2 and 3)
• Mental retardation
Metachromatic ↓ arylsulfatase A • Gaucher cells (enlarged macrophages with
leukodystrophy ↑ sulfatide
fibrillary cytoplasm)
Hurler syndrome (MPSI) ↓ α-l-iduronidase
↑ dermatan sulfate • Renal failure
↑ heparan sulfate • Telangiectasias
• Angiokeratomas
Hunter syndrome (MPSII) ↓ l -iduronate-2-sulfatase • P eripheral neuropathy with pain in extremities
Krabbe disease ↓ dermatan sulfate
↑ heparan sulfate • Ataxia
• Dementia
↓ galactocerebrosidase • Seizures
↑ galactocerebroside
• Coarse facial features
• Corneal clouding
• Hepatosplenomegaly
• Skeletal deformities
• Upper airway obstruction
• Recurrent ear infections
• Hearing loss
• Hydrocephalus
• Mental retardation
• Death <10 years
• B oth mild and severe forms
• S evere similar to Hurler but retinal degeneration instead of
corneal clouding, aggressive behavior, and death <15 years
• M ild form compatible with long life
• Defective myelin sheaths
• P eripheral neuropathy
• Severe seizures
►►Eicosanoid Metabolism
Membrane
Phospholipids
Phospholipase A2 – Corticosteroids
Arachidonic Acid
NSAIDs
–
Cyclooxygenases Lipoxygenase – Zileuton
COX1
COXibs – COX2
Endoperoxides Hydroperoxides
PGI2 TXA2 Leukotrienes
PGE1 PGE2 Receptors blocked
PGF2α by – Lukasts
Definition of abbreviations: AR, autosomal recessive; COX, cyclooxygenase; NSAIDs, nonsteroidal anti-inflammatory drugs; XR, X-linked recessive.
*Common in Ashkenazi Jews
54
►►Enzyme Kinetics
Whereas the thermodynamic equilibrium of a chemical reaction is determined by its free energy (∆G), the rate at which the reaction GENERAL PRINCIPLES │ 2. Biochemistry
reaches equilibrium is determined by its activation energy (∆G‡). Enzymes increase the rate of a reaction by reducing the energy of
activation without affecting the equilibrium constant.
Michaelis-Menten Equation Lineweaver-Burk Equation Classes of Inhibitors
V = Vmax [S] 1 = Km 1 1 Competitive, Reversible
Km + [S] V Vmax [S] + Vmax
(often substrate analogs that compete for
V = initial rate or velocity of reaction Reciprocal form of the Michaelis-Menten the enzyme’s binding site)
equation to achieve a straight line plot
[S] = substrate concentration Vmax: no effect
Km: ↑
Vmax = maximum rate of enzyme
Km = substrate concentration at Vmax/2
In a typical enzyme-catalyzed reaction, the enzyme (E) is thought to bind reversibly to a 1
substrate (S), forming a complex (ES), from which the product (P) dissociates as the reaction V + Competitive Inhibitor
proceeds.
No Inhibitor Present
E + S ↔ E – S → E + P, 01
where E is the enzyme, S is the substrate and P is the reaction product
[S]
Noncompetitive, Reversible
The rate of a reaction as determined by both the concentration of enzyme (E) and substrate (bind outside active site but affects
(S) is described by the Michaelis-Menten equation. enzyme activity, possibly allosterically)
Vmax: ↓
Km: no effect
Michaelis-Menten Plot Lineweaver-Burk Plot
50 Vmax (sec/µmole) 0.06 1 + Noncompetitive Inhibitor
V
V (µmoles/sec) 0.04
No Inhibitor Present
25 1 01
v [S]
0.02 1 Irreversible (Inactivator)
Vmax (binds and inactivates enzyme
permanently)
Km -1.0 -0.5 0 0.5 1.0
Vmax: ↓
2468 10 – 1 1 (mM-1) Km: no effect
[S] (mM) Km
[S]
55
►►Water-Soluble Vitamins
GENERAL PRINCIPLES │ 2. Biochemistry Vitamin or Enzyme Pathway Deficiency
Coenzyme Gluconeogenesis
Fatty acid synthesis Causes (rare): excessive consumption of raw
Biotin Pyruvate carboxylase Odd-carbon fatty acids, eggs (contain avidin, a biotin-binding protein)
Acetyl-CoA carboxylase Val, Met, Ile, Thr
PDH Alopecia (hair loss), bowel inflammation,
Propionyl-CoA carboxylase TCA cycle muscle pain
HMP shunt
Thiamine (B1) Pyruvate dehydrogenase Many Causes: alcoholism (alcohol interferes with
α-Ketoglutarate dehydrogenase absorption)
Niacin (B3) Transketolase Thymidine (pyrimidine)
NAD(H) Dehydrogenases synthesis Wernicke (ataxia, nystagmus,
NADP(H) ophthalmoplegia)
Purine synthesis
Folic acid Thymidylate synthase Korsakoff (confabulation, psychosis)
THF Purine synthesis enzymes Methionine, SAM High-output cardiac failure (wet beri-beri)
Odd-carbon fatty acids,
Cyanocobalamin Homocysteine methyltransferase Pellagra may also be related to deficiency
(B12) Methylmalonyl-CoA mutase Val, Met, Ile, Thr of tryptophan (corn major dietary staple),
which supplies a portion of the niacin
Pyridoxine (B6) Aminotransferases (transaminase): Protein catabolism requirement
PLP AST (SGOT), ALT (SGPT)
Pellagra: diarrhea, dementia, dermatitis, and,
if not treated, death
Causes: alcoholics and pregnancy (body
stores depleted in 3 months)
Homocystinemia with risk of deep vein
thrombosis and atherosclerosis
Megaloblastic (macrocytic) anemia
Deficiency in early pregnancy causes neural
tube defects in fetus
Causes: pernicious anemia. Also in aging,
especially with poor nutrition, bacterial
overgrowth of terminal ileum, resection of the
terminal ileum secondary to Crohn disease,
chronic pancreatitis, and, rarely, vegans, or
infection with Diphyllobothrium latum
Megaloblastic (macrocytic) anemia
Progressive peripheral neuropathy
Causes: isoniazid therapy
δ-Aminolevulinate synthase Heme synthesis Sideroblastic anemia
Cheilosis or stomatitis (cracking or scaling of
lip borders and corners of the mouth)
Convulsions
Riboflavin (B2) Dehydrogenases Many Corneal neovascularization
FAD(H2)
Cheilosis or stomatitis (cracking or scaling of
lip borders and corners of the mouth)
Magenta-colored tongue
Ascorbate (C) Prolyl and lysyl hydroxylases Collagen synthesis Causes: diet deficient in citrus fruits and
Dopamine β-hydroxylase green vegetables
Catecholamine synthesis
Scurvy: poor wound healing, easy bruising
Absorption of iron in GI (perifollicular hemorrhage), bleeding gums,
tract increased bleeding time, painful glossitis,
anemia
Pantothenic acid Fatty acid synthase Fatty acid metabolism Rare
CoA Fatty acyl CoA synthetase
Pyruvate dehydrogenase PDH
α-Ketoglutarate dehydrogenase TCA cycle
Definition of abbreviations: mALoTn,oaplahnoisnpehaatmeinsohturnatn;sNfeAraDs(eH;);AnSicTo, tainsapmaritdaeteaadmenininoetradninsufecrleaosetid; eC;oNAA, DcoPe(nHz)y, mniecoAti;nFaAmDid(He 2a)d, eflnaivnine adenine
dinucleotide; HMP, hexose dinucleotide
phosphate; PDH, pyruvate dehydrogenase; PLP, pyridoxal phosphate, SAM, S-adenosylmethionine; TCA, tricarboxylic acid cycle; THF,
tetrahydrofolate.
56
►►Lipid-Soluble Vitamins
Vitamin Important Functions Deficiency GENERAL PRINCIPLES │ 2. Biochemistry
D (cholecalciferol) In response to hypocalcemia, helps normalize serum Rickets (in childhood): skeletal abnormalities
calcium levels (especially legs), muscle weakness
A (carotene) Retinoic acid and retinol act as growth regulators, After epiphysial fusion: osteomalacia
K especially in epithelium
Night blindness, metaplasia of corneal epithelium,
Retinal is important in rod and cone cells for vision dry eyes, bronchitis, pneumonia, follicular
hyperkeratosis
Carboxylation of glutamic acid residues in many Ca2+-
binding proteins, importantly coagulation factors II, VII, IX, Easy bruising, bleeding
and X, as well as proteins C and S Increased prothrombin time, increased INR
Associated with fat malabsorption, long-term
E (α-tocopherol) Antioxidant in the lipid phase; protects membrane lipids
from peroxidation and helps prevent oxidation of LDL antibiotic therapy, breast-fed newborns, infants
particles thought to be involved in atherosclerotic plaque of mothers who took anticonvulsants during
formation pregnancy
Hemolysis, neurologic problems, retinitis
pigmentosa
57
Molecular Biology, Genetics,
and Cell Biology
Chapter 3
Nucleic Acid Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60
Nucleotide Synthesis and Salvage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61–62
DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Transcription and RNA Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Protein Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Post-Translational Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Collagen Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Recombinant DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69–70
Genetic Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Patterns of Inheritance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Single-Gene Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Chromosomal Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Other Chromosomal Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75–76
Additional Diseases Resulting from Chromosomal Abnormalities . . . . . . . . . . . . 77
Population Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Subcellular Organelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78–79
Plasma Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Cell Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Cell Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84–85
59
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Nucleic Acid Structure
Nucleic acids, including DNA and RNA, are assembled from nucleotides, which contain a five-carbon sugar, a nitrogenous base, and
phosphate. The sugar may be ribose (RNA) or deoxyribose (DNA). The base can be a purine (adenine or guanine) or pyrimidine
(cytosine, uracil, thymidine). Phosphate groups link the 3′ carbon of one sugar to the 5′ carbon of the next, forming phosphodiester
bonds. Base sequences are conventionally written in a 5′ → 3′ direction. Nucleotides lacking phosphate groups are called nucleosides.
In prokaryotes and eukaryotes, RNA is generally single-stranded, while DNA is generally double-stranded in an antiparallel orientation,
with two hydrogen bonds between base pairs A and T and three between G and C. Nuclear DNA forms a double-helix, which undergoes
supercoiling via topoisomerase activity, and is generally associated with histones and other proteins to form nucleosomes, the basic
packaging unit of chromatin.
Hydrogen Bonding in DNA The B-DNA Double Helix Nomenclature
5´- Phosphate 3´- Hydroxyl Base Nucleoside Nucleotide
Adenine
O H3C O H N OH T Guanine Adenosine Adenylate
OP O HN AT Cytosine Adenosine
3´ Thymine
O TN H NA N O 3´ AT Uracil monophosphate (AMP)
5´ CG
5´ 5´CH2 N N 5´CH2 GC
O O O
3´
O
OP O H OP O AT Guanosine Guanylate
NH O TA Guanosine
O ON GC
5´CH2 O CN TA monophosphate (GMP)
H NG N 3´ }Major Groove
3´ N H N TA
O O N O GC } Minor Groove
H 5´CH2
OP O CG Cytidine Cytidylate
O AT Cytidine monophosphate
H O CH3 O
5´CH2 N NH OPO GC (CMP)
O TH N GC
AN N N O AT
3´ N O TA
O
OP O 3´ Thymidine Thymidylate
O Thymidine
5´CH2 NO H O
5´CH2 monophosphate (TMP)
O GN N H HN O
N Uridine Uridylate
NH NC OP O Uridine monophosphate
N
O O (UMP)
3´ H 3´ AT
AT
3´ OH O
5´CH2
O
OP O
3´- Hydroxyl O
5´- Phosphate
Eukaryotic Chromatin Structure 5-Carbon Sugar
Disease Association 5 CH 2OOHOH 5 CH2OOHOH
4 1 41
Antihistone antibodies are characteristic of drug-induced SLE. 32
Drugs include hydralazine and procainamide. 32 OH
OH OH
2-Deoxyribose
+H I Without H I sensitive Ribose
to nuclease
10 nm Nitrogenous Bases
30 nm PPuurriinneess PPyyrriimmiiddiinneess
NNHH22 NN OO NN NNHH22 OO OO CCHH33
NN HHNN NN HHNN HHNN
H2A NN NHNH HH22NN NN NN OO NN OO NN OO NN
H2B AAddeennininee HH HH HH HH
H4 H3 H1 GGuuaannininee CCyyttoossininee UUrraaccilil TThhyymmininee
Chromatin
Expanded View H2A H3 Expanded Euchromatin Heterochromatin
H2B H4 View of a
Nucleosome Loosely packed Tightly packed
Transcriptionally active Transcriptionally inactive
60
Ribose 5-Phosphate
PRPP Synthetase GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
AMP PRPP P PP Allopurinol
►►Nucleotide Synthesis and Salvage IMP _ R _ 6-Mercaptopurine
PRPP 1
amidotransferase
GMP
Nucleotides for DNA and RNA synthesis cHaMn Pbeshguennte.rDateednobvyodseynnotvhoessiysntohcecsuisrsomr saainlvlya5-gipnehothpspeahtolhirviwbeoasryyalsanm, dbinoegtehnoePfrawtehRsiNchHn2erweqpuuirreinPeRaPnPd generated
from ribose-5-phosphate derived from the pyrimidine
bases from precursors. In contrast, salvage pathways reuse preformed bases derived from nucleotidGelsycdinuer, iAnsgpanrtoatrem, GallutRamNinAe turnover or released
from dying cells or transported from the liver. Ribonucleotides are converted to deoxyribonucleotides for DNA synthesis by ribonucleotide
reductase. Antineoplastic drugs that target ribonucleotide reductase (hydroxyurea), or an enzyme in the dTMP branch of pyrimidine synthesis
(5-FU), or reduction of folate (methotrexate) preferentially inhibit DNA synthesis without compromiTsHinFgasRCNarAbonsyDnonthoresis and gene expression.
Excretion of purine bases occurs in the form of uric acid from the kidneys. Inosine monophosphate P Hypoxanthine
(IMP) R
De Novo Purine Synthesis AmDineo fNromovo PyAmriinmo firdomine Synthesis
glutamine aspartate
Ribose 5-Phosphate
GMP AMP
PRPP Synthetase
AMP PRPP P PP Allopurinol
6-Mercaptopurine
R Carbamoyl
phosphate
IMP _ PRPP 1 _ CO2 + Glutamine synthetase-2 Aspartate
Carbamoyl
GMP amidotransferase +ATP (Cytoplasm) Phosphate Orotic Acid
PRPP
P NH2
5-phosphoribosylamine R
Glycine, Aspartate, Glutamine CO 2
UMP
THF as Carbon Donor Hydroxyurea _ Ribonucleotide UDP
reductase
Hypoxanthine dUDP CTP
R
Inosine monophosphate P dUMP
(IMP)
N 5N10 Methylene THF Thymidylate
Amino from Amino from synthase _
glutamine aspartate
THF DHF 5 - Fluorouracil
Dihydrofolate
reductase dTMP
GMP AMP
_
Regulation Methotrexate (eukaryotic)
Trimethoprim (prokaryotic)
Carbamoyl Pyrimethamine (protozoal)
CO2 + Glutamine spyhnotshpehta1asteeP-2RPPCaarmbaimdooytrl aAnspsafretaratese Orotic Acid
+ATP Phosphate PRPP
(Cytopl−asAmM) P, IMP, GMP
Purine Analogs Pyrimidine Analogs
Allopurinol CO2 5-fluorodeoxyuridine;
• I nhibits PRPP amidotransferase UMP 5-bromodeoxyuridine
• T reatment of gout • Inhibit thymidylate synthase
• Inhibits xanthine oxidaHsyedroxyurea _ Ribonucleotide UDP • Antineoplastic
6-mercaptopurine; 6-thioguanine reductase
Folic Acid Analogs
• Inhibits PRPP amidino-transferase dUDP CTP
• Antineoplastic dUMP Methotrexate; aminopterin
N 5N10 Methylene THF Thymidylate • Inhibit eukaryotic DHFR
THF DHF synthase _
• Antineoplastic
Dihydrofolate Trimethoprim
5 - Fluoroura•c ilInhibits bacterial DHFR
reductase dTMP • Antibacterial
_ Pyrimethamine
• Inhibits protozoal DHFR
Methotrexate (eukaryotic) • Antiprotozoal
Trimethoprim (prokaryotic) Hydroxyurea
Pyrimethamine (protozoal)
• Inhibits ribonucleotide reductase
• Antineoplastic
Definition of abbreviations: AMP, adenosine monophosphate; dTMP, deoxythymidine monophosphate; 5-FU, 5-flurouracil;
IMP, inosine monophosphate; dUDP, deoxyuridine diphosphate; CTP, cytosine triphosphate; THF, tetrahydrofolate.
(Continued)
61
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Nucleotide Synthesis and Salvage (Cont’d.)
Purine Salvage Pathway and Excretion
DNA, RNA, and high-energy
compounds (GTP, ATP)
IMP AMP GMP
Adenosine Guanosine HGPRT
deaminase Guanine (salvage)
HGPRT Inosine
(salvage)
Hypoxanthine
Xanthine
Xanthine oxidase (excretion)
Uric acid
Definition of abbreviations: DHFR, dihydrofolate reductase; HGPRT, hypoxanthine-guanine phosphoribosyl pyrophosphate transferase;
NSAID, nonsteroidal anti-inflammatory drug; PRPP, phosphoribosylpyrophosphate; THF, tetrahydrofolate; SCID, severe combined
immunodeficiency disorder.
Disease Association
Adenosine Deaminase Deficiency
• SCID (no B- or T-cell function)
• Multiple infections in children
• Autosomal recessive
• Tx: enzyme replacement, bone marrow transplant
Gout
↑ production or ↓ excretion of uric acid by kidneys
Lesch-Nyhan Syndrome
• HGPRT deficiency
• Mental retardation (mild)
• Spastic cerebral palsy
• Self-mutilation
• Hyperuricemia
• X-linked recessive
62
►►DNA Replication GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
DNA replication involves the synthesis of new DNA molecules in a 5′ → 3′ direction by DNA polymerase using the double-stranded DNA
template. One strand (leading strand) is made continuously, while the other (lagging strand) is synthesized in segments. Prokaryotic
chromosomes are closed, double-stranded circular DNA molecules with a single origin of replication that separates into two replication
forks moving away in opposite directions. Eukaryotic chromosomes are double-stranded and linear with multiple origins of replication.
DNA Replication by a Semiconservative, Regulation Eukaryote
Bidirectional Mechanism Prokaryote
Prokaryotes Eukaryotes Recognizing dna A Unknown
Origin of Replication Multiple Origins of Replication replication Helicase Helicase
origin
Unwinding
double helix
Strand SSB SSB
stabilization
RNA primer Primase Primase
synthesis
Centromere Leading strand DNA pol III DNA pol α
synthesis and δ
Sister Chromatids Are Lagging strand DNA pol III DNA pol α
Separated During Mitosis synthesis
Events at the Replication Fork RNA primer DNA pol I (5′→ 3′ RNase H
removal exonuclease) Unknown
Replacing RNA DNA pol I
with DNA
5′ 3′ Single-strand Joining of DNA ligase DNA ligase
binding proteins Okazaki
Primase fragments
Helicase
Removing DNA topo II (DNA DNA topo II
positive gyrase)
supercoils
3′ DNA pol III ahead of
(holo) replication fork
3′
5′ DNA 5′ Telomere Not required Telomerase
pol I 3′ synthesis
Leading strand
Ligase fragOmkaeznat ki Pharmacology
Pyrimidine Analogs
Lagging strand
Cytosine arabinoside
• Incorporation stops chain elongation
• Antineoplastic
Definition of abbreviations: DNA, deoxyribonucleic acid; DNA pol, DNA polymerase; DNA topo, DNA topoisomerase; RNA, ribonucleic acid;
SSB, single-stranded DNA-binding protein.
63
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►DNA Repair
DNA sequence and structure may be altered either during replication or by exposure to chemicals or radiation. Mutations include point
mutations such as the substitution of one base with another. Substitution mutations in the third position of a codon (wobble position) are
usually benign because several codons code for the same amino acid. Other types of mutations include (1) deletion or addition of one or two
nucleotides (frameshift mutations), (2) large segment deletions (e.g., unequal crossover during meiosis), (3) mutations of 5′ or 3′ splice sites, or
(4) triplet repeat expansion, which can lead to a longer, more unstable protein product (e.g., Huntington disease).
Damage Cause Recognition/ Repair Types of Mutations
UV radiation Excision Enzyme Enzymes Transition: A:T → G:C or G:C → A:T
Thymine dimers Transversion: A:T → T:A or G:C → C:G
(G1) Excision endonuclease DNA
(deficient in xeroderma polymerase
pigmentosum)
DNA ligase
Cytosine Spontaneous/ Uracil glycosylase DNA Silent No change Sub
deamination chemicals AP endonuclease polymerase Missense in AA
(G1) Nonsense
Spontaneous/ AP endonuclease DNA ligase Frameshift Change AA to Sub
Apurination or heat another
apyrimidination A mutation on one of two DNA
(G1) DNA replication genes, hMSH2 or hMLH1, polymerase Early stop Sub or
errors initiates defective repair of codon Ins/Del
Mismatched DNA mismatches, resulting DNA ligase
base (G2) in a condition known as Misreading Ins/Del
hereditary nonpolyposis DNA of all codons
colorectal cancer—HNPCC. polymerase downstream
DNA ligase
DNA Repair Defects
Xeroderma Pigmentosum
(defect in nucleotide excision-repair)
• Extreme UV sensitivity
• Excessive freckling
• Multiple skin cancers
• Corneal ulcerations
• Autosomal recessive
Ataxia Telangiectasia
(defect in ATM gene product, a member of PI-3 kinase family involved in mitogenic signal transduction, detection of DNA damage, and
cell cycle control)
• Sensitivity to ionizing radiation
• Degenerative ataxia
• Dilated blood vessels
• Chromosomal aberrations
• Lymphomas
• Autosomal recessive
HNPCC
(defect in mismatch repair; usually hMSH2 or hMLH1 gene)
• Colorectal cancer
• 2 3 occur in right colon
• Autosomal dominant
• Part of Lynch syndrome
(a multi-cancer syndrome)
Definition of abbreviations: AA, amino acid; HNPCC, hereditary nonpolyposis colorectal cancer; Ins/Del, insert or deletion; Sub, substitution.
64
►►Transcription and RNA Processing GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
Transcription involves the synthesis of an RNA in a 5′ → 3′ direction by an RNA polymerase using DNA as a template. An important class
of RNA is messenger RNA (mRNA). Initiation of transcription occurs from a promoter region, which is the binding site of RNA polymerase,
and stops at a termination signal. In prokaryotes, a single mRNA transcript can encode several genes (polycistronic), and no RNA
processing is required, allowing transcription and translation to proceed simultaneously. In eukaryotes, all mRNAs are monocistronic, but
often include coding segments (exons) interrupted by noncoding regions (introns). Eukaryotic mRNAs must therefore undergo extensive
processing, including a 5′ cap, a 3′ tail, and removal of introns followed by exon splicing. Ribosomal RNA (rRNA) and transfer RNA (tRNA)
are also produced by transcription.
Prokaryotic Transcription Properties
Transcription Prokaryote Eukaryote
Promoter ATG TGA GC rich TTTTTT Gene regions May be polycistronic Always
–35 –10 Coding Region Continuous coding monocistronic
DNA 5´ 3´
3´ Exons and
TATA 5´ introns
Box
5´ Untranslated 3´ Untranslated RNA pol I: rRNA
RNA pol II: mRNA,
Region (UTR) Region (UTR)
Transcription snRNA
+1 Terminates RNA polymerase Core enzyme: α2ββ′ RNA pol III: tRNA,
Transcription 5S rRNA
Shine-Dalgarno AUG Coding Region UGA GC rich Promoter (−25)
Sequence TATA and (−70)
mRNA UUUUUU 3´ Initiation Promoter (−10) CAAT; transcription
5´ TATAAT and (−35) factors (TFIID) bind
sequence; sigma promoter
5´ UTR 3´ UTR initiation subunit
Not well
Eukaryotic Transcription and Processing characterized
Transcription Termination Stem loop and 5′ cap (7-MeG)
Postprocessing UUUU or stem loop 3′ tail (poly-A)
DNA 5´ Promoter ATG TAG Poly A Addition Site and rho factor Intron removal and
3´ –70 –25 Exon 1 Intron Exon 2 Signal AATAAA
None exon splicing in
3´ pre-mRNA
CAAT TATA 5´
Box Box
5R´ eUgniotrnan(UslTaRte)d 5´ Splice 3´ Untranslated
Site Region (UTR)
+1 3´ Splice Transcription
Terminates
Site
Transcription
Primary AUG UAG Poly A Addition
Transcript Exon 1 Intron Exon 2 Signal AAUAAA
RNA 5´ 3´ Types of RNA
5´ UTR 5´ Splice 3´ Splice 3´ UTR mRNA Messenger RNA carries sequence info from
rRNA DNA to ribosomes to be translated
Site Site tRNA
snRNA Ribosomal RNA is a component of ribosomes
Capping and Poly A
Addition (Nucleus) Transfer RNA carries amino acids to ribosomes
for protein synthesis
Me AUG UAG Poly A Addition
Exon 1 Intron Exon 2 Signal AAUAAA Small nuclear RNA plays role in RNA processing
5´ Gppp
AAPAoAlyAAAATaAil3´
Cap 5´ UTR 5´ Splice 3´ Splice 3´ UTR
Site Site
hnRNA Splicing by Spliceosome (snRNA) (nucleus)
3´ Regulation of Gene Expression
Excised Intron Transcription Transcription factors bound to enhancer
(Lariat) Degraded initiation or silencer DNA regions affect
transcription
in Nucleus
AUG Exon 1 Exon 2 UAG Poly A Addition
Me Signal AAUAAA
mRNA Splicing Alternative splicing of primary transcript yields
5´ Gppp AAPAoAlyAAAATaAil3´ variants of protein products
Cap Transport to Cytoplasm 3´ UTR
and Translation
5´ UTR
H2N–Protein–COOH mRNA Various RNA-binding proteins determine stability
degradation of mRNA
Definition of abbreviations: hnRNA, heterogeneous nuclear RNA; 7-MeG, 7-methylguanosine; RNA pol, RNA polymerase;
UTR, untranslated region.
65
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio►►Protein Translation
First base
Translation involves the synthesis of protein from mRNA templates in ribosomes (complexes of proteins and ribosomal RNAs [rRNA]).
Protein synthesis begins from an initiation codon (AUG = methionine) and ends at a stop codon (UAA, UGA, or UAG). Elongation involves
transfer RNAs (tRNA), which have an anticodon region at one end to recognize the codon on the mRNA and an amino acid attached at
the other end for covalent linkage to the growing polypeptide chain. Several ribosomes can simultaneously transcribe an mRNA, forming
a polyribosome, or polysome.
tRNA Genetic Code Properties
3' Amino acid Second base Prokaryote Eukaryote
OH attachment site
A TψC loop U C AG
C
C CC UUU Phe UCU UAU Tyr UGU Cys U Ribosomes 30S + 50S 40S + 60S =
Phosphorylated A G GT ψ C UUC Leu UCC UAC UGC C = 70S 80S
5' terminus 5' p G C U UUA UCA Ser UAA Stop UGA Stop A
"Extra arm" UUG
(variable) UCG UAG Stop UGG Trp G
CUU CCU CAU His CGU U
CUC CCC CAC CGC C Initiation 30S binds 40S
DHU loop C CUA Leu CCA Pro CAA CGA Arg A Third base to Shine- associates
CAG CGG G Dalgarno with 5′ cap
A U CUG CCG Gln U sequence; on mRNA;
G UH2 C fMet Met
AUU ACU AAU Asn AGU Ser A
G AUC Ile ACC AAC AGC G
A AUA ACA Thr U
AAA AGA C
AUG Start/ ACG AAG Lys AGG Arg A
fMet G
GUU GCU GAU Asp GGU
GUC GCC GAC GGC
G GUA Val GCA Ala GAA GGA Gly
U GUG GCG GAG Glu GGG Termination Protein released at
Anticodon stop codon
Formation of Aminoacyl-tRNA Polyribosome Pharmacology
Step I R RO AUG UAA Tetracyclines
Step 2 ATP+NH2 CH COOH NH2 CH C~AMP+PPi
5' 3' (tetracycline, doxycycline, minocycline)
RO RO • Prevent binding of aminoacyl-tRNA to ribosome
NH2 CH C~AMP+tRNA NH2 CH C~tRNA+AMP NH2 • For Chlamydia, Mycoplasma, H. pylori,
RO NH2 NH2 Rickettsia, Brucella, Vibrio, and acne
NH2 CH C~tRNA+AMP+PPi • Prophylaxis in chronic bronchitis
Sum R NH2 • Bacteriostatic
NH2 CH COOH+ATP+tRNA • Phototoxicity, GI distress, tooth discoloration,
Steps In Translation ↓ bone growth in children
Small Ribosomal INITIATION Linezolid
Subunit • Blocks initiation complex formation
5' PA 3' • For VRSA, VRE, drug-resistant pneumococci
UAC GTP AAA • Headache, GI distress
3' AUUAGC CUG
Aminoglycosides
5' met (streptomycin, gentamicin, neomycin)
• Cause misreading at initiation
5' cap or Shine met-tRNA; (Eu) Large • Accumulates intracellular via O2-dependent
(Eu) Dalgamo or Subunit
(Pr) uptake; anaerobes are resistant
fmet-tRNA; (Pr) • For gram – rods, enterococci
• Bactericidal
5' PA 3' ELONGATION 5' PA 3' • Nephrotoxicity, ototoxicity
5' P A 3'
Macrolides
A AA (erythromycin, clarithromycin, azithromycin)
• Interfere with translocation
1. Aminoacyl-tRNA Binds to A Site 2. Peptide Bond Forms. Peptidyl 3. Translocation of Ribosome 3 • For gram ⊕ cocci, Chlamydia, Mycoplasma,
GTP Transferase in Large Subunit Nucleotides
EF-TU and EFTS (Pr) Uses Energy Captured in Activation Along mRNA Ureaplasma, Legionella, Campylobacter
eEF-1(Eu) (2 High Energy Bonds) GTP • Bacteriostatic
EF-G(Pr) • GI distress, inhibits P450, auditory
eEF-2(Eu)
dysfunction at high doses
Elongation Cycle Repeats for Each Amino Acid Added
Clindamycin
5' PA TERMINATION Completed Protein Released From Ribosome • Interferes with translocation
Ribosomal Subunits • For gram ⊕ cocci, B. fragilis
Separate • Pseudomembranous colitis
mRNA Released
Chloramphenicol
STOP CODON • Inhibits ribosomal peptidyl transferase
in A Site • For Salmonella, B. fragilis, Rickettsia, and
bacterial meningitis (used as backup)
• Bacteriostatic
• Bone marrow suppression; aplastic anemia,
“gray baby” syndrome (neonates), optic
neuritis (children)
Definition of abbreviations: AA, amino acid; EF-2, elongation factor 2; fMet, formylmethionine; Met, methionine; VRE, vancomycin-resistant
enterococci; VRSA, vancomycin-resistant Staphylococcus aureus.
66
►►Post-Translational Modifications GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
Whereas cytoplasmic proteins are translated on free cytoplasmic ribosomes, secreted proteins, membrane proteins, and lysosomal
enzymes have an N-terminal hydrophobic signal sequence and are translated on ribosomes associated with the rough endoplasmic
reticulum (RER). After translation, proteins acquire more complex structures by being folded with the help of molecular chaperones.
Misfolded proteins are targeted for destruction by ubiquitin and digested in cytoplasmic protein-digesting complexes called proteasomes.
Co- and Postranslational Covalent Modifications Protein Structure
Glycosylation Addition of oligosaccharides Primary Amino acid sequence
Phosphorylation Addition of phosphate groups by protein kinases Secondary α-Helix or β-sheets
γ-carboxylation Creation of Ca2+ binding sites Tertiary Higher order 3D structure
(vitamin K dependent)
Prenylation Addition of farnesyl/geranyl lipid groups to peripheral Quaternary Multiple subunits
membrane proteins
Mannose phosphorylation Addition of phosphates onto mannose residues to
target protein to lysosomes
Synthesis of Secretory, Membrane, and Lysosomal Proteins
5' 3'
N-Terminal Cytoplasm Translation Begins
Hydrophobic in Cytoplasm
Involves Signal
Signal Recognition Signal Sequence Causes
Sequence Ribosome to Attach to ER
Particle (SRP) to
NH2 Position on ER Signal Peptidase Removes
5' 3' the Signal Sequence
5' 3'
NH2 Translation Continues
ER Lumen on RER
3' NH2 N-Glycosylation Glycosylation in ER
(Dolichol-P) (Continues in Golgi)
5'
NH2 NH2
HOOC
GOLGI CIS to Lysosomes
PhosphobryylaPthioonspohf oMtraannnsofesrease
trans
To Cell
Membrane or
Secretion
DFiMisgeeumraebsrI-ae4n-1eA1, a.sSnsdyonLtcyhsieaossitsoiomofnaSl ePcrroetteoinrys,
I-Cell Disease
(defect in mannose phosphorylation, causing lysosomal enzyme release into extracellular space and
accumulation of undigested substrates in cell)
• Coarse facial features, gingival hyperplasia, macroglossia
• Craniofacial abnormalities, joint immobility, club-foot, claw-hand, scoliosis
• Psychomotor and growth retardation
• Cardiorespiratory failure
• Death in first decade
• 10–20-fold increase in lysosomal enzyme activity in serum
67
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio►►Collagen Synthesis
Plasma MembraneCollagen is a structural protein composed of a triple helix of amino acid chains containing a repeating tripeptide
Gly-X-Y-Gly-X-Y, where the unique amino acids hydroxyproline and hydroxylysine are frequently found in the X position. Hydroxylation
of proline and lysine requires ascorbate (vitamin C), deficiency of which leads to scurvy.
Synthesis of Collagen Disease Association
Synthesis of Prepro-α Chain with 1 Scurvy
Hydrophobic Signal Sequence 2
3 (reduced hydroxylation due to
Removal of Signal ascorbate/vitamin C deficiency)
Sequence by 4
Signal Peptidase • Petechiae, ecchymoses
• Loose teeth, bleeding gums
Rough Pro-α Chain • Poor wound healing
Endoplasmic • Poor bone development
Hydroxylation of Selected
Reticulum Prolines and Lysines Osteogenesis Imperfecta
(RER) (Vitamin C)
OH OH (collagen gene mutations)
OH • Skeletal deformities
Glycosylation of • Fractures
Selected Hydroxylysines • Blue sclera
Cytoplasm OH 5 Ehlers-Danlos Syndrome
Triple Helix
ss Formation s (collagen and lysine hydroxylase gene
s mutations)
OH
• Hyperextensible, fragile skin
OH • Hypermobile joints, dislocations
• Varicose veins, ecchymoses
Secretion From Cell 6 Menkes Disease
OH s (deficient cross-linking secondary
ss s to copper deficiency)
OH • Depigmented (steely) hair
• Arterial tortuosity, rupture
Cleavage of Propeptides 7 • Cerebral degeneration
• Osteoporosis, anemia
OH • Mutation in gene for an
Collagen
ATP-dependent copper
(Tropocollagen) transport protein
OH • X-linked recessive
Assembly into Fibrils 8
Stabilized by
Lysyl Oxidase (Cu+)
Aggregation to Form 9
a Collagen Fiber
68
►►Recombinant DNA GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
Recombinant DNA technology allows DNA fragments to be copied, manipulated, and analyzed in vitro. Eukaryotic DNA fragments may be
genomic DNA containing both introns and exons, or complementary DNA (cDNA), which is reverse-transcribed from mRNA and contains
exons only. DNA fragments may be amplified by polymerase chain reaction (PCR), cut with specific restriction endonucleases, and
ligated into a DNA vector. These vectors can then be used for further manipulation or amplification of the DNA to produce genomic
DNA or cDNA (expression) libraries, to generate recombinant proteins, or for incorporation into humans (gene therapy) or other animals
(transgenic animals).
Formation of a Recombinant Plasmid Polymerase Chain Reaction
GAATTC GAATTC • Amplifies large amounts of DNA
from even just one DNA molecule
CTTAAG CTTAAG
• Used for recombinant DNA
Human DNA manipulation, genetic testing, or
disease screening
• Can be used to make cDNA from
mRNA with reverse transcriptase
(RT-PCR)
EcoRI tetr gene cut tetr with internal
G AATTC by EcoRI GAATTC EcoRI site
AATTC G CTTAAG Heat to 94°C
G CTTAA CTTAA G
EcoRI
ori ori
Anneal ampr Add primers
Ligate Plasmid (Vector) pBR322 Anneal at 50°C
dNTPs and Taq Polymerase
ampr ampr tetr Polymerization at 72°C
GAATTC CTTAGAAGATTC tets (tetracycline resistance
CTTAAG gene split by human DNA insert)
ori Completion of cycle 1.
+
ampr
Recombinaanmt pPrlatestmsid pBR322 Repeat cycle
again
(Continued)
69
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Recombinant DNA (Cont’d.) Restriction Endonucleases
Screening a DNA Library • Recognizes palindromes in dsDNA and
cuts, leaving sticky or blunt ends
1 Agar growth plate
with bacterial colonies • Used to make restriction maps of DNA or to
produce fragments for manipulation
2 Blot 5' GAATTC 3' 5' GGCC
3' CTTAAG 5' 3' CCGG
EcoRI HaeIII
5' GG 3' + 5' CC
Replica of growth 5' G + 5' AATTC 3' 3' CC 5' 3' GG
plate on filter 5'
3 Lyse bacteria, denature 3' CTTAA 5' G Blunt Ends
DNA, and add a
32P-DNA probe for gene; Sticky Ends
make autoradiogram
Lyse bacteria, add 3' 5' GGCC 3'
125I-antibody for protein; 5' 3' CCGG 5'
make autora5'diogramGAATTC
3' CTTAAG
4 EcoRI HaeIII
5' G + 5' AATTC 3' 5' GG 3' + 5' CC 3'
5' 3' CC 5' 3' GG 5'
3' CTTAA 5' G
Sticky Ends Blunt Ends
Pick positive colony
Pick positive colony from original plate
from original plate
Incorporation of Cloned DNA DNA Vectors
Cloned DNA Fragments Circular, self-replicating DNA to carry and
amplify DNA fragments in bacteria or yeast
Gene Therapy (Somatic) Transgenic Animals (Germ Line) ~100−12 kb Plasmid
Cloned gene inserted into DNA of Fertilized OVA Bacterial; restriction sites,
selected somatic cells Micro-inject replication origin, selection
Gene not passed to offspring cloned DNA marker (e.g., antibiotic
Vector used to introduce cloned gene New gene incorporated resistance)
into host DNA/nuclei into germ line DNA
~10−25 kb Phage
• retrovirus Implant in foster mother Up to 45 kb Packaging virus that infects
• adenovirus Up to 10 Mb bacteria; e.g., lambda (λ)
• liposome Offspring are transgenic
Examples New gene inserted is a transgene Cosmid
• SCID (severe combined immuno- Design animal model for human Plasmids with λ cloning sites
deficiency); interleukin receptor gene disease this way
• Cystic fibrosis; CFTR gene BAC, YAC
Bacterial or yeast artificial
chromosomes
Definition of abbreviation: dsDNA, double-stranded DNA.
70
►►Genetic Testing GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
The presence of specific DNA, RNA, and proteins can be identified by first separating these molecules by gel electrophoresis,
transferring to a membrane by blotting, and finally detecting with radioactive nucleic acid probes (for DNA and RNA) or antibodies (for
proteins). Direct detection in cells or tissues can also be performed using similar tools to identify mRNA (in situ hybridization) or proteins
(immunostaining). In vitro detection of proteins can also be achieved by enzyme-linked immunosorbent assay (ELISA). Using these
methods of detection, diversity between individuals or genetic mutations manifested by different restriction endonuclease sites (restriction
fragment length polymorphisms [RFLP]) or expansion of highly repetitive sequences (e.g., satellites, minisatellites, and microsatellites)
may be employed for genetic testing.
DNA RNA Protein Repeated Unit Length of Repeat
20−175 bp 0.1−1 Mb
Separation Gel electrophoresis Satellites 20−70 bp Up to 20 kb
Minisatellites
Blotting Southern Northern Western
(probe) (32P-DNA) (32P-DNA) (125I or antibody)
—
Other In situ Immunostaining or Microsatellites 2−4 bp <150 bp
detection hybridization ELISA
Sickle Cell Disease (Southern Blot; RFLP) Paternity Testing (PCR; STRs or Microsatellites)
M stII restriction digest of patient sample, followed by Southern blotting PCR amplification of STRs or microsatellite sequences can
using a probe against β-globin gene, allows identification be used to match the banding pattern to each parent. The
of either the normal or sickle allele. child should share one allele with each parent.
Mst II Restriction Map of the β-Globin Gene Tested Male
Child
5' 3' Mother
Tested Male
Normal (A) 1.15 kb 0.2 kb Child
Mother
5' 3'
Sickle (S) 1.35 kb
AS = carrier Fragment Size
SS = sickle-cell patient
AA = normal 1.35 kb
1.15 kb
AS SS AA
Possible Paternity
paternity disproved
Cystic Fibrosis (PCR; ASO Dot Blot) HIV Detection (ELISA and Western Blot)
The most common CF mutation, ∆F508, can be detected by comparing Serum antibodies to HIV are first detected by ELISA
PCR product sizes by gel electrophoresis or hybridization with allele- and then confirmed by Western blot.
specific oligonucleotide (ASO) probes on a dot blot (a simplified form of
Southern blot with no electrophoresis required). Serum to Test for HIV Infection
Position of Mutation ELISA
Bracket site of the potential Antibodies against
∆F508 mutation with PCR HIV antigens in serum
primers. The PCR product from
Primer 2 the mutant gene is 3 nucleotides
shorter than the product from the
Primer 1 normal gene. Heat – inactivated
HIV antigens coated
Homozygous on plate
Normal
CF Patient Positive – Confirm with Western Blot (shown below)
CF Carrier
Gel Electrophoresis of PCR Products Control: Control:
HIV+ Test HIV–
Serum Serum Serum
63 bp Normal ASO gp 120
60 bp F508 ASO
p55
gp41
= Sample reacts with probe p24
= Sample does not react with probe
Homozygous CF Patient CF Carrier
Normal
Definition of abbreviation: STR, short tandem repeats
71
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Patterns of Inheritance Autosomal Recessive Pedigree Analysis
Autosomal Dominant • A ffected individuals usually have unaf- Symbols Dead
fected (carrier) parents Mating
• A ffected individuals have an affected Male Consanguineous or
parent • Either sex affected Incestuous Mating
• Early uniform onset (infancy/childhood) Female Sibship
• Either sex affected • Often encode catalytic proteins
• Variable to late onset (may be delayed Unknown Sex Dizygous Twins
to adulthood) Affected Monozygous Twins
• Often encode structural proteins Carrier of an Autosomal
Recessive (Optional)
Carrier of an X-linked
Recessive (Optional)
Stillborn or Abortion
• Familial hypercholesterolemia • Sickle cell anemia Mitochondrial Inheritance
• Huntington disease • Cystic fibrosis
• Neurofibromatosis • Phenylketonuria (PKU) • I nherited maternally because only mother
• Marfan syndrome • Kartagener syndrome contributes mitochondria during conception
• von Hippel-Lindau disease
X-Linked Recessive • Either sex affected
X-Linked Dominant • U sually neuropathies and myopathies
• A ffected individuals usually have unaf-
• A ffected individuals have an affected fected (carrier) parents because brain and muscle are highly depen-
parent dent on oxidative phosphorylation
• Usually affect males only
• Either sex affected • No male-to-male transmission
• No male-to-male transmission • Female carriers sometimes show mild
• Females often have more mild and
symptoms (manifesting heterozygote)
variable symptoms than males
• Fragile X syndrome • Duchenne muscular dystrophy • Leber hereditary optic neuropathy
• Hypophosphatemic rickets • Lesch-Nyhan syndrome • M ELAS: mitochondrial encephalomyopathy,
• G6PD deficiency
• Hemophilia A and B lactic acidosis, and stroke-like episodes
• M yoclonic epilepsy with ragged red muscle
fiber
Decision Tree for Determining Mode of Inheritance*
Male–Male Transmission?
NO YES
May Be X-Linked Must Be Autosomal
Excess Number of Males? Multiple Generations of Affecteds?
NO YES YES NO
X-Linked Dominant Autosomal Recessive
X-Linked Recessive Autosomal Dominant (25% Recurrence Risk for
(Check Also for (50% Average Recurrence
Carrier x Carrier
Skipped Generations) Risk for Typical Mating) Mating; Consanguinity
May Be Present)
Definition of abbreviation: G6PD, glucose-6-phosphate dehydrogenase.
*Note: If transmission occurs only through affected mothers and never through affected sons, the pedigree is likely to reflect mitochondrial inheritance.
72
►►Single-Gene Disorders von Hippel-Lindau Disease Features GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
Neurofibromatosis (NF) Type 1 (mutation in tumor-suppressor gene on Variable expression—
(von Recklinghausen Disease) chromosome 3) differences in severity of symptoms
for same genotype; allelic
(mutation in NF1 tumor-suppressor gene on • Hemangioblastomas in CNS and retina heterogeneity can contribute to
chromosome 17) • Renal cell carcinoma variable expression
• Cysts in internal organs
• Multiple neurofibromas • Autosomal dominant Incomplete penetrance—
• Café-au-lait spots (pigmented skin lesions) some individuals with disease
• Lisch nodules (pigmented iris hamartomas) Cystic Fibrosis genotype do not have disease
• I ncreased risk of meningiomas and phenotype
(mutation in CFTR chloride channel
pheochromocytoma gene on chromosome 7q, leading to Delayed age of onset—
• 90% of NF cases thick secretion of mucus plugs) individuals do not manifest
• Autosomal dominant phenotype until later in life
• Recurrent pulmonary infections
Neurofibromatosis (NF) Type 2 (P. aeruginosa and S. aureus) Pleiotropy—
(Bilateral Acoustic Neurofibromatosis) single disease mutation affects
• Pneumonia, bronchitis, bronchiectasis multiple organ systems
(mutation in NF2 tumor-suppressor gene on • Pancreatic insufficiency; steatorrhea
chromosome 22) • Fat-soluble vitamin deficiency Locus heterogeneity—
• Male infertility same disease phenotype from
• Bilateral acoustic neuromas • Biliary cirrhosis mutations in different loci
• Neurofibromas and café-au-lait spots • Meconium ileus
• Increased risk of meningiomas and • Most common mutation: ∆F508 Anticipation—
• D x: ↑ NaCl in sweat; PCR and ASO earlier age of onset and increased
pheochromocytoma disease severity with each
• 10% of NF cases probes generation
• Autosomal dominant • Tx: N-acetylcysteine, respiratory therapy,
Imprinting—
Marfan Syndrome enzyme replacement, vitamin supplement, symptoms depend on whether
inhaled bronchodilator, antibiotics mutant gene was inherited from
(mutation of fibrillin gene on chromosome 15) • Autosomal recessive father or mother; due to different
DNA methylation patterns of parents
• Skeletal abnormalities (tall build with (e.g., Prader-Willi versus Angelman
hyperextensible joints) syndrome)
• Subluxation of lens
• C ardiovascular defects (cystic medial
necrosis, dissecting aortic aneurysm, valvular
insufficiency)
• Autosomal dominant
Definition of abbreviations: ASO, allele-specific oligonucleotides; CFTR, cystic fibrosis transmembrane conductance regulator;
CNS, central nervous system; Dx, diagnosis; PCR, polymerase chain reaction; Tx, treatment.
73
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Chromosomal Abnormalities
Aneuploidy refers to having a chromosome number that is not a multiple of the haploid number. It is the most common type of chromosomal
disorder, and its incidence is related to increasing maternal age. Most arise from a nondisjunction event, when chromosomes fail to
segregate during cell division. Nondisjunction during either phase of meiosis usually leads to spontaneous abortion, but sometimes results
in live birth, often with severe physical deformities and mental retardation. Trisomies are the most common genetic cause of pregnancy
loss. Nondisjunction during mitosis in the developing embryo can lead to cells in a single individual carrying different karyotypes, a
condition known as mosaicism. Based on the Lyon hypothesis, females are naturally mosaics for genes on the X chromosome because
one X chromosome in every cell is randomly inactivated to form a Barr body. Fluorescence in situ hybridization (FISH) can detect DNA
sequences to identify deletions, translocations, and aneuploidies.
Nondisjunction During Meiosis I Autosomal Trisomies
Gametes Trisomy 21 (Down Syndrome)
Metaphase • Most common chromosomal disorder
of Meiosis 2 • E picanthal folds, brachycephaly, flat
Metaphase nasal bridge, low-set ears, and short,
of Meiosis 1 broad hands with single transverse
palmar crease
S, G2 • Mental retardation
Prophase • Early-onset Alzheimer disease
• Congenital septal defects in heart
Nondisjunction During Meiosis 1 • ↑ risk of acute leukemia
• Incidence: 1/800 births (1/25 if age >45
Disjunction During years)
Meiosis 2 • 9 5% nondisjunction; 4% Robertsonian
translocation
This figure shows the result of nondisjunction of one homologous pair (for example,
Trisomy 18 (Edwards Syndrome)
ccherllo. mTwosoomofeth2e1)gdFauimgriuenrtgeesmII-ae3r-ioe2Bsdi.siNp1loo.niAddlilfsoojurthncechrtriohonommDoosuolroimnggseMs2ee1gi.orsWeigsha1eten (disjoin) normally in the
fertilization occurs, the • Intrauterine growth retardation
• Mental retardation
conception will be a trisomy 21 with Down syndrome. The other gametes with no copy of • Failure to thrive
• Short sternum, small pelvis, rocker-
chromosome 21 will result in conceptions that are monosomy 21, a condition incompatible
bottom feet
with a live birth. • Cardiac, renal, and intestinal defects
• Usually death <1 year
Nondisjunction During Meiosis II • Incidence: 1/8,000 births
Gametes Trisomy 13 (Patau Syndrome)
Metaphase of • Microcephaly and abnormal brain
Meiosis 2 development
Metaphase of • Cleft lip and palate, polydactyly
Meiosis 1 • Cardiac dextroposition and septal defects
• Incidence: 1/25,000 births
S, G2
Prophase Sex Chromosome Aneuploidy
Turner Syndrome (45,XO)
• S hort stature, webbed neck, shield chest
• Primary amenorrhea, infertility
• Coarctation of aorta
• Incidence: 1/6,000 female births
Normal Disjunction Klinefelter Syndrome (47,XXY)
during Meiosis 1
• Eunuchoid body with lack of male
Nondisjunction secondary sex characteristics
during meiosis 2
• Hypogonadism, testicular atrophy
This figure shows the result of nondisjunction during meiosis 2. In this case, the sister • Incidence: 1/2,000 male births
chromatids of a chromoFsigoumre I(I-fo3-r2eCx. aNmonpdleis, juchnrcotimonosDoumrieng2M1)eifoasilisto2 segregate (disjoin). XYY Syndrome
The sister chromatids of all other chromosomes segregate normally. One of the gametes
• Excessively tall with severe acne
is diploid for chromosome 21. When fertilization occurs, the conception will be a trisomy • ↑ risk of behavioral problems
• Incidence: 1/1,000 male births
21 with Down syndrome. One gamete has no copy of chromosome 21 and will result in a
conception that is a monosomy 21. The remaining two gametes are normal haploid ones.
74
►►Other Chromosomal Abnormalities GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
In addition to aneuploidy, large segments of chromosomes may also undergo structural aberrations, including deletions, inversions, and
translocations. Deletions occur when a chromosome loses a segment because of breakage. Inversions are rearrangements of the gene
order within a single chromosome due to incorrect repair of two breaks. An inversion that includes a centromere is called a pericentric
inversion, while one that does not involve the centromere is paracentric. Finally, translocations involve exchange of chromosomal
material between nonhomologous chromosomes. Reciprocal translocations result when two nonhomologous chromosomes exchange
pieces, while Robertsonian translocations involve any two acrocentric chromosomes that break near the centromeres and rejoin with
a fusion of the q arms at the centromere and loss of the p arms.
Reciprocal Translocation
When one parent is a reciprocal translocation carrier:
• A djacent segregation produces unbalanced genetic material and a likely loss of pregnancy
• A lternate segregation produces a normal haploid gamete (and diploid conception) or a liveborn who is a phenotypically
normal translocation carrier
Parent with reciprocal
translocation
Reciprocal translocation in the germline
(translocation carrier) may result in a high
rate of pregnancy loss when the carrier tries
to have children.
2 t(2;8) 8
Alternate Segregation Adjacent Segregation
Fertilization with Normal Egg
Normal Translocation Partial Trisomy 8 Partial Trisomy 2 Near-complete Near-complete
Carrier Partial Monosomy 2 Partial Monosomy 8 trisomy 8 trisomy 2
Near-complete Near-complete
monosomy 2 monosomy 8
Live births Probable loss of pregnancy
Reciprocal Translocations in Somatic Cells May Result in Cancer
t(9;22) CML, ALL bcr-abl fusion produces a fusion protein with tyrosine kinase activity.
t(8;14) Burkitt lymphoma c-myc oncogene (chromosome 8) placed near Ig heavy chain locus. Activating Ig heavy
chain gene activates c-myc.
t(11;14) Mantle cell lymphoma bcl-1 (chromosome 11) encodes cyclin D. Translocation places bcl-1 near Ig heavy chain
locus (chromosome 14).
Definition of abbreviations: ALL, acute lymphocytic leukemia; CML, chronic myelogenous leukemia.
(Continued)
75
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Other Chromosomal Abnormalities (Cont’d.)
Consequences of a Robertsonian Translocation in One Parent
Approximately 5% of Down syndrome cases result from a Robertsonian translocation affecting chromosomes 14 and 21. When a
translocation carrier (in this case, a male) produces gametes, the translocation can segregate with the normal 14 or the normal 21.
Although adjacent segregation usually results in pregnancy loss, it can result in a trisomy 21. Alternate segregation produces a normal
haploid gamete (and diploid conception) or a liveborn who is a phenotypically normal translocation carrier.
Translocation carrier father
45,XY,-14,-21,+t(14;21)
t(14;21) 21
21 14
14 t(14;21) Adjacent Segregation
Alternate Segregation
Conception Product with Normal Egg
Normal Translocation Trisomy 21 Monosomy 21 Trisomy 14 Monosomy 14
diploid carrier Down Loss of pregnancy
Down syndrome Down syndrome
(nondisjunction during meiosis) (parent carries a Robertsonian translocation)
• 47,XX,+21 or 47,XY,+21 • 46,XX,-14,+t(14;21), or 46,XY,-14,+t(14;21)
• No association with prior pregnancy loss • May be associated with prior pregnancy losses
• Older mother • May be a young mother
• Very low recurrence rate • Recurrence rate 10–15% if mom is translocation carrier; 1–2% if
dad is translocation carrier
76
►►Additional Diseases Resulting from Chromosomal Abnormalities GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
Disease Abnormality Characteristics
Cri-du-chat Terminal or interstitial • Mental retardation
syndrome deletion of 5p • Cat-like cry
• Microcephaly, low-set ears, micrognathia
• Epicanthal folds
DiGeorge Deletion of 22q11 • Hereditary absence of thymus and parathyroid glands due to abnormal
syndrome development of 3rd and 4th pharyngeal pouches
• T-cell deficiency
• Cardiac outflow tract abnormalities
• Abnormal facies
• Hypoparathyroidism
Wilms tumor Deletion of 11p13 • Malignant urinary tract tumors
• ⅔ diagnosed by age 4
• Tx: surgical removal
Angelman Deletion of 15q11-q13 • “Happy puppet” syndrome
syndrome in mother • Always smiling but lacks speech
• Hyperactive, hypotonic
• Mental retardation, seizures
• Dysmorphic facial features
• Ataxic, puppet-like gait
Prader-Willi Deletion of 15q11-q13 • Short stature and obese with small hands and feet
syndrome in father • Hyperphagia
• Dysmorphic facial features
• Mental retardation
NOTE: Angelman and Prader-Willi are both examples of the effects of a deletion
in an area affected by imprinting. A minority of cases are caused by uniparental
disomy.
►►Population Genetics
The Hardy-Weinberg equilibrium states that under certain conditions, if the population is large and randomly mating, the genotypic
frequencies of the population will remain stable from generation to generation.
Hardy-Weinberg Conditions Factors Affecting Equilibrium
1. No mutations Natural Selection
2. No selection against a genotype
3. No migration or immigration of the population Increases frequencies of genes that promote
4. Random mating survival or fertility (e.g., malaria protection in
sickle cell heterozygotes)
If: frequency of A allele = p Genetic Drift
frequency of a allele = q
Gene frequency change due to finite
Then: allele frequencies can be expressed as: population size
p+q=1 Gene Flow
genotypic frequencies at that locus can be expressed as: Gene exchange between different populations
p2 + 2pq + q2 = 1 Linkage Disequilibrium
where p2 = frequency of genotype AA Preferential association of an allele at one
locus with another allele at a nearby locus
2pq = frequency of genotype Aa more frequently than by chance alone
q2 = frequency of genotype aa
77
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Subcellular Organelles
In contrast to simple prokaryotic cells that have a cell wall but no membrane-bound nucleus or organelles, eukaryotic cells are,
in general, larger and lack a cell wall, but are composed of various subcellular membranous organelles with distinct functions.
Nucleus
• S ite of DNA replication
and transcription
Euchromatin • Enclosed by nuclear
envelope
Peroxisomes
Heterochromatin • C ontains nucleolus
Mitochondrion (site of ribosome
Lysosome synthesis); no
Golgi
apparatus Nucleolus membrane surrounds
the nucleolus
Nucleus • C ontains DNA
packaged with histones
to form chromatin
Rough Endoplasmic Reticulum (RER)
Smooth Rough • C ontains ribosomes for
endoplasmic endoplasmic synthesizing proteins
reticulum (SER) reticulum (RER) destined for RER,
SER, Golgi, lysosomes,
Microtubule cell membrane, and
& filaments secretion
Prokaryotic Eukaryotic Copyright 2000 Gold Standard Multimedia, Inc. All rights reserved. • Cotranslational
modifications, including
N-linked glycosylation
(proteins synthesized on
free ribosomes are not
usually glycosylated)
Small (1−10 µm) Large (10−100 µm) Ribosomes
Thick, rigid cell wall No cell wall Types of Ribosomes
No membrane-bound Various subcellular • Site of protein synthesis RER- Free
organelles membranous organelles Bound Cytosolic
• Composed of ribosomal RNA (rRNA)
Non-membrane−bound Nucleus with double- and proteins forming large 60S + small Proteins Cytosolic,
nucleoid region membrane envelope 40S subunits for RER, mitochondrial,
SER, Golgi nuclear, and
Proteasome • Single mRNA simultaneously translated apparatus, peroxisomal
by several ribosomes is a polysome lysosomes, proteins
cell
• Small complexes of proteolytic enzymes in cytosol membrane,
• Digest (usually misfolded) proteins that are marked with and
secretion
ubiquitin
• P eptides produced are presented along with MHC I
at the cell surface
(Continued)
78
►►Subcellular Organelles (Cont’d.) Smooth Endoplasmic Reticulum (SER) GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
Golgi Apparatus
Plasma Secretory
membrane granules
Secretory Condensing
vesicles vacuole
TGN
trans
Membrane CIS Rough
vesicles Endoplasmic
Reticulum
Nucleus
Golgi Image copyright 1984 Lippincott Williams & Wilkins. Used with permission.
apparatus
• Involved in detoxification reactions, including phase I
• S ite of post-translational modifications and protein sorting hydroxylation (via cytochrome P450) and phase II
conjugation (addition of polar groups)
• Consists of disk-shaped cisternae in stacks
• Synthesis of phospholipids, lipoproteins, and sterols
• Cis (forming) face associated with RER • S equesters Ca2+; known as sarcoplasmic reticulum
• Trans (maturing) face oriented toward plasma membrane in striated and smooth muscle cells
Mitochondria Lysosomes
Cristae
Outer
membrane
Inner membrane
Intermembrane
compartment
• Major function is ATP synthesis Copyright 2000 Gold Standard Multimedia, Inc. All rights reserved.
• Similar to bacteria in size and shape; self-replicating
• Contain their own double-stranded circular DNA • E nzymatic degradation of extracellular or intracellular
• S mooth, permeable outer membrane; heavily infolded, macromolecules
impermeable inner membrane • P rimary lysosome fuses with phagosomes or cellular organelles
to form secondary lysosomes
Endosomes
• Acidic hydrolytic enzymes with optimal activity at pH 5
• Degradation of intracellular organelles known as autophagy
Peroxisomes
Mitochondria
• F ormed from endocytosed vesicles acquired by receptor- Peroxisome
mediated endocytosis involving clathrin-coated pits
• C an fuse with primary lysosomes to form secondary lysosomes
to degrade extracellular materials
• Exogenous peptides presented on membrane with MHC II on
antigen presenting cells
Copyright 2000 Gold Standard Multimedia, Inc. All rights reserved.
• Synthesis and degradation of hydrogen peroxide
• β-oxidation of very long chain fatty acids (>C24)
• Phospholipid exchange reactions
• Bile acid synthesis
79
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Plasma Membrane
The plasma membrane of a cell is a bilayer of lipids and proteins. The lipids include phospholipids, unesterified cholesterol,
and glycolipids and are amphipathic (polar head to interact with aqueous environment, and nonpolar tail to interact with the
bilayer interior). Proteins may act as adhesion molecules, receptors, transporters, channels, or enzymes. Proteins embedded
in the bilayer are integral proteins, whereas those loosely associated with the membrane are peripheral proteins. In general,
N-glycosylation of proteins and lipids is associated with location on the external surface, whereas N-myristoylation, prenylation, and
palmitoylation of proteins are associated with location on the cytoplasmic face of the plasma membrane.
Structure of Biologic Membranes
Integral Peripheral
protein protein
(N-glycosylation)
Extracellular
space
Hydrophilic end Phospholipid
Hydrophobic end
Cytoplasm Peripheral
protein
(N-myristoylation,
prenylation,
palmitolyation)
Types of Integral Membrane Proteins
Extracellular side
N CN
C N Type III C
Type I Type II
Intracellular side
Simple Diffusion Types of Transport Active Transport
• M ovement of highly permeable Facilitated Diffusion • Movement of molecules from a region
molecules from a region of higher to of lower to higher concentration with 1)
lower concentration • Passage of poorly permeable molecules energy expenditure via ATP hydrolysis,
from a region of higher to lower or 2) cotransport using another
• e.g., O2, CO2, NO concentration via a carrier molecule’s chemical gradient
• e.g., glucose transporter • e .g., 1) Na+/K+ pump, Ca2+-ATPase
• e .g., 2) Na+/glucose symporter
80
►►Cytoskeleton GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
The cytoskeleton consists of a supportive network of tubules and filaments in the cytoplasm of eukaryotic cells. It is a dynamic structure
responsible for cellular movement, changes in cell shape, and the contraction of muscle cells. It also provides the machinery for
intracellular movement of organelles. The cytoskeleton is composed of three types of supportive structures: microtubules*, intermediate
filaments, and microfilaments.
Microtubules* Intermediate Filaments Microfilaments Disease Association
Tubulin (hollow cylindrical • Keratin (epithelium) Actin (double-stranded Chediak-Higashi Syndrome
polymer of tubulin dimers) • V imentin (nonepithelial) polymer twisted in helical (defect in microtubule polymerization in
• N eurofilament (neurons) pattern) leukocytes)
• M ovement of Function • Structural • Recurrent pyogenic infections of
chromosomes in Structural • Muscle contraction via respiratory tract and skin
mitosis or meiosis
interaction with myosin • Partial albinism
• Intracellular transport
via motor proteins • Photophobia, nystagmus, peripheral
neuropathy, motor dysfunction, seizures
• C iliary and flagellar
motility • Presents early in childhood
Axoneme Structure Kartagener Syndrome
(immotile cilia due to defect in axonemal
proteins, such as dynein arms)
Spoke 9 BA Dynein arms • Chronic cough, rhinitis, and sinusitis
8 1 • Situs inversus
Central Nexin link • Fatigue and headaches
singlet 7 2 3 • Male infertility from immotile spermatozoa
4 • Autosomal recessive
5
6 Plasma Pharmacology
Bridge membrane
Colchicine
Central • Inhibits tubulin polymerization
sheath • Used for gout
Vincristine/Vinblastine
• Inhibits tubulin polymerization
• Antineoplastic
Taxol
• Promotes tubulin polymerization
• Antineoplastic
Motor Proteins
Kinesins for – → ⊕ anterograde direction
Dyneins for ⊕ → – retrograde direction
*Microtubules are polarized structures with assembly/disassembly occurring at the ⊕ ends, which are oriented toward the cell’s periphery.
81
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Cell Adhesion
A cell must physically interact via cell surface molecules with its external environment, whether it be the extracellular matrix or basement
membrane. The basement membrane is a sheet-like structure underlying virtually all epithelia, which consists of basal lamina (made of
type IV collagen, glycoproteins [e.g., laminin], and proteoglycans [e.g., heparin sulfate]), and reticular lamina (composed of reticular fibers).
Cell junctions anchor cells to each other, seal boundaries between cells, and form channels for direct transport and communication between
cells. The three types of junctional complexes include anchoring, tight, and gap junctions.
Cell Junctions Extracellular Matrix
Apical Microvilli Proteoglycans
surface Plasma • 90−95% carbohydrate; 5−10%
membrane
protein
• Forms hydrated gel for embedding
fibrous proteins
• P rovides shock absorption and
lubrication
Tight Intermediate Collagen
junction filaments • T riple helix of polypeptide chains
(keratin)
Adhesion Gap junctions rich in glycine and proline
belt Cell D • Collagens I−III: fibrous form for
Hemidesmosome
Desmosome structure
Gap • Collagen IV: sheet-like meshwork
Cell A Cell B Cell C Junctions
Gap junctions specific to basal lamina
Basal direct passage for small particles
lamina and ions between cells via Elastin
connexon channel proteins • C ross-linked fibers rich in glycine and
Anchoring Tight
Junctions Junctions Connexon LIniptriadcbeillaluylearr space proline
Tight junction (zonula 2–4 nm of cell A • Provides elasticity to tissues (e.g.,
• Adherens occludens)fusion of apposed
junction (zonula cell membranes LIniptriadcbeillaluylearr of cell B lungs and large arteries)
adherens) space
band-like junction Sealing Fibronectin
near apical region Strands 1.5 nm • L arge, fibrous protein with disulfide
for attachment to
adjacent epithelial Image copyright 1984 Lippincott Williams & Wilkins. Used with permission. 7 nm crosslinks
cells, forming an • P rovides adhesion between cells
“adhesion belt” Function Allows direct intercellular
Provides a tight seal to communications (e.g., allowing and extracellular matrix
• Desmosome ions to pass for synchronous
(macula prevent fluid leak between firing of cardiac pacemaker Laminin
adherens) compartments (e.g., between cells) • T hree polypeptide chains in shape
juxtaposition of intestinal lumen and intestinal
two disk-shaped villi) of a cross and connected by
plaques from disulfide bonds
adjacent cells, with • Major glycoprotein in basal lamina
IFs radiating away • Provides adhesion between cells
from the plaques; and extracellular matrix
hemidesmosomes
anchor cells to the Disease Association
extracellular matrix
Pemphigus Vulgaris
For structural integrity (autoantibodies against desmosomal
of large sheets proteins in skin cells)
of tissues (e.g.,
providing tensile • Painful flaccid bullae (blisters) in
strength of epithelial oropharynx and skin that rupture
tissues); adhesion easily
belt also allows
epithelial tissue • Postinflammatory hyperpigmentation
contractions • Treatment: corticosteroids
Bullous Pemphigoid
(autoantibodies against basement-
membrane hemidesmosomal proteins)
• Widespread blistering with pruritus
• L ess severe than pemphigus
vulgaris
• Rarely affects oral mucosa
• C an be drug induced (e.g., middle-
aged or elderly patient on multiple
medications)
• Treatment: corticosteroids
Definition of abbreviations: IF, intermediate filament.
82
►►Cell Cycle GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
The cell cycle consists of the mitosis phase (M), the presynthetic gap (G1), the DNA synthesis phase (S), and the postsynthetic gap (G2).
Mitosis is the shortest phase, consisting of prophase, metaphase, anaphase, and telophase. Both G1 and G2 phases are variable in
duration, with most cells spending much of their time in a stable, nondividing G0 phase. Cells in G2 have twice the amount of DNA as
those in G1.
Phases of Mitosis
PPrroopphhaassee
• Chromosomes coil
• Nuclear envelope
disappears
• Spindle apparatus forms
Mettaapphhaassee
Chromosomes align
AAnapphhaassee
Chromatids separate
Regulators of the Cell Cycle Teloopphhaassee • Chromosomes uncoil
• Nuclear envelope
Cyclins Cyclin levels rise and fall with stages of cell cycle.
CDKs reappears
APC Cyclin-dependent kinases with various substrates promote • Spindle apparatus
SPF cell-cycle progression.
MPF disassemble
Anaphase-promoting complex triggers chromatid separation; • Cell divides in two
p53 degrades M-phase cyclins.
RB (cytokinesis)
p21 S-phase promoting factor includes CDKs and cyclins, which
prepare cell for DNA replication. Disease Association
M-phase/maturation-promoting factor includes CDKs and Retinoblastoma
cyclins, which promote assembly of mitotic spindle and nuclear
envelope breakdown. (mutation in the RB1 tumor-suppressor gene on
chromosome 13)
p53 is a tumor suppressor that blocks cell cycle if DNA is damaged.
• Most common childhood eye tumor
Retinoblastoma susceptibility protein is a substrate of CDKs that • Leukocoria (white reflex in pupil)
promotes cell division. • Strabismus
• “Two-hit model” of carcinogenesis:
p21 is a CDK inhibitor that also blocks cell-cycle progression.
1) inherited mutation of one allele
2) somatic mutation of second allele
83
GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio ►►Cell Signaling
In order to act on a cell, external molecules, such as hormones and neurotransmitters, must interact with a receptor. In general, small
hydrophobic molecules (e.g., cortisol, sex hormones, thyroid hormone, and retinoids) can readily penetrate the plasma membrane to
bind intracellular receptors, which often act as transcription factors to affect gene expression. Most other molecules bind to cell surface
receptors, which include ion-channel−linked receptors (e.g., transmitter-gated channels), G-protein–linked receptors (the largest
family), and enzyme-linked receptors (e.g., tyrosine kinase receptors). These cell surface receptors usually transmit their signal via a
number of downstream second messengers, leading to a signal transduction cascade. One exception is the gaseous nitric oxide
(NO), which readily diffuses across the plasma membrane to activate soluble guanylate cyclase, generate cGMP, and promote smooth
muscle relaxation.
G-Protein−Coupled Receptor Systems
cAMP System PIP2 System Trimeric G Protein Cycle
+
+ NH3 1
NH3
Receptor for:
Receptor for: • Vasopressin αβ αβ
• Glucagon • Epinephrine α1 GDP GTP
• Epinephrine βα2(G(Gs)i)
• Epinephrine γ γ 2
Inactive G Protein Active G Protein
Membrane PIP2 DAG
Cytoplasm
Adenylate + Protein α
Cyclase Kinase C
β GTP
– + ATP
COO– α COO– α Phospho- 4γ Enzyme (e.g., Adenylate
CREB γβ γβ lipase C Cyclase)
IP3 Ca2+
Gs or Gi Gq 3
cAMP PDE
α
+ + Gene GDP Pi
+ ER Ca2+ Expression
Protein Kinase A
CREB P
Nucleus CREB P +
DNA CRE +1 + Protein Kinase
Gene Enzymes P
Dephosphorylated Enzymes
+ Phosphorylated
Gene Expression in Nucleus (Phosphatase)
Tyrosine Kinase Receptor
1 ss ss
Insulin Binding
Activates Tyrosine
s s s s Kinase Activity
P P P P 3 Insulin receptor
ADP ADP substrate
(IRS) binds receptor
and is phosphorylated
IRS-1 on tyrosine residues
ATP Tyrosine ATP
2 Kinase 4 SH2-domain proteins
bind phosphotyrosine
Autophosphorylation P P P residues on IRS
of Receptor SH2 SH2 SH2
PI-3 Kinase
Protein Kinase Protein
Enzymes P Protein Translocation
Dephosphorylated Enzymes of GLUT-4 to
Phosphorylated + membrane in:
p21ras
G protein • Adipose
• Muscle
Protein +
Phosphatase
Gene Expression in Nucleus
Definition of abbreviations: See next page. (Continued)
84
►►Cell Signaling (Cont’d.) GENERAL PRINCIPLES │ 3. Mol. Bio/Genetics/Cell Bio
Guanylate Cyclase Examples of Receptors
Adrenergic Receptors
Receptors for + Produced from Arginine by Drugs: • α1 (Gq)smooth muscle contraction
Atrial Natriuretic NH3 Nitric Oxide Synthase in • Nitroprusside • α2 (Gi/o)inhibits NT release
Factor (ANF) Vascular Endothelial Cells • Nitroglycerine • β1 (Gs)↑ heart rate and contractility
• β2 (Gs)smooth muscle relaxation
• Isosorbide
Muscarinic Acetylcholine Receptors
Nitric Oxide (NO) dinitrate
• M1 (Gq) affects CNS, PNS, gastric
Membrane parietal cells
Cytoplasm COO– NO • M 2 (Gi/o)↓ heart rate and contractility
cGMP + • M 3 (Gq) stimulates glandular secretions
GTP Soluble Guanylate Cyclase (heme) • M4 (Gi/o)CNS only; role unclear
• M5 (Gq)role unclear
+ GTP
Protein Kinase G Dopamine Receptors
Vascular Smooth
Muscle • D1 (Gs)s mooth muscle relaxation;
natriuresis; CNS effects
Relaxation of Smooth Muscle
(Vasodilation) • D2 (Gi/o) inhibits sympathetic
transmitter release; CNS
effects
• D3 (Gi/o)similar to D2
• D4 (Gi/o)similar to D2
• D5 (Gs)similar to D1
Vasopressin Receptors
Pharmacology • V1 (Gq)smooth muscle contraction
• V2 (Gs)↑ H2O reabsorption in kidney
Nitrates Sildenafil
Other Receptors
(nitroglycerin, nitroprusside, • Inhibits cGMP-dependent
isosorbide dinitrate) phosphodiesterase (PDE5), leading to • I nsulin (TK)↑ glycogen synthesis;
cGMP buildup, causing smooth muscle ↓ glycogenolysis
• Converted to NO, which activates relaxation and dilation of blood vessels
guanylate cyclase, leading to ↑ in leading to the corpus cavernosum • G lucagon (Gs) ↑ glycogenolysis;
cGMP. cGMP causes smooth muscle ↓ glycogen synthesis
relaxation of blood vessels. • F or erectile dysfunction and Raynaud
phenomenon • I GF (TK)↑ proliferation of various cell
• For angina and pulmonary edema types
• Adverse effects: headache, hypotension
• A dverse effects: headache, hypotension • P DGF (TK)↑ proliferation of connective
tissue, glial, and smooth
muscle cells
• EGF (TK) ↑ proliferation of
mesenchymal, glial, and
epithelial cells
• ANF (GC) smooth muscle relaxation;
↑ Na+ and H2O excretion in
kidney
• NO (GC)smooth muscle relaxation
Definition of abbreviations: ANF, atrial natriuretic factor; ATP, adenosine triphosphate; cGMP, cyclic guanosine monophosphate; DAG,
diacylglycerol; EGF, epidermal growth factor; ER, endoplasmic reticulum; GC, guanylate cyclase−coupled receptor; Gi/o, cAMP-inhibiting
GPCR; Gq, PLC-activating GPCR; Gs, cAMP-activating GPCR; IGF, insulin-like growth factor; PDE, phosphodiesterase; PDGF, platelet-
derived growth factor; PIP2, phosphoinositol biphosphate; PLC, phospholipase C; NO, nitric oxide; TK, tyrosine kinase receptor.
85
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