THE NITROGENOUS METABOLISM OF THE EARTHWORM (LUMBRICUS ...

THE NITROGENOUS METABOLISM OF THE EARTHWORM

(LUMBRICUS TERRESTRIS)

II. ARGINASE AND UREA SYNTHESIS

BY STANLEY COHEN AND HOWARD B. LEWIS

(From the Department of Biological Chemistry, Medical School, University of

Michigan, Ann Arbor)

(Received for publication, October 22,1949)

Negligible amounts of urea were excreted by fresh’ earthworms &urn- Downloaded from http://www.jbc.org/ by guest on February 8, 2016
bricus terrestris) ; however, on fasting, the quantity of urea in the excreta
increased.notably (1). The oral administration of arginine was followed
by an increased elimination of urea. The excretion of urea was also aug-
mented, to a lesser extent, by the ingestion of citrulline with certain
other amino acids. The data presented here were obtained in an attempt
to clarify the mechanism of urea synthesis in the earthworm by studies

in vitro. Arginase has been found to occur in the intestinal tissue of the
earthworm and to be present in increased amounts in fasted animals.

EXPERIMENTAL

Animals-Mature earthworms, ranging in weight from 3 to 5 gm. each,
were collected from the soil of the University campus and used for study
within 24 hours after collection.

PhosphateHomogenatesof Whole Worms-The S$rensenphosphate buffer
(pH 7.5) was added to 40 gm. of worms to give a total volume of 100ml.
and the mixture was cooled and homogenized in a Waring blendor. The
homogenate was centrifuged briefly and the supernatant fluid was used as
the enzyme preparation.

Phosphate Homogenatesof Intestines-After the worms had been nar-
cotized with gaseouscarbon dioxide (l), the section of the gastrointestinal
tract which contained the chlorogogue tissue was excised and washed with
saline solution.2 2 ml. of the phosphate buffer (pH 7.5) were added for
each gm. of tissue, and the mixture was homogenized in the apparatus of
Potter and Elvehjem (3). The homogenate was diluted with the phos-
phate buffer to give a final concentration of 5 to 15 per cent of tissue, wet

1 The term “fresh” as used in this discussion refers to worms which had been col-
lected within 24 hours prior to the experimental procedures.

2 The term “saline” is used to designate a 0.16 osmolar (0.08 N) solution of sodium
chloride. This solution has an osmotic pressure approximately equivalent to that
of the coelomic fluid of the earthworm (2).

479

480 ARGINASE IN EAR!l’FIWORMS

weight. Aliquots of these homogenates contained approximately 0.059 Downloaded from http://www.jbc.org/ by guest on February 8, 2016
mg. of nitrogen per mg. of dry tissue.

Incubation-3 ml. aliquots of the homogenized tissue, warmed to 29”,
were added to 3 ml. of the desired substrate in a test-tube (24 X 200 mm.).
After careful mixture, the solutions were adjusted to pH 7.5, and the in-
cubation was begun. The concentrations of the substrate are indicated
in the text. The incubation was carried out in a water bath at 29” for
1 hour; the tubes were shaken (115 oscillations per minute) during the
incubation.

When a solution of cobaltous chloride was added to the medium, it
replaced an equivalent quantity of water.

Procedure after Incubation-At the completion of an incubation period,
the enzymatic activity was stopped by the addition of 1 drop of 10 per
cent hydrochloric acid. The mixture was then heated for 2 minutes in a
boiling water bath and was centrifuged. Aliquots of the supernatant

fluid were adjusted to pH 6.4 by a solution of potassium hydroxide; bro-
mocresol purple was employed as an internal indicator. 1 ml. aliquots
were used for the determination of urea (1); all analyses were done in

duplicate.
Xubstrates-The preparations of arginine and hydantoin were those

used in work reported in our earlier paper (1). Glycocyamine was pre-
pared ,by the method of Wheeler and Merriam (4) and hydantoic acid by
the procedure of West (5).

Heidermanns had reported (6) that after incubation of the intact in-
testine of the earthworm with peptone there was an increased formation
of urea. Since a similar incubation with arginine did not effect a synthesis
of urea, the absence of arginase was assumed. We have confirmed these
results with arginine by the use of intact intestine of fresh earthworms.
However, we have not been able to demonstrate an increased synthesis
of urea after incubation with Witte’s peptone. We are unable to explain
the discrepancy between our observations and those of Heidermanns.

It was possible, however, to demonstrate the presence of arginase in
phosphate homogenates of the whole worm. In further studies, the in-
testinal tissue was removed and the arginase activities of the homoge-
nates of the intestine and of the carcass (the tissue remaining after the re-
moval of the intestine) were determined (Table I). Practically all of the
arginase activity was localized in the wall of the intestine; the slight ac-
tivity of the remaining tissues was probably due to small amounts of in-
testinal tissue not removed. No urease activity could be demonstrated
with phosphate homogenates of whole worm.

Under our conditions of incubation, the optimal pH for arginase activity
was found to lie between 7.2 and 8.6. The addition of Co++ in a concen-

S. COHEN AND H. B. LEWIS 481

tration of lo+ Mincreased the activity of the arginase. The extent of this
increase in activity was dependent, to some degree, upon the length of
time the enzyme was allowed to remain in contact with the Co++ before
the substrate was added. For example, the relative activities of the
arginase in homogenates of intestine from fresh worms when measured
without the addition of cobalt ions, with Co++ at a concentration of 1OV
M, and when the same concentration of Co++ was allowed to remain in
contact with the enzyme for 30 minutes at room temperature before the
substrate was added, were, respectively, 1.0, 1.3, and 2.1. When the
Co++ was allowed to remain in contact with the enzyme for 3 hours before
the addition of the substrate, the relative activity rose only to approxi-

TABLE I Downloaded from http://www.jbc.org/ by guest on February 8, 2016

Localization of Arginase in Intestine of Fasted Earthworms

Final concentration of arginine, 200 mg. per cent; of Co++, lo+ M. The values

are calculated as micrograms of urea N formed per 100 mg. of tissue (wet weight)

in 1 hour. -

Experiment No. Incubation mixture Arginase activity

1 Intestine + arginine 3:6
2 “ +“ 364
13
3 Carcass + arginine
4 “+ “ 13
5
5 Intestine + water
6 “ +“ 5
4
7 Carcass + water 4
8 ‘I + “

mately 2.2. In the absenceof cobalt ions, the arginaseactivity of homoge-
nates of the intestinal tract decreasedrapidly on standing at room tempera-

ture. These observations are similar to those of Hunter and Downs (7),
who studied the arginase of beef liver.

The arginase of the intestine of fresh worms (homogenates) failed to
form urea from guanidoacetic acid (glycocyamine), hydantoin, or hydantoic

acid.
In a seriesof experiments, preformed urea was determined in homoge-

nates of the whole worm as fasting progressed up to 14 days. The urea
content of the worms was calculated as micrograms of urea nitrogen per
100 mg. of whole worm (Series 1, Table II). Arginase activity was also
measured in the homogenates of this series, the activity being calculated
as micrograms of urea formed per 100 mg. of worm per hour. In another
series (Series 2, Table II), arginase activity of intestinal homogenateswas

482 ARGINASE IN EARTHWORMS

determined and the results are expressed as micrograms of urea nitrogen
formed per mg. of tissue nitrogen per hour.

For the determination of the urea content of the tissues, aliquots of the
phosphate homogenates of the whole worm were heated, immediately
after preparation, in a boiling water bath for 2 minutes, the solutions were
centrifuged, and the centrifugates were analyzed for urea. In the deter-
minations of arginase activity the incubation procedure was used with a

TABLE II

Urea Content and Arginase Activity of Homogenates of Intestines and Whole Worms

See the text for the details of procedure and calculation of results. The basis

of calculation of arginase activity is different in the two series. - Downloaded from http://www.jbc.org/ by guest on February 8, 2016
-

SeriesNo. Emxpeenrti- Condition of animal foPrRm-ed Aargcitniavsiety
NO. ureaN

--

YY

1. Whole animal 1 Fresh 1 11
2“ 1 11
3“ 1 12

4 Fasted 7 days 7 16
5 “ 7 ‘I
6 ‘L 7 “ 10 19
9 15

7 “ 14 “ 52 36
“ 14 “
8 ‘I 14 “ 57 33
9 60 41

2. Intestine 10 Fresh 26
11 “
12 ‘I 54
62

13 Fasted 29 days 504

14 ‘( 29 “ 626
15 “ 29 “ 600’

16 “ 37 “ 594
17 “ 37 (‘ 618
-

final arginine concentration of 500 mg. per cent. No Co++ was added to
homogenates of whole worm; in the studies with the intestinal homoge-
nates, Co* was added to activate the enzyme as already described. As
shown in Table II, not only was there a progressive increase in the urea
nitrogen of the whole organism during fasting, but there also occurred a
parallel increase in arginase activity of homogenates of both the whole
worm and the intestine. The increased arginase activity of homogenates
prepared from the intestine of fasted worms compared with those from
fresh worms could be demonstrated in the absence of Co++, in the presence
of Co++ (lo+ M), and also when Co++ was allowed to activate the enzyme

8. COHEN AND H. B. LEWIS 483

for periods up to 2.5 hours before the activity of the arginase was meas- Downloaded from http://www.jbc.org/ by guest on February 8, 2016
ured.

DISCUSSION

It has been shown in a previous paper (1) that the pattern of nitrogenous
excretion in earthworms underwent a marked change during prolonged
fasting, a change characterized by a progressive increase in the quantity
of urea synthesized. The data here presented demonstrate that after 14
days of inanition the urea nitrogen content of the tissues had increased
from negligible amounts to 57 mg. per 100 gm.

The presence of the enzyme arginase has been demonstrated in the
intestinal tissue of both fresh and fasted earthworms. The arginase ac-
tivity of the intestinal tract of worms after 4 weeks of inanition was ap
proximately 10 times that of fresh worms. In other experiments it was
observed that under conditions of incubation in which the intact intestine
of fresh worms showed negligible arginase activity intact preparations of
the intestine of fasting animals showed significant arginase activity. It
may be noted that a 2-fold increase in arginase activity of the livers of
normal rats either after a prolonged period of fasting or after the inges-
tion of a high protein diet has been reported (8).

Urea may be formed from uric acid by the activity of the enzymes
uricase, allantoinase, and allantoicase (9) or the synthesis may involve
the ornithine-citrulline-arginine cycle (10). Since neither uricase, allan-
toinase, nor allantoicase is stated to be present in the tissue of the earth-
worm (11, 12), the first mode of synthesis of urea is precluded. An essen-
tial factor in the ornithine cycle of Krebs and Henseleit is the enzyme
arginase. The presence of this enzyme in tissues of the earthworm and
the increased activity during fasting may indicate that the ornithine cycle
functions in urea synthesis by the earthworm.

SUMMARY

1. The urea content of the earthworm increased markedly during inani-
tion.

2. Arginase was present in homogenates of the intestinal tissue of the
earthworms.

3. The arginase activity of homogenates of the intestine of the earth-
worm was increased about lo-fold after prolonged inanition (24 to 30 days).

BIBLIOGRAPHY’

1. Cohen, S., and Lewis, H. B., J. Biol. Chem., 180, 79 (1949).
2. Adolph, E. F., J. Exp. Zool., 47,31 (1927).
3. Potter, V. R., and Elvehjem, C. A., J. Biol. Chem., 114,495 (1936).

484 ARGINASE IN EARTHWORMS

4. Wheeler, H. L., and Merriam, H. F., Am. Chem. J., 29,478 (1903).
5. West, C. J., J. Biol. Chem., 34, 187 (1918).
6. Heidermanns, C., 2001. Jahrb., Abt. allgem. 2001. u. Physiol., 68,57 (1937).
7. Hunter, A., and Downs, C. E., J. Biol. Chem., 166,173 (1944).
8. Lightbody, H. D., and Kleinman, A., J. Biol. Chem., 129, 71 (1939); Proc. Sot.

Exp. Biol. and Med., 46, 25 (1940).
9. Florkin, M., L’evolution du metabolisme des substances azotees chez les animaux,

Actualites biochimiques, Liege and Paris, No. 3 (1945).
10. Krebs, H. A., and Henseleit, K., 2. physiol. Chem., 210, 33 (1932).
11. Truszkowski, R., and Chajkin6wna, S., Biochem. J., 29,236l (1935).
12. Florkin, M., and Duchateau, G., Arch. internat. physiol., 63, 267 (1943).

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THE NITROGENOUS METABOLISM OF Downloaded from http://www.jbc.org/ by guest on February 8, 2016
THE EARTHWORM (LUMBRICUS
TERRESTRIS): II. ARGINASE AND
UREA SYNTHESIS
Stanley Cohen and Howard B. Lewis

J. Biol. Chem. 1950, 184:479-484.

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