NITROGENOUS WASTE PRODUCTS AND EXCRETORY ENZYMES
IN THE MARINE POLYCHAETE CIRRIFORMIA SPIRABRANCHA
(MOORE, 1904)
INTRODUCTION
Although some information is available on nitrogenous
excretion in terrestrial oligochaetes, little is known about
the subject in marine polychaetes. Bahl did the first consis¬
tent study on oligochaete excretion. He found that ammonia,
urea, and creatinine were excreted through nephridia in the
body wall. Cohen and Lewis (1950) found high arginase activity
in the gut wall of Lumbricus and almost no activity in the body
wall. They concluded that the earthworm utilizes the ornithine¬
urea cycle as part of its nitrogen metabolism. More recently,
Needham (1960) has compared arginase activity in two oligochaetes,
Lumbricus and Eisenia, in relation to the amount of urea: ammon
ia excreted during feeding and fasting regimes. Needham's work
supported the study done earlier by Cohen and Lewis on the earth¬
worm. In the present study done on the cirratulid polychaete,
C. spirabrancha, nitrogenous excretion products have been deter¬
mined and levels of enzymes involved in nitrogen metabolism have
been measured. The major waste product was found to be ammonia,
which is excreted through the tentacles and anterior body wall.
Urea and traces of taurine were also found in the aqueous excre¬
ta. The tissues exhibit all of the purine catabolism enzymes,
uricase, allantoinase, allantoicase, and urease. Three enzymes
in the ornithine-urea cycle were assayed: ornithine transcar¬
bamylase, argininosuccinate lyase, and arginase. Argininosuc¬
cinate synthetase and carbamyl phosphate synthetase were not
studied.
EXPERIMENTAL PROCEDURES
MATERIALS
Animals: Worms were collected daily from the mudflats
under Fisherman's Wharf, Monterey, California. Each worm weigh¬
ed from 0.3 to 1.3 grams. The tentacles, dorsal blood vessel,
body wall and gut wall were selectively removed for study.
Tissues were washed in sterile sea water to remove any coelomic
fluid, mucus, and gametes. The tissues were kept cold and weigh¬
ed in tared flasks.
Chemicals: 1-arginine (HCl), 1-lysine (HCl), 1-aspartic
acid, 1-cysteine (HCl), dl-asparagine, glycine, glycylglycine,
1-serine, 1-ornithine (HCl), 1-leucine, dl-histidine (HCl), 1¬
proline, hydroxy-1-proline, 1-cysteic acid, dl-tyrosine, 1-citrul¬
line, 1-valine, and argininosuccinate were obtained from Calbio¬
chem. L-phenylalanine, 1-glutamic acid, 1-tryptophan, alanine,
cystine, dilithium carbamyl phosphate, 2,3-butanedione-2-oxime,
allantoin, cetyltrimethylammonium bromide, tris (hydroxymethyl)
aminomethane, and uric acid were ordered from Sigma. Ninspray
and taurine came from Nutritional Biochemicals. Allantoic acid
was obtained from K and K.
GENERAL METHODS
Collection of aqueous excreta: Fresh worms were dried
for 30 seconds on Whatman no. 1 filter paper, weighed, and placed
in plastic snap top vials along with 1.0 ml. of sterile sea
water containing streptomycin and penicillin at 1 mg. and 0.3
mg., respectively, per ml. At every collection interval 1 ml.
was drawn off and replaced by fresh sterile sea water. The sam¬
ples were analyzed for ammonia, urea, uric acid, alkaline labile
amides, and hot trichloroacetic acid labile amides. Excretion
1å
of free amino acids was also investigated.
Analysis of free amino acids in aqueous excreta: Approx¬
imately 100 ml. of aqueous excreta (the term urine will be used
to refer to aqueous excreta collected from around the worms.
Bahl (1947) used similar terminology) was desalted by passing
the solution through a column of Dowex-50 (H' form, 200-400
mesh). Enough Dowex-50 was used to desalt two times the number
of millequivalents of monovalent cations in 100 ml. of sea water.
The column was eluted with 1 N NHAOH and the pH of the effluent
was monitored at all times. When the pH of the effluent changed
froml.2 to 4.4, the column was washed with three bed volumes
of 1 N NH,OH and all effluents combined. This amino acid frac¬
tion was evaporated to dryness in a flash evaporator and the cry¬
talline residue taken up in 1.0 ml. of distilled water. 10
ul, of amino acid extract was spotted on a 20 cm. by 20 cm. piece.
of Whatman no. 1 filter paper and developed in two dimensions
with n-butanol-acetic acid-water (4:1:5, the top layer of this
two layer system was used as the mobile phase) as the first sol
ventand n-butanol-2-butanone-water (2:2:1), with the chamber sat¬
urated with cyclohexylamine (Mizell and Simpson) as the second
solvent system. A standard map was made using arginine, lysine,
aspartic acid, cysteine, glutamine, asparagine, cystine, glycine
hydroxyproline, alanine, proline, cysteic acid, histidine, valine,
tyrosine, leucine, tryptophan, phenylalanine, glutamic acid, ser¬
ine, taurine, ornithine and citrulline. All chromatograms were
developed with Ninspray. It was necessary to use only one'ad¬
ditional identification procedure: the reaction between taurine
and o-phthalaldehyde followed by alcoholic KOH (Smith).
Analytical methods: Ammonia was determined by a modif-
16
ication of Ternberg's (1964) method. Erlenmeyer flasks (125 ml.)
fitted with rubber serum stoppers, with sleeves, were substit¬
uted for the side arm flasks suggested by Ternberg. Saturated
sodium carbonate was introduced into the sealed vessel by means
of a syringe. The development tabs were cut from Whatman no. 1
filter paper and in all procedures handling the paper rubber
gloves were worn to avoid contamination from the skin. The method
proved to be very consistent (+ 0.015 u mole NHa was the standard
deviation for the urease solution used in the development of sam¬
ples containing urea and the standard deviation for the standard
ammonium sulfate solution was + 0.004 u mole NH3 )and ten times
more sensitive than Nesslerization.
Urea was determined as the difference (endogenous ammon¬
ia + ammonia liberated by urease) minus (endogenous ammonia a¬
lone). Samples were incubated with 100 ul. of a 10% solution
of urease (Matheson, Coleman, and Bell) in 30% ethanol for fif¬
teen minutes on a rotating platform. The modified Ternberg method
was employed for ammonia determinations.
Uric acid was determined by the colorimetric method of
Sobrino-Simoes (1965). Interfering substances are glucose and
ascorbic acid.
Citrulline was determined by the method of Archibald (1945),
It was necessary to use technical grade sulfuric acid (Ratner)
filtered through glass wool. It was also found that wrapping the
developing tubes in aluminum foil preserved the optical density
for up to four hours. The problem of interference by allantoin
and urea was avoided by filtering all samples through Whatman no.
1 filterpaper directly into a column (1 cm. by 5 cm.) of Dowex¬
50 and isolating the citrulline according to Watts, et al.
7
Glyoxylic acid was determined by the method of Brown
(1968). 2 ml. of 0.5 M HClO followed by 1 ml. of 2,4-dinitro¬
phenylhydrazine solution (50 mg./100 ml. 95% ethanol) were added
to 2 ml. of sample to be analyzed and this mixture was allowed to
stand at room temperature for 10 minutes to effect formation of the
2,4-dinitrophenylhydrazone of glyoxylic acid. The final color
was developed by the addition of 2.50 ml. of 2.5 N NaoH with
rapid mixing. The color was transient (change in O.D. of 0.200
in one hour) but reproducible when readings were taken after a
fixed interval (15 minutes) for all samples.
Two different methods were utilized in the determination
of labile amides in the tissues of the worm. The first method
was a general alkaline hydrolysis carried out in 1 N NaoH for
30 minutes at room temperature. The second method was an acid
hydr; lysis taken from Harris' (1943) glutamine hydrolysis pro-
cedure. Samples were digested in 10% TCA at 70 degrees for 75
minutes, cooled, brought to pH 11 in the sealed microdiffusion
chamber and analyzed for ammonia. This method hydrolyzes glu¬
tamine completely. Under the same conditions asparagine gives
11% hydrolysis and urea 0.2%, whereas arginine, guanidine, guanine,
adenine, creatinine, and glutamic acid liberate no appreciable
amount of ammonia. The difference between total ammonia measur¬
ed after one of the two procedures and the endogenous ammonia
gave a measure of the amount of bound or labile ammonia pres¬
ent in the tissue of the worm. A control was run for each method:
for the alkaline hydrolysis, a tissue sample was brought to pH
11 and analyzed for ammonia. For the Harris method, hydrolysis
in 10% TCA for 75 minutes was carried out at 5 degrees.
Tissue homogenates: After dissection and weighing, tis-
sues were extracted with 1 ml. of homogenizing media per 0.125 g
of tissue. For some assays, 10% homogenates were used. Body
Walls and tentacles were homogenized in a motorized teflon pes¬
tle homogenizer; guts and blood were homogenized in ground glass
homogenizers.
Ornithine transcarbamylase assay: The method of Brown
and Cohen (1959) was modified for use with polychaete tissues.
0.5 ml. of CTB supernatant Sj and 0.25 ml. of supernatant S9
were added to 0.5 ml. 1-ornithine, pH 8.0, and 0.5 ml. Na gly¬
cylglycine buffer, pH 8.3, and the reaction started by addition
of 0.5 ml. carbamyl phosphate solution. The final u molar amounts
of substrates and buffers were the same as those used by Brown and
Cohen. The incubation was carried out at 24 degrees (as were
all enzyme assays to be described) and stopped by the addition
of 5.0 ml of 0.5 M HClOA. The deproteinized sample was filter¬
ed through Whatman no. 1 filter paper and directly applied to a
column of Dowex-50. Citrulline was eluted and four ml. of the
effluent was analyzed according to Archibald.
Argininosuccinate lyase enzyme assay: Activity was
assayed by a method similar to Needham's (1960) arginase assay,
with 2.0 ml. of 1.5% argininosuccinate in place of arginine as
the substrate. No attempts were made to substitute a C. spira¬
brancha "Ringer" for the appropriate amount of earthworm Ringer
suggested by Needham. The M/4 phosphate buffer (pH 7.4) was used
for all dilutions of tissue homogenates. The endogenous arginase
split any arginine formed during the incubation and the resulting
urea was determined as previously described.
Arginase assay: The method above was used only with the
substrate being 2 ml. of a 2% solution of arginine HCl.
72
Urease assay: Some modifications of the preceding method
afforded a good urease assay. Tissues were homogenized in M/4
phosphate buffer (pH 7.4), diluted to 8 ml. and 2 ml. of 1%
urea were added to start the reaction. Addition of the arginase
cofactor Cocly was unnecessary in this procedure.
Uricase assay: The method for determining uricase was
adapted from Jorgenson's (1963) procedure for the enzymatic de¬
termination of uric acid and xanthine.
Tissue homogenates
were prepared in 0.06 M glycine buffer (pH 9.3). 0.2 ml. of
homogenate was diluted to 2.0 ml. with the same glycine buffer,
The reaction was initiated by the addition of 1 ml. of 1.5 X
10-4 M uric acid solution and the resulting change in optical
density was recorded on a strip chart connected to a Beckman DU
Spectrophotometer with Gilford Linear Absorbance attachments.
Allantoinase assay: The method of Brown (1968) was em-
ployed for this assay. Homogenates were prepared with tris (hy¬
droxymethyl)aminomethane buffer (50 u moles per ml.), pH 7.8,
and a standard curve was prepared with known concentrations of
allantoic acid. All samples went through a heat treatment in or¬
der to cleave the allantoic acid into urea and glyoxylate and the
glyoxylate was measured as previously described.
Allantoicase assay: Tissues were homogenized in M/4
phosphate buffer, pH 7.4, diluted to 8.5 ml. with more phosphate
buffer, and the reaction initiated by the addition of 1.5 ml.
of 2% allantoic acid solution. The standard assay for urea was
applied to 1 ml. samples every hour over a four hour period,
Blanks run in parallel gave no increase in the amount of ammonia
during the incubation period.
Specific activity: specific activity will refer to
7
the number of u moles of substrate catalytically changed, or
u moles of product produced, per hour under assay conditions,
per gram of wet tissue.
RESULTS
Analysis of urine: Ammonia was found to be the primary
constituent of the aqueous excreta. The level of urea ranged
from 0% in most samples analyzed (73 of 75) to 3.2% (in 2 of
75 samples). It is doubtful that urea is excreted. Uric acid
was below detection (less than 0.012 u mole per ml.). Incuba¬
tion of the urine in 1 N NaoH for 30 minutes gave no increase
over the level of endogenous ammonia. Similarly, the glutamine
hydrolysis in hot 10% TCA gave no more ammonia than in the nor¬
mally developed sample. Thus, labile amides in the urine are not
detectable and are assumed to be negligible. The rate of ammonia
output, in u moles/hour/g wet tissue, does not significantly
fluctuate over a 36 hour period nor does it fluctuate over a
shorter time period of 14 hours. Table 1 summarizes these results.
The results of the chromatographic study done on the
urine indicate that amino acids are not actively excreted by
C. spirabrancha. Only one compound gave a positive ninhydrin
test after the desalted urine concentrate was separated in two
dimensions: taurine was identified by matching it with the a¬
ppropriate spot on the map and proof of identification was made
by the unknown reacting with o-phthalaldehyde in acetone followed
by alcoholic KOH, giving the red color characteristic of taurine,
One other detectable substance with a very low Re (less than
0.05) was not identified, but it was assumed to be an arginine
type compound (Re arginine=0.07) such as taurocyamine.
Anatomical location of nitrogenous waste products in
different tissues;
The blood, tentacles, body wall, gut wall and aqueous gut con¬
tents of C. spirabrancha were analyzed for ammonia and urea in
both fresh worms and in worms fasting for one week. The same
tissues were assayed for uric acid, citrulline and two kinds of
labile amides. Results are summarized in Table 2. The tentacles
and gut wall show marked drops in ammonia levels upon inanition
while the level in the body wall shows a small decrease and the
level in the blood rises. The urea level in the tentacles, body
wall, and gut wall drops greatly, while the level of urea in the
blood drops less markedly. The level of ammonia and both al¬
kaline and acid labile amides are conspicuously high in the tent¬
acles. The gut wall, blood and body wall have much lower levels
of ammonia, descending in that order, and the ammonia content of
the gut contents is very low. Basic hydrolysis of the blood, body
wall and tentacles released more bound ammonia than the acid hy¬
drolysis, whereas the opposite situation is true in the gut wall.
Urea in the blood proved to be approximately twice the level found
in the three other tissues. The uric acid was very high in the
blood, much lower in the tentacles and gut wall, and below detec¬
tion in the body wall and aqueous gut contents (less than 0.095
u mole per gram of tissue).
Enzyme activities in different tissues: Three enzymes of
the ornithine-urea cycle were examined. Ornithine transcarbamyl¬
ase has the highest specific activity (71.6) of any of the three
enzymes. The blood contains the greatest cumulative activity of
all three of the enzymes. Table 3 summarizes the specific activ¬
ities of ornithine transcarbamylase, argininosuccinate lyase, and
arginase.
All of the enzymes involved in purine catabolism were
examined and allantoinase in the blood has the highest specif¬
ic activity (66.50). It appears that both uricase and allanto¬
icase are both limiting in C. spirabrancha. Table 3 summarizes
the specific activities of these enzymes.
DISCUSSION
It is clear that the ammonia excreted by C. spirabrancha
comes from two sources: the first being the deamination of amino
acids, such as glutamine or other labile amides, which occur in
especially high levels in the tissues associated with excretion
(predominantly the tentacles and, to a lesser extent, the anter¬
ior body wall. This topic will be discussed in detail later in
the discussion.). The second source is purine catabolism. This
polychaete seems to have beneficially compartmentalized various
steps in the degradation of purines to ammonia. The blood poss¬
esses the highest uricase and allantoinase activities and the
lowest allantoicase activity, 9.13, 66.50, and 3.72, respect¬
ively. This suggests that mainly non-toxic degradation occurs
within the main body of the worm, while at the site of excretion,
the high levels of allantoicase and urease convert the allantoic
acid completely to ammonia, which then leaves the body or becomes
bound as a non-toxic amide such as glutamine. The latter fate of
the ammonia is highly likely because the rate of ammonia excre¬
tion is fairly constant and this would demand a regulation sys¬
tem which would internally control the endogenous ammonia.
It is of interest to note that the two limiting en¬
zymes in the purine cycle, uricase and allantoicase, have very sim¬
ilar maximum rates, 9.13 and 9.15, respectively, and occur in the
ratio of about 1:6 when compared to the non-limiting enzyme, allan¬
toinase. The study done by Brown, et al. (1966) on the purine cycle
enzymes in the liver of the African lungfish yields similar re¬
176
lationships. Both limiting enzymes have very similar activities,
1.27 and 1.25, and the ratio between limiting enzyme and the en¬
zyme in excess is roughly 5. Perhaps these similarities are
characteristic of organisms utilizing a purine cycle. This
speculation will require much comparative work for justification.
The possibility that the ornithine-urea cycle is function¬
al in C. spirabrancha cannot be dismissed. Ornithine transcar¬
bamylase in the blood has the highest activity of any enzyme
assayed and argininosuccinate lyase and arginase are also def¬
initely present. Neither carbamyl phosphate synthetase nor ar¬
gininosuccinate synthetase were studied. The unusually high ac¬
tivity of ornithine transcarbamylase compared with the other two
enzymes studied and the multiplicity of metabolic functions of
the intermediates in the ornithine-urea cycle suggest many other
possibilities for the presence of these enzymes than for strictly
the synthesis of urea. The arginase may be present in conjunc¬
tion with arginine phosphagens or it may serve to catalyze the
breakdown of ingested arginine to urea. In addition, 1-ornithine
can react with + -keto-glutarate (Bernfield) to eventually form
1-proline and carbamyl phosphate can act as a precursor in pyr¬
imidine biosynthesis through the intermediate uridylic acid.
The high concentration of endogenous citrulline strongly
suggests a conventional role for the ornithine cycle enzymes.
The presence of the cleavage enzyme strengthens the argument and
also suggests that the limiting enzyme is the condensing enzyme
a high citrulline concentration).
The distribution of urease in the four tissues studied
illustrates a rational scheme for choosing the sight of ammonia
excretion. The internal tissues, the blood and gut wall, are
very low in urease activity and very high in enzymes necessary
(thus
for biosynthesis of urea or its immediate precursors, while the
external tissues, the tentacles especially and the anterior body
wall, have very high urease activity.
The proposed anatomical sites of excretion correlate
well with the general behaviour of the worm and the descriptive
anatomy of the nephridia. The worm lives in a mucus burrow im¬
pacted in black, sulfide mud, and it waves its tentacles in the
circulating fresh sea water above the mud. Excretion of nitrogen
ous wastes through the tentacles, which have a very large sur¬
face area, would not expose the worm to high concentrations of
toxic ammonia within the stationary burrow.
The single pair of nephridia (Gilbert) are located ven¬
trally behind the first septum and extend posteriorly 3 to 5 seg¬
ments. The internal funnel is highly ciliated, giving each
nephridium the power to circulate a great quantity of fluid.
Each nephridium is intensely vascularized and possesses a large
nephridiopore (25-30 u in diameter). This proposed site of ex¬
cretion, the anterior body wall, would also allow dilution of tox¬
ic ammonia by the shifting sea water above the worm.
The maintainence of the ammonia level in the blood of a
fasting worm suggests that the blood contains most of the bio¬
synthetic machinery involved in nitrogen homeostasis, whereas the
body wall and tentacles mainly rids the body of nitrogenous wastes.
The finding of taurine as the only constituent besides
ammonia and a small amount of urea in the urine is perfectly
reasonable when one considers that 3% of the body wall's weight
is taurine (Luck) and some leakage can be expected.
There is no comparative literature on this subject ex¬
cept for the studies done on oligochaetes. Since the environ¬
126
ments are so diverse, a comparison would not be meaningful.
SUMMARY
1. C. spirabrancha primarily excretes ammonia, abng with
trace amounts of urea and taurine. No amino acids were found to
be excreted.
2. Studies of uricase, allantoinase, allantoicase, and
urease suggest that the ammonia primarily results from a urico-
lytic pathway.
3 The presence of ornithine transcarbamylase, arginino¬
guccinate lyase, arginase and citrulline in the tissues strongly
suggest a functional ornithine-urea cycle.
4. Glutamine-like labile amides present in the greatest
amounts in the tissues associated with excretion support the idea
that amino acid deamination may also play a role in the formation
of ammonia.
5. The rate of ammonia excretion in this sedentary
polychaete is relatively constant.
6. The ammonia content of the aqueous gut contents is
very low, which suggests that there is no enteronephric excretion.
7. The most probable sites of excretion are the tent¬
acles and, to a lesser extent, the anterior body wall, which con¬
tains a single pair of large nephridia. This hypothesis is sup¬
ported by biochemical assays done on the tissues, by behavioural
studies and by descriptive anatomy of the nephridia.
8. Following inanition, the levels of ammonia and urea
drop markedly in the tissues associated with excretion and main¬
tain their levels in the tissues associated with the bulk of the
catabolic machinery.
17
ACKNOWLEDGEMENTS
I would like to thank Prof. David Epel and John Phillips
for the time they spent with me and for their helpful suggestions,
Tom Gilbert's description of C. spirabrancha nephridia was greatly
appreciated as was the allantoinase assay procedure sent to me
by Prof. G.W. Brown. This work was supported by the Undergrad¬
uate Research Program of the National Science Foundation, Grant
4369.
86
time of
collect¬
ion¬
in hours
12
18
24
30
36
number
of
samples
5
5
5
TABLE 1
u moles
time of
collect¬
of ammon
ion¬
ia excre
ted per
in hours
hour per
gram wet
tissue
0.128
0.164
0.155
6
8
0.210
0.166
10
0.168
12

14
number
samples

5
u moles
of ammon¬
ia excre¬
ted per
hour per
gram wet
tissue
0.214
0.142
0.143
0.150
0.096
0.101
—
0.128
18
Tissue:
u mole of
endogenous
compound /
gram wet tis
sue
ammonia
ammonia-af¬
ter fasting
lor / days
urea
urea-after
fasting for
days
citrulline
uric acid
alkane
drolysis
1 N NaoH
alkaline con
trol-pH 1l
acid hydrol¬
vsis in hot
10% TCA
acid control
cold 10% TCA
entacles
30.35
23.94
3.32
1.22
4.36
1.34
85.76
19.01
67.65
8.30
TABLE 2
blood
body wall
11.48
7.88
12.48
7.13
6.38
3.74
4.88
O.37
5.82
2.62
below
4.15
detection
24.39
32.28
no increaße
2.94
over en¬
dog. NII3
11.36
3.71
no incr.
no incr.
gut wall
14.94
9.77
3.56
0.71
2.23
1.01
18.18
no incr.
39.98
no incr.
aqueous
gut
contents
2.65
—
below
detectior
6.06
no incr.
5.00
no incr.
182
90
8
7
60
50
•—
J
40
30

20
10
oo

FIGURE 1

k
40
20
minutes
60
BLOOD
GUT WALL
BODY WALL
TENTACLES
8
O
E
1.80
1.6(
0.20
0

FIGURE 2
ENTACLES

40
20
60
minute:
6.00
03

E2.0
1.33
O.67
0

O

20
min

FIGURE 3


„BLOOD
TENTACLES
GUT WALL

60
40
10
5.

7.(
6.0
O
O
3.00
2.00
1.00
18
FIGURE 4
TENTACLES
BODY WALL
„BLOOD




40
60
20
minutes
9.00
8.0
6.0
5.0
4.0
9
O
-3.00
2.00
1.00
oL

FIGURE 5




40
20
minutes


60
BLOOD
BODY WALL
TENTACLES
GUT WALL
2.
6.(

518.0
12.0
6.0
0

FIGURE 6

40
BLOOD
SUE NLL
BODY WALL
TENTACLES
—
60
20
minutes
25
o4.0
0
2.00
1.00
0
—
40
60
20
minutes
FIGURE 7
8
„BODY WALL
TENTACLES
BLOOD-GUT
WALL
Tissue:
Enzyme:
ornithine
ranscar
bämylase
argininosuc-
cinate split
ing enzyme
arginase
urease
uricase
allantoinase
allantoicase
tentacles
10.3
1.16
3.62
9.89
3.81
5.25
4.50
blood
71.6
5.55
2.66
9.13
66.50
TABLE 3
body wall
25.4
0.39
1.34
8.52
4.81
5.25
9.15
gut wall
31.3
3.07
2.30
1.75
23.87
blood
and
guts
—
3.72
96
Figure and Table Legends
Table 1. Rates of ammonia excretion (in u moles/hour/gram
live body weight) in C. spirabrancha. The data
shown are the average of the number of experiments
done within each collection period.
Table 2.
u molar amounts of endogenous nitrogen containing
compounds in the fractionated tissues of C. spira¬
brancha.
The specific activity (defined as u moles of sub¬
Figure 1.
strate catalytically changed, or u moles of product
produced, per hour under assay conditions per gram
wet tissue.) of ornithine transcarbamylase given
in u moles of citrulline produced per hour per
am wet tissue weight.
The specific activity of argininosuccinate cleavage
Figure 2.
enzyme given in u moles of arginine produced per
hour per gram wet tissue weight.
Figure 3.
The specific activity of arginase in u moles of
arginine utilized per hour per gram wet tissue.
Figure 4.
The specific activity of urease in u moles of urea
utilized per hour per gram wet tissue.
Figure 5.
The specific activity of uricase in u moles of uric
acid utilized per hour per gram wet tissue.
The specific activity of allantoinase in u moles
Figure 6.
of allantoic acid produced per hour per gram wet
tissue.
The specific activity of allantoicase in u moles
Figure 7.
of urea produced per hour per gram wet tissue.
A numerical summary of the specific activities of
Table 3.
the enzymes shown in figures 1 through 7. All
values have the units u moles substrate or pro¬
duct per hour per gram wet tissue.
10
92
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156, 121-141.
Bahl, K.N. (1947) Excretion in the Oligochaetes. Biol. Rev
22, 109-147.
Bergmeyer, H.U., et al. (1963) Methods of Enzymatic Analysis.
Academic Press
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