Distribution of arginase activity in mollusks

Distribution of arginase activity in mollusks

Comp. Biochem. Physiol., 1966, Vol. 17, pp. 259 to 270. Pergamon Press Ltd. Printed in Great Britain D I S T R I B U T I O N OF ARGINASE ACTIVITY IN ...

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Comp. Biochem. Physiol., 1966, Vol. 17, pp. 259 to 270. Pergamon Press Ltd. Printed in Great Britain

D I S T R I B U T I O N OF ARGINASE ACTIVITY IN M O L L U S K S S U Z A N N E G A S T O N and J A M E S W. C A M P B E L L Department of Biology, Rice University, Houston, Texas

(Received 15 June 1965) Abstract--1. In the seventy species of mollusks examined, arginase activity was of most common occurrence in the pulmonate gastropods, being present in terrestrial, fresh-water and marine species. Only in this group of mollusks did levels of activity exceed those found in vertebrate liver. Arginase occurred less frequently in bivalves and was present in low levels only in some fresh-water and estuarine species. It was not detected in eleven marine species. Activity was not detected in a single amphineuran, but was present in two species of cephalopods. The occurrence of arginase in mollusks appears *o be influenced more by taxonomic relationships than by either habitat or feeding habits. 2. In several species possessing arginase activity, the enzyme was not restricted to the hepatopancreas tissue. Arginase activity was present in all tissues examined from a land snail and octopus. 3. The levels of activity in the hepatopancreas of the land snail Otala were extremely variable among individuals of a given population kept under uniform laboratory conditions. The cause of this variation was not established. Prolonged starvation appeared to cause a slight increase in activity, while injury or estivation caused a decrease. INTRODUCTION ARClNASE (E. C. 3.5.3.1 L-arginine ureohydrolase) catalyzes the hydrolytic cleavage of L-arginine to L-ornithine and urea, which is recognized as the terminal step in urea biosynthesis. In this latter connection, arginase is felt to have played an important role in biochemical evolution by allowing animals to detoxify ammonia as urea during their invasions of the land (Cohen & Brown, 1963). T h e general absence of arginase and the ubiquity of the arginine synthesizing system in most microorganisms suggest that arginine synthesis was a primary, and urea synthesis a secondary, function of the ornithine-urea cycle. T h e presence of arginase in some animals, such as birds and insects, which apparently do not have a functional urea cycle (Bowers & Grisolia, 1962; T a m i r & R a t n e r , 1963; Porembska & Mochnacka, 1964) suggests that it has persisted in cases where other enzymes of the cycle have been lost. At the cellular level, arginase may function to control the intracellular level of arginine and consequently its synthesis (Schimke, 1964 a, b). T h e catabolic function of arginase has also been recently shown to be affected by carbohydrate availability (Eliasson, 1965). Arginase occurs in most, if not all, invertebrate phyla. It has been reported from mollusks, crustaceans and echinoderms (Baldwin, 1935); flatworms (van Grembergen & Pennoit-DeCooman, 1944; Campbell & Lee, 1963); annelids 259

260

SUZANNEGASTONANDJAMESW. CAMPBELL

(Cohen & Lewis, 1950; Needham, 1960); insects (Kilby & Neville, 1957; Szarkowaka & Porembska, 1959) ; and the brachiopod, Lingula reevi (Hammen et al., 1962) which is reputedly one of the older living animal species. In what are now classical studies of arginase in invertebrates, Baldwin & Needham (1934)and Baldwin (1935) first investigated the function and distribution of the enzyme in the phylum Mollusca. Of the twelve mollusks examined, the activity occurred in terrestrial and fresh-water, but not marine, species. It was suggested that a more detailed survey of the phylum might reveal a strict correlation of arginase activity and habitat. Because such a correlation would be of interest from a comparative viewpoint, we have re-examined the distribution of the enzyme in the classes Gastropoda and Bivalvia and have also attempted to determine what physiological factors influence the level of activity in individuals of the land snail, Otala lactea. MATERIALS AND METHODS Most of the mollusks used in the survey were from the Texas Gulf coast area. The Pacific species were collected from southern California. Individuals of Otala lactea were purchased from commercial suppliers of edible snails or, in some cases, were collected in Houston, Texas. The hepatopancreas was generally the tissue chosen for examination except in small species where the whole body or visceral mass was used. The tissue was homogenized in nine vol 0.1~}/o cetyltrimethylammonium bromide in a TenBroeck homogenizer. The homogenate was used both undiluted and diluted with 0.02 M sodium glycinate, pH 9"5. In all cases, sufficient dilutions were used to insure measurement of activity under conditions where urea formation was linear with respect to incubation time and enzyme concentration. Generally, 0.1 and 0.2 ml portions of the homogenate and 0-1 ml portions of 1:10, 1:25, 1:50 and 1:100 dilutions were incubated in the assay mixture previously described (Campbell, 1961) for 10, 20, 30 and, in some cases, 60 min at 25 °. The reaction was stopped with 0.5 M HC10~ and urea was determined in a portion of the deproteinized reaction mixture. Controls consisted of 0time incubation, heat-inactivated enzyme, and the complete reaction mixture plus urease. A tissue activity of approximately 10-50,000 t~moles/g/hr could be measured reliably in this manner. Urea, arginine, uric acid and protein were determined as previously described (Campbell, 1961; Lee & Campbell, 1965). A unit of enzyme activity represents 1/*mole urea formed/hr at 25 ° and specific activity refers to units/mg protein. RESULTS AND DISCUSSION Distribution of activity The distribution of arginase activity among seventy species of mollusks is shown in Table 1. The general classification used is that of Morton & Yonge (1964) and feeding habits, where not known, were taken from Morton (1960), Fretter & Graham (1962) or Pilsbry (1939-1948). A sufficient number of gastropod and bivalve species were available to represent several combinations of taxonomic group,

DISTRIBUTION OF ARGINASE ACTIVITY IN MOLLUSKS

261

habitat and feeding habits. N o scaphopods and only one a m p h i n e u r a n and two cephalopods were e x a m i n e d and a m o r e detailed survey of these groups should provide interesting information in terms of the distribution of the e n z y m e in the phylum. Arginase activity was of most c o m m o n o c c u r r e n c e in the p u l m o n a t e gastropods, being present in f o u r t e e n of the sixteen species examined. T h e s e included terrestrial (T), m a r i n e (M) and fresh-water ( F W ) forms. As was first s h o w n b y Baldwin (1935), the levels of activity in some species of pulmonates exceed those of vertebrate TABLE 1 - - D I S T R I B U T I O N

Species

OF ARGINASE ACTIVITY IN MOLLUSKS

Arginase activity Habitat Food Tissue No. of animals Units/g tissue Units/mg protein

AMPHINEURA POLYPLACOPHORA

Mopalia muscosa

M-2

D

HEP

1

0

T-4 M-2

D D

WA HEP

14

0-60

3

0

BE M-2

D D

WA HEP

7 1

2152 0

M-1 M-1

D D

HEP HEP

1 1

0 0

M-5 M-5 M-5 M-5 M-5 M-1 M-1 M-1 M-5 M-5 M-5

C F F F H D D D C C C

HEP HEP WA WA HEP HEP HEP HEP HEP HEP HEP

2

2 4 3 2 8 10 6 1 2 1

FW-1

D

HEP

3

0

M-5 M-5 M-5 M-5

C C C C

HEP HEP HEP HEP

1 2 3 2

0 0 0 0

M-4

C

HEP

2

M-2

C

HEP

7

GASTROPOA PROSOBRANCHIA

Arehaeogastropoda Helicina orbiculata Megathura crenulata Neritina ( Theodoxus) virginea Haliotis cracherodii Lottia gigantea Acmaea sp. Mesogastropoda Bursa californica Crepidula fornicata Crepidula plana Crepidula sp. Cypraea spadicea Littorina irrorata Littorina nebulosa Littorina ziczac Polinices draconis Polinices duplieatus Polinices lewisii Pomacea (Ampullaris) flagellata Neogastropoda Busycon sp. Forreria beleheri Kelletia kelletia Nassarius fossatus Oliva sayana Thais haemastoma haysae

0-0.6 22.5

0

122 258 62 0 0-101 240 115 0 0 0

1-3 4"2 0.9 0-0.8 1.4 0.7

0

0-148

0-1-0

SUZANNE GASTON AND JAMES W . CAMPBELL

262

TABLE 1--DISTRIBUTION OF ARGINASE ACTIVITY IN M O L L U S K S - - c o n t / ~ d Arginase activity Species

Habitat

Food

Tissue No. of

animals U n i t s / g tissue U n i t s / m g p r o t e i n OPISTHOBRANCHIA Cephalaspidea

Bulla occidentalis

M-4

D

HEP

9

0

M-5

H

HEP

3

0

--

M-6

C

WA HEP GON VM

1 5 5 3

108 0 0 440

2"7 --N.D.

FW-1

D

M- 1

D

FW- 1 H M-1 D FW-1 H , D

WA WA WA HEP HEP

3 2 4 3 4

1706 504 942 0 0

4"8 8'1 3-0 ---

T-1 T-2 T-2 T-2 T-3 T-4 T-1 T-3 T-2 T-2 T-2

H H H H D D D O H H H

HEP WA HEP WA HEP HEP WA HEP HEP HEP HEP

3 6 2 3 3 13 3 3 2 4 78

797 596 128 97 3018 44200 72 274 47400 2160-14380 2000-45000

M-4 BE BE M-4

F F F F

HEP HEP HEP HEP

1 2 3 2

0 156 228 0

BE BE M-5

F F F

HEP WA HEP

3 7 2

0-trace ? trace ? 0

FW-2 M-4 FW-2

F F F

HEP HEP HEP

1 1 several

0 0 36-100

Anaspidea

Aplysia sp. Acoela

Scyllaea pelagica

PULMONATA Basommatophora Helisoma sp.

Melampus coffeus Physa sp. Siphonaria pectinata Australorbis glabratus Stylommatophora

Bulimulus alternatus Deroceras laeve Limax flavus Limax valentianus Mesodon thyroidus Polygyra sp. Practicollela sp. Rumina decollata Helix pomatia Helix aspersa Otala lactea BIVALVIA PROTOBRANCHIA Atrina sp.

Brachidontes sp. Crassostrea virginica Noetia ponderosa Volsella (Modiolus) demissus Mytilopsis leucophaeta Aquipecten irradians LAMELLIBRANCHIA Anodonta sp.

Barnea truncata Carunculina parva

6"0 6.6 1-2 1-3 24'0 398'0 0"8 2'0 948.0 N.D. 26"5-551.0

-1-4 2.4 -0-<0"1 N.D. 6 --0'1-2.0

263

DISTRIBUTION OF ARGINASE ACTIVITY I N MOLLUSKS T A B L E 1 - - D I S T R I B U T I O N OF ARGINASE ACTIVITY I N

Species

Donax variabilis Chione cancellata Ensis minor •Laevicardium mortoni Lampsilis teres Lucina floridana Mercenaria campechiemis Mulinia lateralis Petricola pholadiformis Quadrula quadrula Rangia cuneata Rangia flexuosa Spisula solidissima Tagelus gibbus CEPHALOPODA Octopoda Octopus bimaculatus Deeapoda Lolliguncula brevis

MOLLUSKS--continued

Arginase activity Habitat Food Tissue No. of animals Units/g tissue Units/mg protein M-3 M-5 BE M-4 FW-2 M-5 BE M-4 M-4 FW-2 BE BE M-4 BE

F F F F F F F F F F F F F F

HEP 13 HEP 7 WA 3 WA 2 HEP several HEP 1 HEP 3 WA 3 WA 3 HEP 3 HEP 2 HEP 2 HEP 1 HEP 10

0 0 0 0 0-364 0 trace 0 0 0 trace ? 0 0 0

-N.D. -N.D. ---N.D. ----

M-6

C

HEP

2

17

0.1

M-6

C

HEP VM

5 5

3120 2640

30.0 41 "0

Abbreviations used: Units,/~mole/hr at 25°; for habitat, T - l , arid terrestrial; T-2, hot humid terrestrial; T-3, moderate terrestrial; T-4, ubiquitous terrestrial; M-l, rocky supratidal marine; M-2, rocky intertidal marine; M-3, beach marine; M-4, neritic burrowing marine; M-5, subtidal marine; M-6, pelagic marine; BE, bracldsh-estuarine; FW-1, quiet fresh-water; FW-2, running fresh-water; for food, H, herbivorous; C, carnivorous; O, omnivorous; D, detritus; F, filterfeeder; for tissue, HEP, hepatopancreas or liver; GON, gonad; WA, whole animal; VM, visceral mass; and, N.D,, no data. liver. I n addition to those species listed in T a b l e 1, Baldwin (1935) reported levels of activity in the snails Viviparus fasdatus (FW) and Limnaea stagnalis ( F W ) comparable to or higher than those in m a m m a l i a n liver and lower levels for the snail Planorbis corneus (FW), the slug Arion ater (T) and the mussel Anodonta cygnaea (FW). L o w levels of arginase activity occurred in a few species of fresh-water and estuarine bivalves. Activity could not be detected in the eleven marine species which were examined. H a m m e n et al. (1962) have reported tissue activities of 2.4 and 3.8, respectively, for the estuarine bivalves Volsella demissus and Crassostrea virginica. Since arginase activity was detected in nine marine gastropods and two cephalopods, there does not appear to be a strict correlation between the absence of activity and the marine environment per se. T h e presence or absence of arginase

264

SUZANNE G A S T O N AND JAMES W . C A M P B E L L

activity in mollusks thus appears to be m o r e closely related to t a x o n o m i c relationships t h a n to either feeding habits or e n v i r o n m e n t . Because of the variation enc o u n t e r e d in the levels of activity in m a n y species and also because of the " e x t r a h e p a t i c " distribution of the enzyme, generalizations c o n c e r n i n g arginase activity in mollusks w o u l d be, at this time, premature. T h e activity does appear to be m o s t c o m m o n in those f o r m s w h i c h are generally considered to be the most highly evolved.

"Extrahepatic" occurrence of arginase I t b e c a m e obvious d u r i n g the course of the survey that arginase activity was not restricted to the h e p a t o p a n c r e a s tissue. F o r example, activity was detectable with either the whole b o d y or visceral mass of Scyllaea b u t not with the hepatopancreas and, in Lolliguncula, the specific activity obtained with the visceral mass TABLE 2--TISSUE DISTRIBUTION

OF ARGINASE ACTIVITY IN OTALA AND OCTOPUS

Arginase activity (units/g) Tissue

Kidney Pancreas Reproductive tract Testes Muscle : columellar tentacle Salivary gland Albumin gland Crop Intestine Blood Skin Sole of foot Gills "Lung" Branchial heart Eye (whole) * Units/ml. t Pooled lung tissue from three

Otala

Octopus

490-2820 -442-1840 --

1328 74 --

144 -1402 154-1010 234-488 1872 0-240 * -5112 -977 t ---

--

113

15

60 --51 -88 -235 -62 40

Helix aspersa gave 3053 units/g.

was greater t h a n that obtained with hepatopancreas tissue (Table 1). As s h o w n in T a b l e 2, arginase activity was detected in every tissue e x a m i n e d of b o t h Otala and Octopus. W i t h Otala, the tissues were pooled f r o m several individuals for m o s t assays and, because of the individual variation discussed below, a general ranking

DISTRIBUTION OF ARGINASE ACTIVITY IN MOLLUSKS

265

of the tissues in order of activity is not possible. However, in one estivating individual, the levels of activity for the hepatopancreas, kidney, albumin gland, blood, reproductive tract and foot sole were 2080, 2280, 900, 21, 442 and 5112 units/g tissue (or ml blood), respectively. Porembska & Heller (1952) have previously reported both ornithine transcarbamylase and arginase from kidney and foot muscle as well as hepatopancreas of Helix pomatia. They report specific activities, using cetyltrimethylammonium bromide extracts of acetone powders and an assay temperature of 37 °, of 166, 88 and 46.6 for foot muscle, hepatopancreas and kidney, respectively. In the one octopus examined by us, the levels of activity in all tissues were in excess of that in the liver. Arginine phosphate is the major phosphagen of mollusks (Roche et al., 1957) and, although arginase is not active on arginine phosphate (see Greenberg, 1951) nor has any connection between the two ever been shown, the presence of high levels of activity in all tissues, especially muscle, seems an interesting circumstance. In vertebrates, no comparable degradative enzyme exists for creatine and the excess is excreted as creatinine after dehydration (Borsook & Dubnoff, 1947). It seems possible that arginase might in some way function in a control system relating to the phosphagen role of arginine in mollusks. Because of the importance of arginine as a protein constituent, as well as a phosphagen, such a control mechanism would also presumably involve the arginine synthesizing system.

Variability of arginase activity A remarkable amount of variation was found in the levels of arginase in many of the mollusks examined and because of this variation, physiological experiments using individuals of Otala* were difficult or impossible to interpret. In other species, such as Helicina, Thais, Littorina irrorata, Volsella and Lampsilis (Table 1), the activity ranged from below limits of detection to trace amounts or more with individuals collected on different occasions from either the same or different populations. In attempts to determine possible physiological factors affecting the levels of activity in Otala, some 200 individuals were assayed under various conditions. Some of these experiments are described below. An arginase inhibitor (Ceska & Fisher, 1959; Reifer & Morawska, 1963) appears not to be involved in this variation since mixing experiments with high and low activity individuals and those feeding and in estivation resulted in 98-103 per cent recovery of the total activity. Also, since certain enzymes are known to be distributed between two cell compartments and thus may be affected independently by physiological factors (Nordlie et al., 1965), it was established that the * Because different populations of Otala have been used, interpopulation differences in enzyme activity might be expected. There may also be a problem of speciation with Otala: while all the snails we have used for these and other studies appear to be O. lactea according to Pilsbry (1939-1948) and other authors, some show a banding pattern similar to that depicted by him (Vol. 1, pt. 1, p. 12, Fig. 6(d)) for O. vermiculata. The variation referred to here, however, exists among individuals from a given population showing almost identical banding patterns and which are kept under uniform conditions of temperature, humidity, diet and photoperiod.

266

SUZANNE GASTONANDJAMESW. CAMPBELL

arginase in Otala hepatopancreas is localized mainly, if not exclusively, in one compartment: fractionation of the tissue in 0.44 M D-mannitol-0"005 M tris chloride, pH 7-4, gave 90 per cent or more of the total homogenate activity in the 105,000g supernatant fraction. Localization studies under various physiological conditions were not, however, carried out in detail. With snails ranging in weight from 3.31 to 12.10 g, no correlation of size and arginase activity was found. These size differences presumably also reflect some age differences. With individuals of approximately the same size, the marked weight fluctuations previously reported for Helix pomatia (Howes & Wells, 1934) were also noted with Otala. A series of ten snails which had been feeding for 8 days

FEEDING STARVED

I

DAY

STARVED

3 DAYS

STARVED

7

DAYS

E3 STARVED DAYS

E3 Z

ESTIVATING

14

23 °

B ESTIVATING

I I

I 2

I 3

3=

I I I I I ~) 4 5 6 7 8 SPECIFIC ACTIVlTY, x10 -2

FIG. 1. Hepatopancreas arginase in individual Otala during starvation and estivation. following estivation showed an average increase in total weight of 51.24_+8.13 (S.D.) per cent. In another series of fifteen individuals which were feeding for 18 days, the average weight gain was 15.43 ± 7.13 per cent. In this latter group, ten showed a negative correlation of percentage weight gain and arginase activity during the 18-day period, while five showed the reverse correlation. These weight changes presumably reflect differences in tissue hydration (Machin, 1964) and, because of this, enzyme activity expressed as units/mg protein shows the lesser variation. Within a given group of snails, the protein content of the hepatopancreas is relatively constant and no correlation with arginase activity exists. The average protein content of the hepatopancreas in fully hydrated individuals (feeding, deprived of food, inactive at room temperature, etc.) was 7.40 ± 0-98 per cent for a group of forty-three. In another group of twelve individuals which were estivating in nature or at 3 ° in the laboratory (Fig. 1), the hepatopancreas protein

DISTRIBUTION OF ARGINASE ACTIVITY I N MOLLUSKS

267

content was 13.55 + 3.72 per cent. T h e latter presumably represents the dehydrated value for protein and indicates a tissue weight change of approximately 45 per cent. A change in total body weight of 50 per cent (Howes, Wells, 1934) must, however, reflect a greater hydration or dehydration of other tissues since with snails weighing from 7.16 to 10.34 g, the body weight accounts for 41.84_+2.49 per cent of the total weight. T h e hepatopancreas makes up 5.72 + 0.87 per cent of the total and 13-68 -+ 1.89 per cent of the body weight. T h e kidney makes up 2-58-+ 0.58 per cent of the total weight. Because starvation and estivation had been reported by Baldwin (1935) to lower arginase levels in Helix, assays were carried out on actively feeding individuals, those deprived of food but still active, and those inactive or in estivation. In all experiments, there was a general tendency for at least some in each group which was deprived of food for an extended period of time and which remained active to show increased levels of enzyme activity. This is illustrated with a few individuals

LETTUCE 23 e CABBAGE 23 e

SPINACH

T

I 4

I 8

LETTUCE &

23"

CABBAGE 23 e

LETTUCE & CABBAGE

3e

LETTUCE &

32 e

I ! I 12 16 2 0 SPECIFIC ACTIVITY,

CABBAGE

xlO "!

FIo. 2. Hepatopancreas arginase in individual Otala kept at different temperatures and on different diets. in Fig. 1. In the mammal, starvation increases the level of arginase as well as other urea cycle enzymes (Schimke, 1962). Estivation or inactivity* at room temperature generally had approximately the same effect as prolonged starvation. Natural estivation or estivation at 3 ° in the laboratory generally serves to lower the specific activity although, as has previously been reported (Linton & Campbell, 1962), there is no marked effect on tissue activity. T h e latter is, however, even more variable than the specific activity and it is difficult to draw definite conclusions. * As discussed by Hunter (1964), the physiological states of terrestrial gastropods are not easily defined and estivation may not be an apt term. In some cases, "inactive" seems more appropriate since the animals may not form an epiphragm and are immediately responsive to stimuli although not active. Some individuals of Otala enter this state almost daily even under conditions of high humidity and food availability.

268

SUZANNE GASTON AND JAMES W . CAMPBELL

The range in tissue activity for those animals depicted in Fig. 1 was, for example: feeding, 23,650-37,844; starved 1 day, 9,118-22,038; 3 days, 20,648-33,554; 7 days, 12,680-38,400; 14 days, 10,888-52,800; estivating at 23 °, 18,690-45,388; and at 3 °, 6,130-35,100. As shown in Fig. 2, there was no marked difference in arginase activity among individuals of Otala fed on four different diets. Lettuce, cabbage and spinach were originally chosen because of their differences in free arginine content. For the lettuce used, the average free arginine was 0-27/~mole/g, for spinach, 3-65 and, for cabbage, 4.29 (determined on 5% trichloroacetic acid extracts). Also shown in Fig. 2 are the levels of activity for snails kept at both low and high temperatures under conditions where most remain active (high humidity and with food available). B

4 o

~3 o 0

2

G W

i

0"6

I

0"8 DAILY

I I'0 INTAKE, g

i 1.2

i 60

I 90 KIDNEY

I I 1 120 150 180 URIC AC1D, m g / g

FIG. 3. Average daily food (cabbage) intake (A) and kidney uric acid content (B) and hepatopancreas arginase levels in individual Otala. Survival, in both cases, was low and there was a marked decrease in enzyme activity. This appears to be an injury response since a similar decrease took place in other experiments in which the snails were injured. For example, in an experiment in which both the products and substrate of the arginase reaction were injected into individuals through a small hole in the shell (Lee & Campbell, 1965), a marked decrease in enzyme activity occurred and there was also a high mortality rate in some groups. In snails injected with either 0.15 M NaC1 (0.1 ml) or 5/~mole urea (0.1 ml)/day for 7 days, the mortality was 0. In those injected with 2 t~mole L-arginine/day for 7 days, the mortality was 40 per cent and in those injected with 5/~mole L-arginine/day, the mortality was 67 per cent. L-Arginine thus appears to be fairly toxic to Otala. It is known to be toxic to rats although less so than most of the common amino acids (Gullino et al., 1956). In those injected with 5/~mole

D I S T R I B U T I O N OF ARGINASE ACTIVITY I N M O L L U S K S

269

L-ornithine/day, the mortality was 33 per cent. I n all cases, including the controls (NaC1 injected), the specific activity for arginase was 20 or below. Because all Otala in a group may not feed consistently, it seemed possible that the variation in arginase activity might be due to starvation of some individuals. T o test this, a series of fifteen individuals were isolated in finger bowls and the actual daily intake of cabbage for each snail was recorded for eighteen days. As shown in Fig. 3, no correlation of average daily intake during this period and arginase activity was found. Because a correlation of arginase activity and kidney uric acid has been reported for different species of mollusks (Baldwin, 1935), the kidney uric acid was determined at the end of this experiment. As shown in Fig. 3b, such a correlation does not exist among individuals of Otala. T h u s , because of the individual variation within a group of Otala kept under identical conditions and also because of individual differences in behavior, it has not been possible to establish for certain the effect on the hepatopancreas arginase activity of any of the physiological parameters examined. Tentatively, starvation appears to cause a slight increase in activity, while injury or "estivation" cause a decrease. Acknowledgements--This work was supported by a grant from the U.S. Public Health Service (AI 05006). We thank Dr. T. W. Lee for the uric acid analyses and Dr. S. H. Bishop for the tissue fractionation studies. REFERENCES BALDWIN E. (1935) Problems of nitrogen catabolism in invertebrates. III. Arginase in the invertebrates, w;_th a new method for its determination. Biochem. ft. 29, 252-262. BALDWINE. & NEEDHAMJ. (1934) Problems of nitrogen catabolism in invertebrates. I. The snail (Helix pomatia). Biochem. ft. 28, 1372-1392. BORSOOKH. & DUBNOFFJ. W. (1947) The hydrolysis of phosphocreatine and the origin of urinary creatinine, ft. Biol. Chem. 168, 493-510. BOWERS M. D. & GRISOLIAS. (1962) Biosynthesis of carbamyl aspartate in pigeon and rat tissues. Comp. Bioehem. Physiol. 5, 1-16. CAMPBELL J. W. (1961) Studies on tissue arginase and ureogenesis in the elasmobranch, Mustelus eanis. Arch. Bioehem. Biophys. 93, 448-455. CAMPBELL J. W. & LEE T. W. (1963) Ornithine transcarbamylase and arginase activity in flatworms. Cornp. Biochem. Physiol. 8, 29-38. CESKAM. & FISHERJ. R. (1959) An arginase inhibitor(s) and its possible role in the developmental decrease of arginase activity in chick embryos. Biol. Bull., Woods Hole 117, 611-625. COHEN P. P. & BROWN G. W. JR. (1963) Evolution of nitrogen metabolism. Proc. Fifth Int. Congr. Biochem. 3, 129-138, Pergamon Press, London. COHEN S. & LEWISH. B. (1950) The nitrogenous metabolism of the earthworm (Lumbricus terrestris). II. Arginase and urea synthesis. J. Biol. Chem. 184, 479-484. ELIASSONE. E. (1965) Regulation of arginase in Chang's liver cells in tissue culture. Biochim. biophys. Acta 97, 449-459. Fm~TTERV. & GRAHAMA. (1962) British Prosobranch Molluscs. Ray Society, London. Gm~ENBERG D. M. (1951) Arginase. In The Enzymes (Edited by SUMNER J. B. & MYRBXCK K.) 1, 893-921. Academic Press, New York. VAN G~gBnRGEN G. & PENNOIT-DECooM~ E. (1944) Experimenteele Gegevens over het Stikstofmetabolisme der Plathelminthen. Natuurwet. Tijdschr. 26, 91-97.

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GULLINO P., WINITZ M., BIRNBAUMS. M., CORNFIELDJ., OTEY M. C. • GREENSTEINJ. P. (1956) Studies on the metabolism of amino acids and related compounds in vivo. I. Toxicity of essential amino acids, individually and in mixtures, and the protective effect of L-arginine. Arch. Biochem. Biophys. 64, 319-332. HAMMEN C. S., HANLON D. P. & LUM S. C. (1962) Oxidative metabolism of Lingula. Comp. Biochem. Physiol. 5, 185-191. HowEs N. H. & WELLS G. P. (1934) T h e water relation of snails and slugs. I. Weight rhythms in Helix pomatia L. J. Exp. Biol. 11, 327-343. HUNTER W. R. (1964) Physiological aspects of ecology in nonmarine molluscs. In Physiology of Mollusca (Edited by WILBUR K. M. & YONGE C. M.), 1, 83-126, Academic Press, N.Y. KILBY B. A. & NEVILLEE. (1957) Amino-acid metabolism in locust tissues. J. Exp. Biol. 34, 276-289. LEE T. W. & CAMPBELLJ. W. (1965) Uric acid synthesis in the terrestrial snail, Otala lactea. Comp. Biochem. Physiol. 15, 457-468. LINTON S. N. & CAMPBELL J. W. (1962) Studies on urea cycle enzymes in the terrestrial snail, Otala lactea. Arch. Biochem. Biophys. 97, 360-369. MACHIN J. (1964) T h e evaporation of water from Helix aspersa. I. The nature of the evaporating surface. J. Exp. Biol. 41, 759-769. MORTON J. W. (1960) Molluscs--An Introduction to their Form and Function. Harper & Bros., N.Y. MORTON J. W. & YONCE C. M. (1964) Classification and structure of the Mollusca. In Physiology of Mollusca (Edited by WILBUR K. M. & YONGE C. M.), 1, 1-58. Academic Press, N.Y. NEEDHAM A. E. (1960) The arginase activity of the tissues of the earthworms Lumbricus terrestris L. and Eisenia foetida (Savigny). J. Exp. Biol. 37, 775-782. NORDLIE R. C., VARRICCHIOF. E. & HOLTEN D. D. (1965) Effects of altered hormonal states and fasting on rat-liver mitochondrial phosphoenolpyruvate carboxykinase levels. Biochim. biophys. Acta 97, 214-221. PILSBRY H. A. (1939-48) Land Mollusca of North America (North of Mexico), Vols. 1 and 2. Academy Natural Sciences, Philadelphia, Pennsylvania. POREMBSKAZ. & HELLER J. (1962) Studies on the ornithine cycle in tissues of Helix pomatia during hibernation. Acta Biochim. Polon. 9, 385-390. POREMBSKA Z. & MOCHNACKA I. (1964) T h e ornithine cycle in Celerio euphorbiae. Acta Biochim. Polon. 11, 113-119. REIFER I. & MORAWSKAG. (1963) An arginase inhibitor from sunflower seeds. Biochem. J. 89, 51. ROCHE J., THOAI N. V. & ROBIN Y. (1957) Sur la pr6sence de cr6atine chez les invert6br6s et sa signification biologique. Biochim. biophys. Acta 24, 514-519. SCHIMKE R. T. (1962) Differential effects of fasting and protein-free diets on levels of urea cycle enzymes in rat liver. J. Biol. Chem. 237, 1921-1924. SCHIMKE R. T. (1964a) Enzymes of arginine metabolism in mammalian cell culture. I. Repression of argininosuccinate synthetase and argininosuccinase. J. Biol. Chem. 239, 136-145. SCmMKE R. T. (1964b) Enzymes of arginine metabolism in cell culture: studies on enzyme induction and repression. U.S. Natl. Cancer Institute Monograph 13, 197-217. SZARKOWSKA L. 8,: POREMBAKA Z. (1959) Arginase in Celerio euphorbiae. Acta Biochim. Polon. 6, 273-276. TAMIR H. & RATNER S. (1963) Enzymes of arginine metabolism in chicks. Arch. biochem. Biophys. 102, 249-258.