arginine and urea metabolism in the south american land snail, strophocheilus oblongus

Comp. Biochem. Physiol., 1972, Vol. 421:1, pp. 439 to 449. Pergamon Press. Printed in Great Britain

ARGININE AND AMERICAN

UREA METABOLISM IN THE SOUTH LAND SNAIL, S T R O P H O C H E I L U S

OBLONGUS*

P A U L R. T R A M E L L t and JAMES W. C A M P B E L L ~

Department of Biology, William Marsh Rice University, Houston, Texas 77001

(Received 15 October 1971)

Abstract--1. Using 1*C-labeled bicarbonate, L-ornithine and L-citrulline, the South American land snail Strophocheilus oblongus was shown to be capable of arginine biosynthesis de novo.

2. The relative rates of incorporation of bicarbonate-l'C into arginine, aspartate and glutamate by hepatopancreas tissue in vitro indicates that the amount of arginine synthesized is nutritionally significant in this species.

3. Three enzymes of the arginine pathway, ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinate lyase, are present in most tissues of S. oblongus.

4. The tissues also contain arginase activity and injected L-arginine-(guanidino-14C) is converted to urea-14C in tqvo.

5. A major portion of urea-14C injected into individual snails is released as carbon dioxide-l'C and ureolytic activity, was detected in hepatopancreas and also kidney.

6. These findings are discussed relative to the ability of S. oblongus to accu- late blood urea for osmotic water retention.

INTRODUCTION

THE METABOLISM of the helicid pulmonates Otala lactea and Helix aspersa, is unique among animals in that these species are capable of urea synthesis de novo and also possess a distinctive urease. There appears to be no compartmentat ion of the biosynthetic and degradative systems and urea formed via the arginine-urea pathway does not accumulate for excretion in these snails as in ureotelic organisms but rather is broken down by urease to ammonia and carbon dioxide (Campbell & Speeg, 1968a; Speeg & Campbell, 1968, 1969). This enigmatic metabolism of urea may be directed toward an acid-base balance prerequisite for calcium carbonate deposition (Campbell & Speeg, 1969). Among the indirect evidence for this suggestion is the observation that acetohydroxamic acid, an inhibitor of urease, decreases urea turnover in vivo (Speeg & Campbell, 1969) and also inhibits shell

*Supported by grants from the U.S. Public Health Service (AI 05006; 5-T1-GM-884; and 5-K3-GM-6780).

tPresent address: Abbott Scientific Products Division, 820 Mission Street, South Pasadena, California.

.~To whom to write for reprints.

439

440 PAUL R. TRAMELL AND JAMES W. CAMPBELL

regeneration (Campbell & Speeg, 1969). Although some urea accumulates in hepatopancreas tissue in vitro during acetohydroxamic acid inhibition (Speeg & Campbell, 1969), this has not been observed to occur naturally in O. lactea and H. aspersa. Urea accumulation has, however, recently been observed in the terrestrial snail Strophocheilus oblongus during estivation (De Jorge & Petersen, 1970; Camp- bell et al., 1972). If the type of metabolism found in O. lactea and H. aspersa is common to other terrestrial pulmonates, then the accumulation of urea under special circumstances indicates that the breakdown of urea by urease in these snails may be a regulated process. Urea accumulation in body fluids of terrestrial snails would be highly advantageous for water conservation since they lose water by evaporation as though from a free surface (Machin, 1964). The utilization of urea to decrease the vapor pressure of lower vertebrate body fluids to conserve water in both the marine (Goldstein, 1970) and terrestrial (Janssens, 1964; Shoe- maker et al., 1969) environments is well known but this had not previously been described in invertebrate animals. Because of the possible physiological significance of urea metabolism in S. oblongus, the work reported here was directed toward the origin of this compound via the arginine-urea pathway and its subsequent metabolism in actively feeding snails.

M A T E R I A L S AND M E T H O D S 3Iaterials

Specimens of Strophocheilus oblongus were obtained from Porto Alegre, Brazil through the courtesy of Dr. C. P. Jaeger. They were kept under humid conditions and fed lettuce ad lib.

z4C-Substrates were purchased from New England Nuclear Corp., Boston, Mass. with the exception of o-glucose-(U-t4C) which was obtained from CalBiochem, Los Angeles, Calif. These substrates were used without additional purification except for L-citrulline- (ureido-14C) which was purified as described by Hall et al. (1960). Unlabeled compounds were added to the l*C-substrates to obtain the final specific radioactivities used. The substrates, co-factors and supplementary enzymes used in the enzyme assays and their sources were as previously described (Campbell & Speeg, 1968b).

Analytical methods The methods described by Campbell & Speeg (1968a) were used to determine incorpora-

tion of bicarbonate-t4C into free and protein amino acids. For the incorporation of L~C- substrates into free and protein arginine, this amino acid was isolated from free amino acid fractions and protein hydrolysates by the method of Benson & Patterson (1965), modified by the substitution of a 5-cm column of Beckman Custom Resin PA-35 and a temperature of 55°C. Incorporation into urea was determined enzymatically as described by Speeg & Campbell (1969).

Enzyme assays

The assays for ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinate lyase activities were those described by Campbell & Speeg (1968b) and for urease, by McDonald et al. (1971). The arginase assay was that of Linton & Campbell (1962). The latter system was also used for y-guanidinobuD'rate ureohydrolase by substituting 50 mM y-guanidinobutyrate (sodium salt, pH 9"5) for L-arginine.

UREA METABOLISM IN A LAND SNAIL 441

RESULTS 1. Tracer studies

The first approach to establish the metabolic origin of arginine and urea via the arginine-urea pathway in S. oblongus was to demonstrate the incorporation of 14C-labeled precursors into these compounds by whole animals in vivo and hepatopancreas tissue in ¢:itro.

Incorporation in vivo of 14C-labeled arginhze and urea precursors. As shown in Table 1, the more direct precursors of arginine (bicarbonate, ornithine and citrulline) are incorporated into this amino acid following their injection into individual S. oblongus. There were, however, certain inconsistencies in the labeling patterns obtained with these precursors. For example, the highest specific radioactivity of free arginine was given by citrulline-14C although no radioactivity was detected in protein arginine with this precursor. Ornithine-14C, which gave rise to a relatively lower specific radioactivity in free arginine, was, on the other hand, a very good precursor of protein arginine. Because individual snails were used with each precursor, these inconsistencies could reflect individual differences with respect to protein turnover.

In addition to the three tissues listed in Table 1, incorporation into total protein arginine of five other tissues was also determined following the injection of bicarbonate-14C. Incorporation into protein arginine of heart and blood was neg- ligible; in reproductive tract, foot and kidney, the specific radioactivities of isolated protein arginine were 90, 140 and 130 dpm//~mole, respectively.

The only known route for ornithine biosynthesis is from glutamic acid (Rod- well, 1969). However, neither free nor protein arginine was labeled from glutamate- ~4C in the one S. oblongus tested. On the other hand, protein arginine of mantle and albumen gland was labeled from glucose-t4C. Ornithine is svnthesized de novo in O. lactea, as evidenced by its labeling from bicarbonate-14C, and ornithine 3-transaminase, an enzyme that may be responsible for ornithine synthesis in animals (Volpe et al., 1969), is present in this species (Campbell & Speeg, 1968a). The failure to obtain incorporation from glutamate-14C in S. oblongus could thus again reflect an individual variation or possibly a compartmentation of glutamate metabolism such that there is a preferential utilization of endogenously synthesized (from bicarbonate-14C or glucose-t4C) glutamate for ornithine formation. Naka- gawa et al. (1964) did not detect incorporation of glucose-14C into either free or protein arginine of the frog Rana catesbeiana, a species known to synthesize urea by the arginine-urea pathway.

The incorporation of bicarbonate-t4C, ornithine-t4C and citrulline-14C into arginine by S. oblongus indicates that this snail, like O. lactea and H. aspersa, is capable of arginine biosynthesis de novo. That arginine is converted to urea in z'i~.o is shown bv the data in Table 2. Following the injection of arginine-(guanidino-a4C), urea-X4C was present in all tissues examined; it was highest in mantle tissue and lowest in hepatopancreas. The small amount of urea-a4C present in hepatopancreas is consistent with the subsequent demonstration of ureolvtic

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TABLE 2 - - C O N V E R S I O N in i:ivo OF L - A R G I N I N E - ( G U A N I D I N O - l a C ) TO UREA BY Str@hocheilus

Radioactivity in urea (dpm/g tissue)

Hepatopancreas 320 Reproductive tract 2060 Foot 1060 Blood 2430 (per ml) Kidney 1400 Albumen gland 2000 Mantle ("lung") 56,000 Heart 10,600

Incorporation into urea was determined as described in the text 13 hr after injecting 10/amoles (1/zCi//zmole) L-arginine-(guanidino-14C) into the fleshy part of the foot.

activity in this tissue. In the exper iment described in Table 2, only 0.45 per cent of the injected arginine-14C was recovered as urea-14C. This low recover), is most likely due to the rapid breakdown of urea-14C in vivo and not to a slow rate of arginine hydrolysis.

Synthesis of amino acids from bicarbonate-14C by hepatopancreas tissue. T h e relative incorporation of bicarbonate-14C into several free and protein amino acids by hepatopancreas tissue in vitro is shown in Table 3. T h e greatest incorporation was into free glycine. However , due to the high concentration of this amino acid in

TABLE 3--INCORPORATION OF BICARBONATE-laC INTO FREE AND PROTEIN AMINO ACIDS BY

Strophocheilus HEPATOP.~CREAS i~t ~'itro

Protein amino acid

Free amino acid Mole °o (dpm/g tissue) dpm//xmole of protein

Alanine 78,200 490 8.01 Arginine 112,600 (34,900) 3000 4"69 Aspartic acid 52,600 3460 11"51 Glutamic acid 135,200 2800 7.07 Glycine 684,900 (30,528) 170 7.84 Proline - - 400 5-89 Serine - - 460 6.34

Hepatopancreas tissue (271 mg) was incubated for 90 min at 30°C in 2 ml modified Chiarandini's (1964) saline containing 5/zmoles (10/zCi//zmole) bicarbonate-14C. The free and protein amino acids were then isolated and counted in an automated chromatographic system coupled with a flow monitor scintillation system. The specific radioactivities (dpm/Fzmole) of free glycine and arginine are given in parentheses.


hepatopancreas, the specific radioactivity of glycine was actually slightly less than that of free arginine. The incorporation of bicarbonate-14C into arginine was comparable to that of either glutamate or aspartate. These two amino acids are synthesized via transamination reactions from tricarboxylic acid cycle intermediates and are therefore seldom if ever nutritional requirements of animals. The amount of bicarbonate-14C incorporated into arginine relative to that into glutamate and aspartate suggests that the synthesis of arginine in S. oblongus is nutritionally significant as it may also be in O. lactea and H. aspersa (Campbell & Speeg, 1968a).

2. Enzymes of arginine biosynthesis A mitochondrial carbamyl phosphate synthetase has previously been described

in S. oblongus hepatopancreas tissue (Tramell & Campbell, 1970b). This enzyme differs from other carbamyl phosphate synthetases in utilizing L-glutamine, but not ammonia, as the amino donor and in showing an absolute requirement for N- acetyl-L-glutamate. The level of carbamyl phosphate synthetase activity in hepatopancreas of actively feeding snails is from 0.2 to 0.5/zmole/g tissue per hr at 30°C. The tissue distribution of the remaining enzymes of arginine biosynthesis, ornithine transcarbamylase, argininosuccinate synthetase and argininosuccinate lyase, is shown in Table 4. For each of these enzymes, the effect of time

TABLE 4 TISSUE DISTRIBUTION OF ORNITHINE TRANSC.A_RBAM'Y'I.,ASE, ARGININOSUCCINATE SYNTHETASE AND ARGININOSUCCINATE LYASE IN Strophocheilus

Tissue activity (~mole/g tissue per hr) .

Ornithine Argininosuccinate Argininosuccinate transcarbamylase synthetase lyase

Hepatopancreas 186 0"21 16-7 Reproductive tract 49 0'05 4.5 Foot 9 0-06 0.6 Blood Trace 0'003 (per ml) 0-08 (per nal) Kidney 40 0"03 14.3 Albumen gland 145 0"03 1"3 Mantle ("lung") 39 Trace ! 3'5 Heart 77 0"01 20.5

and enzyme concentration on the reaction was studied. Except for argininosuccinate synthetase (Fig. 1), product formation was linear with respect to these parameters under the conditions used. The pH optima of ornithine transcarbamylase (in sodium glycylglycinate buffer) and argininosuccinate lyase (in potassium phosphate buffer) were both in the 7.6-8.0 range.

Based on levels of enzyme activity, hepatopancreas tissue has the greatest potential for arginine formation in S. oblongus. This is also reflected by the high

U R E A I X ~ E T A B O L I S M I N A L A N D S N A I L 445

rate of bicarbonate-x4C incorporation into arginine bv this tissue (Table 3). Argininosuccinate synthetase would appear to be rate-limiting in arginine synthesis by hepatopancreas. However, because of the non-linear formation of product with time and enzyme concentration under the conditions of assay (Fig. 1) and the possible complications of enzyme inhibition (Campbell & Speeg, 1968b), it seems

oJ

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FIG. 1. Effect of enzyme concentrat ion and incubation t ime on the argininosuccinate synthetase reaction in S. oblongus. Argininosuccinate- laC formation by hepatopancreas extracts was measured indirect ly as carbon dioxide-14C in the

coupled assay system of Campbel l & Speeg (1968b).

likely that measurements of this reaction in snail tissues are subject to considerable quantitative error. Maximal activity was obtained for the argininosuccinate synthetase reaction in the presence of L-aspartate and ATP and the supplementary enzymes argininosuccinate lyase, arginase and urease.

3. Arginase The levels of arginase activity in S. oblongus tissues are shown in Table 5.

In contrast to most pulmonates (Gaston & Campbell, 1966), the activity in hepatopancreas is remarkably low. Arginase was highest in kidney and mantle tissues followed by heart.

Extracts of all S. oblongus tissues examined were also found to hydrolyze 7-guanidinobutyrate. This activity, which appears to be due to an enzyme distinct from arginase (Porembska et al., 1968), is commonly found in gastropod molluscs (Baret et al., 1965). The possible role of 7-guanidinobutyrate ureohvdrolase in arginine degradation by mollnscs has been discussed (Campbell & Bishop, 1970).

446 PAt'L R. TRAMELL \Xl) JAMES W. CA.'x.~PBELL

4. Ureolytic activity

Considerable skepticism is generally associated with reports of animal urease due, perhaps, to the association of mammalian gastric urease with the gut micro- flora (Delluva et al., 1968). There is, however, considerable evidence for the constitutive nature of urease in helicid snails (Campbell et aL, 1972). That urea is broken down in vivo by S. oblongus was shown by injecting 5 tzmoles urea-HC (105 dpm/tzmole) into each of two individuals and monitoring their formation of carbon dioxide-t4C as described by Speeg & Campbell (1969). At the end of 12 hr, one individual had evolved 28 per cent of the injected urea-t4C as carbon dioxide-~C and the other had evolved 12 per cent.

The eight tissues listed in Table 5 were examined for ureolytic activity. Of these, only hepatopancreas and kidney had detectable activity. The level of ureolvtic activity in hepatopancreas was from 7 to 10 tzmoles/g tissue per hr at

TABLE 5 - - T t s s u E DISTRIBUTION OF ARGINASE AND y-GUANIDINOBUTYRATE UREOHYDROLASE I N Strophocheilus

Tissue activity (/zmole/g tissue per hr)

y-Guanidinobut3-rate A r g i n a s e ureohydrolase

Hepatopancreas 5-27 76-92 Reproductive tract 22 4 Foot Trace 5 Blood 0'3 - Kidney 2580 22 Albumen gland 9 - Mantle ("lung") 2390 25 Heart 238 -

30°C and in kidney, 0.04 tzmole/g tissue per hr. The amount of urea hydrolysis bv S. oblongus hepatopancreas is thus approximately one-half that of O. lactea hepatopancreas. Unlike the latter species, in which urease activity is present in most tissues (Speeg & Campbell, 1968), the activity in S. oblongus appears to be restricted mainly to two tissues. The presence of ureolytic activity in hepatopancreas and kidney is consistent with the small amounts of urea-14C found in these two tissues following the injection of arginine-*tC (see Table 2).

Ureolytic activity was not detected in all individuals of S. oblongus examined. Such qualitative individual variation in other species is indicative of a microbial origin of the activity (Delluva et al., 1968). However, the activity in S. oblongus hepatopancreas was similar to that in O. lactea hepatopancreas in at least two respects. First of all, the hydrolysis of urea by water extracts showed a pH optimum near 9 (Fig. 2). This property has been used to distinguish O. lactea urease from

U R E A M E T A B O L I S M I N A L A N D S N A I L 447

bacterial ureases (Speeg & Campbell, 1968), Secondly, centrifugation of 0.25 M sucrose homogenates (10 per cent, w/v) at 110,000 g for 30 rain does not sediment the activity. Under these conditions, 99 per cent of the activity remains in the supernatant fluid. In tissues whose ureolytic activity is associated with known

I- 1 i

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0 04 - "

i

7 8 9 I0 [I

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Fro. 2. Effect of pH on the ureolytic activity of water homogenates of S. oblongus hepatopancreas. The assay system was that of McDonald et al. (1971) using Tris-hydrochloride and modified by omitting EDTA from the reaction mixture.

symbionts, all of the activity is sedimented by centrifugation at 10,000 g for 10 min (Reddy & Campbell, 1969). Thus the individual variation in ureolytic activity in S. oblongus may not necessarily reflect a microbial origin. Although such variation has not been observed in O. lactea and H. aspersa (Campbell et al., 1972), these species have also never been observed to accumulate urea as has S. oblong'us.

DISCUSSION De Jorge & Petcrsen (1970) report actively feeding specimens of S. oblongus to

contain high concentrations of urea in hepatopancreas (79/~mole/g), lung tissue (99 ~moles/g) and especially kidney (268/xmole/g) . .~ter 90 days' estivation at 17:C, these values increase approximately fourfold in hepatopancreas (to 311 ~mole/g) and threefold in lung (to 279 t~mole/g) ; there is only a slight ( < 20 per cent) increase in kidney. After 75 days at 30°C, hepatopancreas urea increases to 325/xmole/g and lung tissue urea, to 396 p.mole/g; the kidney urea content shows a slight decrease under this condition. Qualitatively, the pulmonate Thaumastus achilles appears to be somewhat similar to S. oblongus in showing high tissue urea


levels which also increase during estivation. Because of the known function of blood urea accumulation for osmotic water retention in vertebrates (Schmidt- Nielsen, 1972), we have previously investigated this possibility in S. oblongus (Campbell et al., 1972). Although De Jorge et al. (1965) reported blood urea values for S. oblongus of around 5/zmole/ml, this compound is not always detectable in actively feeding individuals kept under the same conditions as used in the present work (Tramell & Campbell, 1970a). During food and water deprivation for up to 96 days at around 24°C, some individuals of S. oblongus were found to accumulate blood urea concentrations as high as 2.6 per cent (0.44 M) which is in the range encountered in elasmobranch blood (Campbell et al., 1972). There was, however, considerable individual variation in this response with some individuals showing no detectable urea in the blood. During food and water deprivation, there was little change in the hydration state of the tissues as indicated by the absence of changes in their dry weights but the protein content of the blood increased more than twofold indicating that the major water loss was from the blood. These results are consistent with a role for blood urea in osmotic water retention under desiccating conditions. In those individuals of S. oblongus that accumulated blood urea, the tissue urea contents were, in some cases, also elevated but were not in equilibrium with the blood urea. Blood urea/tissue urea ratios as high as 14 were obtained indicating that, in spite of its accumulation in the blood, this compound may still be turned over bv the tissues. These results are consistent with the ureolytic activity reported here in S. oblongus. The results presented here show that S. oblongus is capable of urea synthesis de novo via the arginine-urea pathway and that this species may also possess a urease. In these respects, its metabolism of urea is similar to that of O. lactea and H. aspersa. However, the metabolic control of urea turnover in S. oblongus which may aUow it to accumulate blood urea for osmotic water retention remains to be established.

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l'acide y-guanidobutyrique et de l'arginine par l'Mpatopancrdas ou le foie de divers invertdbr&. C.r. Sdanc. Soc. Biol. 159, 2446-2450.

BENSON J. V. & PATTERSON J. A. (1965) Accelerated chromatographic analysis of amino acids commonly found in physiological fluids on a spherical resin of specific design. ,4nalyt. Biochem. 13, 265-280.

CA,~IPBELL J. W. & BisHoP S. H. (1970) Nitrogen metabolism in molluscs. In ComparatA'e Biochemistry of Nitrogen 2vletabolism (Edited by CAMPBELL J. W.), pp. 103-206. Academic Press, London.

CAMPBELL J. W., DROTMAN R. B., McDoNALD J. A. & TaAMELL P. R. (1972) Nitrogen metabolism in terrestrial invertebrates. In Nitrogen 3/Ietabolism and the Enzqronment (Edited by CAMPBELL J. W. and GOLDSTEL'q L.). Academic Press, London. (In press.)

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TRAMELL P. R. & CAMPBELL J. W. (1970b) Carbamyl phosphate synthesis in a land snail, Strophoeheilus oblongus. 97. biol. Chem. 245, 6634-6641.

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Key Word Index--Urea; ornithine; citrulline; Strophocheilus oblongus; arginine; osmotic balance.

arginine and urea metabolism in the south american land snail, strophocheilus oblongus

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