Carbamyl Phosphate Synthetases in an Air-Breathing Teleost, Heteropneustes fossilis

Comp. Biochem. Physiol. Vol. 116B, No. 1, pp. 57–63, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0305-0491/97/$17.00 PII S0305-0491(96)00193-9

Carbamyl Phosphate Synthetases in an Air-Breathing Teleost, Heteropneustes fossilis Nirmalendu Saha,* Jacqueline Dkhar,* Paul M. Anderson† and Braja K. Ratha§ *Biochemical Adaptation Laboratory, Department of Zoology, North-Eastern Hill University, Shillong793022, India; †Department of Biochemistry and Molecular Biology, School of Medicine, University of Minnesota, Duluth, MN 55812-2487, U.S.A.; and §Environmental Biochemistry Laboratory, Centre for Advance Study in Zoology, Banaras Hindu University, Varanasi-221005, India

ABSTRACT. The Indian air-breathing teleost fish Heteropneustes fossilis has been shown to have a functional urea cycle and to be able to switch from ammoniotelic to ureotelic nitrogen metabolism when exposed to high levels of ammonia or air. The objective of this study was to identify the type of carbamyl phosphate synthetase (CPS) catalyzing the first step of the urea cycle in H. fossilis. Mitochondrial CPS III [glutamine- and N-acetylL-glutamate (NAG)-dependent] and cytosolic CPS II (glutamine-dependent) activities were found to be present in liver, analogous to that described for two other teleosts that have CPS III activity. The same activities and subcellar localization were found in kidney. Unexpectedly, a CPS I-like activity (ammonia- and NAG-dependent) was found to be present at levels higher than the CPS III activity in the mitochondrial fraction of both liver and kidney. The urea cycle-related CPS III found in invertebrates and fish is considered to be the evolutionary precursor of the urea cycle-related CPS I in ureotelic mammalian and amphibian species. Whether or not this CPS I-like activity 1) is due to the presence of a separate CPS I gene in addition to a CPS III gene or 2) represents an adapted CPS III activity in H. fossilis, these results suggest that the presence of both CPS I-like and CPS III activities may play an important physiological adaptive role in the tolerance of these fish to high concentrations of external ammonia. Copyright  1997 Elsevier Science Inc. comp biochem physiol 116B;1:57–63, 1997. KEY WORDS. Carbamyl phosphate synthetase, ammonia-dependent, glutamine-dependent, urea cycle, ammoniogenesis, ureogenesis, physiological adaptation, air-breathing teleost, Heteropneustes fossilis

INTRODUCTION Most teleost fishes are ammoniotelic in terms of nitrogen excretion and, until recently, the presence of a functional urea cycle was not known to exist in teleosts [for reviews, see references (5), (9) and (33)]. However, interest in the urea cycle and regulation of the expression of urea cycle enzymes in fish has increased in recent years due to 1) reports of a functional urea cycle in several different teleost fishes, including an Indian freshwater air-breathing catfish (Heteropneustes fossilis) (25,26), an alkaline lake-adapted tilapia (Oreochromis alcalicus grahami) (22) and the marine toadfishes Opsanus tau and O. beta (19), and 2) documentation of the expression of a specific type of urea cycle-related carbamyl phosphate synthetase (CPS, which catalyzes the first step of the urea cycle) in liver of largemouth bass (Micropterus salmoides) (1,10,11), in trout (Oncorhynchus mykiss) embryos (34) and liver of the marine toadfishes and midshipman (Porichthys notatus) (2,8,19). CPSs, which catalyze formation of carbamyl phosphate Correspondence to: Dr. Nirmalendu Saha, Biochemical Adaptation Laboratory, Department of Zoology, North-Eastern Hill University, Shillong793022, India. Fax (191) 364-760076; E-mail: [email protected] Received 24 January 1996; revised 12 May 1996; accepted 27 June 1996.

as the first step of both the urea cycle and pyrimidine nucleotide biosynthesis pathways, are important enzymes when considering the evolution of the urea cycle and nitrogen metabolism in vertebrate species. Three types of CPSs have been recognized [for a recent review, see Anderson (6)]. In ureotelic mammalian and amphibian species, the first step of the urea cycle is catalyzed by CPS I, which utilizes only ammonia as the nitrogen-donating substrate, requires Nacetyl-L-glutamate (NAG) as an allosteric activator for activity and is localized exclusively in the mitochondrial matrix in liver and small intestine. Carbamyl phosphate formation for pyrimidine nucleotide biosynthesis in all vertebrates is catalyzed by CPS II, which utilizes glutamine as the physiologically significant nitrogen-donating substrate, does not require NAG for activity (and activity is not affected by the presence of NAG), is subject to allosteric inhibition by UTP and is localized in the cytosol of many tissues as part of a multifunctional enzyme that includes the activities of the next two steps of the pathway, aspartate carbamyl transferase and dihydro-orotase. A third type of CPS, CPS III, was first reported in invertebrates by Trammel and Campbell (29,30) and later in the liver of largemouth bass, a freshwater teleost, and, at much higher levels, in liver of marine ureosmotic elasmobranchs

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by Anderson (1,2). The properties and function of CPS III are very much like those of urea cycle-related CPS I, except that glutamine is utilized as the nitrogen-donating substrate instead of ammonia (3,11). The glutamine- and NAG-dependent CPS III is thought to be the evolutionary precursor to the ammonia- and NAG-dependent CPS I of ureotelic mammalian and amphibian species, (6,9,15,16,19). The function of CPS III in elasmobranchs is related to urea synthesis for the purpose of osmoregulation (4,7) and, as noted above, apparently in some teleosts for reasons that are not yet understood in most cases. The presence of a functional urea cycle and the transition from ammoniotelism to ureotelism under hyperammonia stress and during exposure to air, as the result of a 2- to 3fold increase in the level of activity of all of the urea cycle enzymes except arginase (present at high levels), including CPS activity, have been reported in the air-breathing amphibious freshwater teleost, H. fossilis from India (23,25– 28). However, the type of CPS present in this unique fish has not yet been established. Here we report that liver and kidney from H. fossilis have mitochondrial CPS III and cytosolic CPS II activities, analogous to the liver of largemouth bass (10) and marine toadfish (8). However, there also appears to be a CPS I-like activity in the mitochondria, which has not been previously observed in the other species of fish. MATERIALS AND METHODS Animal The fish, H. fossilis, 40–50 g body wt, were purchased from commercial sources and acclimatized in the laboratory for 4–6 weeks at about 30°C with a 12 hr : 12 hr light and dark period before being used for experiments. No sex differentiation of fish was done while performing these studies. Minced pork liver was given as food, and the water, which was collected from a natural stream, was changed on alternate days. Subcellular Fractionation Subcellular fractionation was carried out by a method described previously with minor modification (13). All steps were carried out at 4°C. Freshly excised liver and kidney were minced and suspended in 4.5 volumes of the fractionating buffer containing 0.01 M Tris-HCl buffer (pH 7.5), 0.3 M mannitol, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 0.1 M KCl. The suspension was homogenized with a motor-driven Potter-Elvehjem glass homogenizer with a loosely fitted Teflon pestle. The homogenate was centrifuged at 600 g for 10 min to remove the cell debris and nuclei. The supernatant was decanted and saved. The loose pellet was again resuspended in an equal volume of the fractionation buffer, homogenized a second time as described above, and centrifuged at 600 g for 10 min. The supernatant was decanted, combined with the first supernatant and centrifuged at 14,000 g for 30 min to give a well-defined and

firm pellet (mitochondrial fraction). The supernatant from this centrifugation step is the soluble fraction. The mitochondrial fraction was resuspended in the same fractionation buffer as described above but without mannitol. Both the mitochondrial and cytosolic fractions were treated with 0.1% Triton X-100. The mitochondrial fraction was also sonicated to facilitate proper breakage of mitochondria. Enzyme Assays The reaction mixture for the assay of CPS contained 50 mM potassium phosphate buffer (pH 7.5), 50 mM NaHCO3 , 10 mM L-ornithine, 15 mM MgSO4 , 10 mM ATP and 10 units of ornithine transcarbamylase (OTC) (Sigma, type IV) and tissue extract in a final volume of 1 ml. The reaction mixture also contained 5 mM NH4Cl, 25 mM L-glutamine, 5 mM NAG and/or 1 mM UTP in different combinations as indicated in Tables 1 and 2. The amount of citrulline formed after incubating at 30°C for 30 min was expressed as CPS activity. Glutamate dehydrogenase (GDH; a mitochondrial marker enzyme) and lactate dehydrogenase (LDH; a cytosolic marker enzyme) were assayed following the method of Olson and Anfinsen (21) and Vorhaben and Campbell (31), respectively. The enzyme activity was expressed as the amount that catalyzed the formation of 1 µmol of citrulline in the case of CPS, and 1 µmol of NADH utilized for GDH and LDH per hr at 30°C. Apparent Km values for ammonia and glutamine were estimated from double reciprocal (Lineweaver-Burk) plots of velocity vs substrate concentration. Reaction conditions were as described above, except the ammonia and glutamine concentrations were varied (0.5–100 mM and 0.1–12 mM, respectively). Preparation and Assay of Extracts of Liver from the Elasmobranch Squalus acanthias (Spiny Dogfish) An extract was prepared by homogenizing 4 g of liver that had been stored at 280°C with 16 ml of fractionation buffer (without mannitol) followed by sonication and then centrifugation at 14,000 g for 10 min. The supernatant (15 ml) was passed through a Sephadex G-25 column equilibrated with fractionation buffer (without mannitol); 5 ml of the middle portion of the eluted peak of protein was collected. The activity of the extract was determined essentially as previously described (2), except that the reaction mixtures were identical to those described in the previous paragraph (however, OTC was not added). Liver Perfusion Technique The fish were anaesthetized in MS 222 for 2 min before operation for liver perfusion. Livers were perfused via the portal vein in a nonrecirculating manner, with haemoglobin-free media as used by French et al. (14) and certain mod-

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TABLE 1. Activity of CPS (units/g wet wt) in liver of H. fossilis in the presence of different nitrogen-donating substrates, NAG

and/or UTP Components in the reaction mixture Ammonia Ammonia 1 UTP Ammonia 1 NAG Ammonia 1 NAG 1 UTP Glutamine Glutamine 1 UTP Glutamine 1 NAG Glutamine 1 NAG 1 UTP Ammonia 1 glutamine 1 NAG Ammonia 1 glutamine 1 NAG 1 UTP GDH LDH

Homogenate

600 6 30 4824 6 450

Mitochondrial fraction

Cytosolic fraction

0.26 6 0.04 0.20 6 0.05 3.11 6 0.33 3.00 6 0.47 0.39 6 0.05 BLD 2.20 6 0.22 2.31 6 0.18 4.47 6 0.27 4.52 6 0.26 410 6 31 (68) 857 6 106 (18)

0.34 6 0.09 0.31 6 0.07 0.35 6 0.02 0.33 6 0.07 2.02 6 0.25 0.34 6 0.06 2.34 6 0.28 0.41 6 0.09 2.41 6 0.18 0.49 6 0.06 150 6 18 (24) 3692 6 403 (77)

Values are expressed as mean 6 SEM (n 5 3–4). The reaction mixture for carbamyl phosphate synthetase (CPS) assay contained everything as mentioned in the Materials and Methods section, plus 5 mM ammonium chloride, 25 mM glutamine, 5 mM N-acetyl-L-glutamate (NAG) and/or 1 mM UTP wherever noted. One unit of enzyme activity was expressed as the amount that catalyzed 1 µmol of citrulline formed in the case of CPS, and 1 µmol of NADH utilized for glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH) per hr at 30°C. BLD, below the level of detection; % activity out of the total in the case of GDH and LDH are given in parentheses.

ifications (28). The isotonic media (265 mOsmol/1) contained 119 mM NaCl, 5 mM NaHCO3, 5.4 mM KCl, 0.35 mM Na2HPO 4, 0.44 mM KH2PO4 , 0.81 mM MgSO4 and 1.25 mM CaCl2 as a basic solution for perfusion. The medium also contained 5 mM L-glucose and 2 mM L-ornithine. Ammonium chloride (2 mM), L-glutamine (5 mM) or a combination of these two was infused along with the perfusion media to study the rate of formation of urea by the perfused liver. The medium was gassed with O2 /CO2 (99 : 1, v/v) before infusing into the liver at a flow rate of

5–6 ml/g liver/min. The pH of the medium was always maintained at 7.4 after gassing. The temperature of the medium was 30°C. The effluent coming out of the perfused liver was collected through a cannula catheterised at the superior vena cava at 1-min intervals for analysis of ammonia and urea-N. The concentrations of ammonia and ureaN in the effluent collected at the 1-min intervals were measured enzymatically, based on the procedure of Kun and Kerney (18). The rate of formation of ammonia from glutamine in the reaction mixture was determined following the

TABLE 2. Activity of CPS (units/g wet wt) in kidney of H. fossilis in the presence of different nitrogen-donating substrates,

NAG and/or UTP Components in the reaction mixture Ammonia Ammonia 1 UTP Ammonia 1 NAG Ammonia 1 NAG 1 UTP Glutamine Glutamine 1 UTP Glutamine 1 NAG Glutamine 1 NAG 1 UTP Ammonia 1 glutamine 1 NAG Ammonia 1 glutamine 1 NAG 1 UTP GDH LDH

Homogenate

260 6 17 2678 6 144

Mitochondrial fraction

Cytosolic fraction

0.14 6 0.03 0.17 6 0.03 2.12 6 0.17 2.09 6 0.18 0.22 6 0.04 BLD 1.46 6 0.05 1.42 6 0.07 3.04 6 0.19 3.12 6 0.15 224 6 16 (86) 189 6 18 (7)

0.20 6 0.02 0.22 6 0.05 0.19 6 0.02 0.25 6 0.04 1.06 6 0.11 0.17 6 0.03 1.18 6 0.15 0.20 6 0.01 0.25 6 0.04 0.29 6 0.03 34 6 5 (13) 2232 6 124 (83)

Values are expressed as mean 6 SEM (n 5 3–4). The concentrations of ammonium chloride, glutamine, NAG and/or UTP in the reaction mixture are the same as noted in Table 1. Abbreviations as in Table 1.

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same enzymatic method as mentioned previously. Protein was precipitated out from the reaction mixture with 2 M PCA at a different time of incubation, and the ammonia was measured in the supernatant after neutralizing with 2 N NaOH. Data collected from 3–4 replicates were statistically analyzed and presented as mean 6 SEM. Chemicals OTC (type IV), GDH, urease, NAG, DTT, UTP and MS 222 were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Sodium [14 C]bicarbonate was obtained from Research Products International Corp. All other reagents used were of the highest quality obtained from indigenous sources. RESULTS CPS III Activity in Mitochondrial Extracts The initial objective of these studies was to assay mitochondrial and cytosolic fractions of liver and kidney under the different conditions that would permit identification of CPS III in the mitochondria and CPS II in the cytosol (the anticipated findings). As noted by Anderson (5), these two enzyme activities can be identified by the fact that CPS III would be localized in the mitochondria (whereas CPS II would be localized in the cytosol), and although CPS III (like CPS II) utilizes glutamine as substrate, unlike CPS II, it would be activated by NAG and would not be inhibited by UTP. As shown in Tables 1 and 2, a significant level of glutamine-dependent CPS activity was present in the cytosolic fractions of both liver and kidney. This activity was inhibited by UTP and was not affected by the presence of NAG. In addition, little activity was observed when glutamine was replaced by 5 mM ammonia, whether or not NAG was present. These kinetic and specificity properties are characteristic of CPS II. It should be noted that CPS II is also active with ammonia as substrate, but at pH 7.5 the ammonia concentration must be much higher (e.g., 100 mM) than the 5 mM used here to attain a maximum velocity comparable to that attained with glutamine. Significant glutamine-dependent activity was also found in the mitochondrial fractions of both liver and kidney, but only when NAG was present; this glutamine- and NAG-dependent CPS activity was not affected by the presence of UTP. These properties are characteristic of CPS III. Several observations made from the activity in liver mitochondrial extracts indicate that the glutamine-dependent activity is not an artifact in which activity is actually due to ammonia as a substrate, present as a contaminant of glutamine or arising from hydrolysis of glutamine by a glutaminase present in the extract. Product formation with either ammonia or glutamine as substrate was linear with time up to 45 min (data not shown). Some ammonia was formed with time in reac-

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tion mixtures, with glutamine (e.g., 0.06, 0.13 and 0.2 mM after 15, 30 and 45 min, respectively), but the levels were too low to account for the rate observed with glutamine as substrate (note the Km values of 2.0 and 0.4 mM for ammonia and glutamine, respectively, below). Thus, these results appear to confirm the expectation that these fish have a urea cycle-related mitochondrial glutamine- and NAGdependent CPS III and a pyrimidine-related cytosolic glutamine-dependent CPS II activity, analogous to the only other two teleosts in which the distribution of these CPSs has been documented, i.e., largemouth bass (10) and marine toadfish (8). The distribution of the activities of GDH and LDH, marker enzymes for mitochondrial and cytosolic fractions, respectively, indicates that although complete separation between the mitochondrial and cytosolic components was not attained, the subfractionation did provide significant enrichment of the two subcellular fractions, sufficient for the purposes described here. CPS I-Like Activity in Mitochondrial Extracts An unexpected finding was the presence, in the mitochondrial fractions of both liver and kidney, of a level of ammonia- and NAG-dependent CPS activity that was not affected by the presence of UTP, but that was higher than the glutamine- and NAG-dependent activity (Tables 1 and 2). Although CPS III from spiny dogfish and largemouth bass will catalyze carbamyl phosphate formation with ammonia as substrate, and the Km is quite low (comparable to that of CPS I, i.e., about 2 mM), the maximal velocity is less than one fourth of that observed with glutamine as substrate (3,11). A possible explanation may be that the ammoniaand NAG-dependent activity is due to a typical CPS III, but that the assay conditions used in this study are different than has been used in previous studies and permits a higher activity with ammonia relative to that observed with glutamine. However, when CPS III in extracts of liver from S. acanthias was determined under the assay conditions used in this study, ammonia- and NAG-dependent activity remained at a level that was about one half of that obtained with glutamine (data not shown). If ammonia- and NAGdependent activity is distinct from glutamine- and NAGdependent activity, then the two should be additive. As shown in Tables 1 and 2, inclusion of both ammonia and glutamine in the reaction mixture does result in an increase in the level of activity that is nearly additive of the two activities obtained separately. When the CPS III from S. acanthias was assayed under these conditions, an additive effect was not observed (data not shown). It should also be noted that this additive effect was not observed in the cytosolic fractions (Tables 1 and 2). The observed additive effect might occur if either ammonia or glutamine can serve as nitrogen-donating substrate and the concentration of each is well below its Km value. This does not appear to be an explanation, however, because the measured Km values

Carbamyl Phosphate Synthetase in an Air-Breathing Teleost

TABLE 3. Rate of urea synthesis from ammonia and gluta-

mine separately, and from ammonia and glutamine together by the perfused liver of H. fossilis Urea synthesis ( mmol/g liver/hr) Control Ammonia (2 mM) Glutamine (5 mM) Ammonia (2 mM) 1 glutamine (5 mM)

BLD 10.7 6 1.70 8.6 6 1.35 17.6 6 2.41

Livers were perfused first with isotonic medium containing 5 mM glucose and 2 mM ornithine as mentioned in the Materials and Methods section for 20 min without ammonium chloride and glutamine, followed by the infusion of ammonium chloride (2 mM), glutamine (5 mM) or a combination of these two substrates. Values were obtained between 26–30 min of infusion and calculated as mean 6 SEM (n 5 3–4). BLD, below the level of detection.

for ammonia and glutamine were found to be 2.0 and 0.4 mM, respectively. Urea Formation from Ammonia and Glutamine by Perfused Liver The observations described in the preceding paragraph suggest that there may be two separate NAG-dependent CPS activities in the mitochondrial fractions from both liver and kidney, one ammonia-dependent and the other glutaminedependent. Additional support for this view was obtained from liver perfusion studies. When ammonium chloride and glutamine were infused separately into liver of H. fossilis, a significant rate of urea formation was observed with either substrate, the rate with ammonium chloride being somewhat higher than that observed with glutamine (Table 3). When ammonium chloride and glutamine were infused together, the rate of urea formation was increased to a level that was almost additive of that observed with each substrate alone. DISCUSSION The results from the limited number of studies reported to date characterizing the presence of CPS activity in ureotelic, ureosmotic and ureogenic (i.e., all of the urea cycle enzyme activities can be detected, but the fish are primarily ammoniotelic) fish have suggested that a mitochondrial CPS III is responsible for catalyzing carbamyl phosphate formation from glutamine as the first step of urea formation via the urea cycle in fish. This has been directly confirmed in liver of S. acanthias (a representative marine ureosmotic elasmobranch), where the glutamine synthetase (GS) is localized exclusively in the mitochondria and mitochondrial formation of citrulline from ammonia has been shown to involve obligatory intermediate formation of glutamine (7). The characteristic of exclusive mitochondrial localization of GS in the liver of elasmobranchs is accompanied by an

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absence of the pyrimidine-related CPS II in the liver, presumably because glutamine is utilized exclusively for urea formation (17). The situation in teleosts is less clear. The presence of CPS III activity has been documented in only a few teleost species, as noted in the Introduction section. In largemouth bass, the liver GS is localized primarily in the cytosol, and available evidence suggests that the liver CPS III may not be functional in the adult (10). GS is also localized primarily in the cytosol in O. tau and P. notatus; in O. beta, however, a significant level of the mitochondrial GS activity is present in the mitochondria as well as the cytosol, and the level of the mitochondrial GS may fluctuate in relation to the ureotelic nature of the fish’s nitrogen metabolism (32). Also, unlike elasmobranchs, cytosolic pyrimidine-related CPS II is present in the liver of those teleost species, where mitochondrial CPS III activity has been reported and the presence of CPS II activity has been documented (2,8,10). In H. fossilis, high levels of GS in liver and kidney are localized primarily in the mitochondria (12), along with the localization of arginase primarily again in the mitochondria (13). In this respect, H. fossilis resembles elasmobranchs. However, in contrast to elasmobranchs and like other teleosts, cytosolic CPS II is present in liver (and kidney). The level of CPS II activity noted here for H. fossilis is higher than has been reported for other teleosts. The level of CPS III activity in H. fossilis is comparable to the level of CPS III activity in those few teleost species where CPS III activity is present and has clearly been shown to be related to a functional urea cycle, i.e., marine toadfish (8,19) and the alkaline lake-adapted tilapia (22). Given the known ureotelic capability of H. fossilis, this observation was not unexpected. However, the presence of significant ammonia- and NAG-dependent CPS activity in liver and kidney mitochondria of H. fossilis is unique from that observed in other teleosts. This activity, of course, is characteristic of a CPS I. The presence of a gene for both a CPS III and a CPS I in H. fossilis would not seem to be a likely explanation for these two activities within the context of the current understanding of the structural relationships between these two enzymes, their currently understood species distributions (CPS I is present only in ureotelic mammalian and amphibian species, CPS III is present only in invertebrates and fishes) and the prevailing view that CPS I evolved from CPS III (5,6,9,15,16,20). Perhaps adaptation in H. fossilis and closely related species was achieved as a separate event in which the CPS III gene underwent duplication and one gene subsequently lost the structural requirements for utilization of glutamine as substrate. Alternatively, perhaps the observed CPS I-like activity represents an adapted form of CPS III with separate ammonia and glutamine binding sites. Either of these possibilities would represent possible intermediate scenarios for a proposed evolutionary transition from CPS III to CPS I. The glutamine-dependent CPS III activity described here does appear to differ from the CPS III activities of S. acanthias and large-

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FIG. 1. Ilustration of the urea cycle in the liver and kidney of H. fossilis. CPS I, II and III, carbamyl phosphate synthetase I, II and III; OTC, ornithine transcarbamylase; ASS, argininosuccinate synthetase; ASL, argininosuccinate lyase; ARG, arginase; NAG, N-acetyl-L-glutamate; GS, glutamine synthetase; GDH, glutamate dehydrogenase; ?, not confirmed.

mouth bass. The dogfish and bass enzymes catalyze carbamyl phosphate formation from high concentrations of glutamine in the absence of NAG at a rate that is over one half of the rate obtained when NAG is present [(3,11); also observed for the CPS III in the dogfish liver extract prepared as described in the Materials and Methods section and assayed under the assay conditions described in this study (data not shown)]. As noted in Tables 1 and 2, significant glutaminedependent activity in mitochondria from either tissue of H. fossilis is not observed unless NAG is present. Elucidation of the nature of the two types of activities will require purification and characterization of the enzyme(s) responsible for these activities and/or the mRNA(s) coding for the enzyme(s). The presence of significant levels of both kinds of CPS activity, enabling H. fossilis to utilize ammonia, either directly or indirectly as glutamine, as substrate for carbamyl phosphate formation in mitochondria, may be an adaptive physiological feature characteristic of these species that is related to their remarkable tolerance to high levels of ambient ammonium chloride (up to 75 mM) (24,27) and their ability to switch from ammoniotelism to ureotelism when exposed to high levels of ammonia and during exposure to air (Fig. 1). The authors thank the North-Eastern Hill University, Shillong for laboratory facilities and financial support. The liver perfusion technique learned in the laboratory of Professor Dr. D. Haussinger in Germany by one of the authors (N. S.) is gratefully acknowledged.

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