Almost all about citrulline in mammals

Summary. Citrulline (Cit, C₆H₁₃N₃O₃), which is a ubiquitous amino acid in mammals, is strongly related to arginine. Citrulline metabolism in mammals is divided into two fields: free citrulline and citrullinated proteins. Free citrulline metabolism involves three key enzymes: NO synthase (NOS) and ornithine carbamoyltransferase (OCT) which produce citrulline, and argininosuccinate synthetase (ASS) that converts it into argininosuccinate. The tissue distribution of these enzymes distinguishes three “orthogonal” metabolic pathways for citrulline. Firstly, in the liver, citrulline is locally synthesized by OCT and metabolized by ASS for urea production. Secondly, in most of the tissues producing NO, citrulline is recycled into arginine via ASS to increase arginine availability for NO production. Thirdly, citrulline is synthesized in the gut from glutamine (with OCT), released into the blood and converted back into arginine in the kidneys (by ASS); in this pathway, circulating citrulline is in fact a masked form of arginine to avoid liver captation. Each of these pathways has related pathologies and, even more interestingly, citrulline could potentially be used to monitor or treat some of these pathologies. Citrulline has long been administered in the treatment of inherited urea cycle disorders, and recent studies suggest that citrulline may be used to control the production of NO. Recently, citrulline was demonstrated as a potentially useful marker of short bowel function in a wide range of pathologies. One of the most promising research directions deals with the administration of citrulline as a more efficient alternative to arginine, especially against underlying splanchnic sequestration of amino acids. Protein citrullination results from post-translational modification of arginine; that occurs mainly in keratinization-related proteins and myelins, and insufficiencies in this citrullination occur in some auto-immune diseases such as rheumatoid arthritis, psoriasis or multiple sclerosis.     

Regulation of nitric oxide production by arginine metabolic enzymes

Nitric oxide (NO) is synthesized from arginine by NO synthase (NOS), and the availability of arginine is one of the rate-limiting factors in cellular NO production. Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by successive actions of argininosuccinate synthetase (AS) and argininosuccinate lyase (AL), forming the citrulline-NO cycle. AS and sometimes AL have been shown to be coinduced with inducible NOS (iNOS) in various cell types including activated macrophages, vascular smooth muscle cells, glial cells, neuronal PC12 cells, and pancreatic beta-cells. Cationic amino acid transporter (CAT)-2 is induced in activated macrophages but not in PC12 cells. On the other hand, arginase can downregulate NO production by decreasing intracellular arginine concentrations. iNOS and arginase activities are regulated reciprocally in macrophages by cytokines, and this may guarantee the efficient production of NO. In contrast, iNOS and arginase isoforms (type I and II) are coinduced in lipopolysaccharide (LPS)-activated macrophages. These results indicate that NO production is modulated by the uptake, recycling, and degradation of arginine.     

NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice

Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino acid and protein kinetics are regulated by NO synthesized by nitric oxide synthase-2 or -3 (NOS2 or NOS3), we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2-/-), and NOS3-deficient (NOS3-/-) mice under control (unstimulated) and lipopolysaccharide (LPS)-treated conditions. Muscle amino acid metabolism was studied across the hindquarter by infusing the stable isotopes L-[ring-2H5]phenylalanine, L-[ring-2H2]tyrosine, L-[guanidino-15N2]arginine, and L-[ureido-13C,2H2]citrulline. Muscle blood flow was measured using radioactive p-aminohippuric acid dilution. Under baseline conditions, muscle blood flow was halved in NOS2-/- mice (P < 0.1), with simultaneous reductions in muscle glutamine, glycine, alanine, arginine release and glutamic acid, citrulline, valine, and leucine uptake (P < 0.1). After LPS treatment, (net) muscle protein synthesis increased in WT and NOS2-/- mice [LPS vs. control: 13 +/- 3 vs. 8 +/- 1 (SE) nmol.10 g(-1).min(-1) (WT), 18 +/- 5 vs. 7 +/- 2 nmol.10 g(-1).min(-1) (NOS2-/-); P < 0.05 for LPS vs. control]. This response was absent in NOS3-/- mice (LPS vs. control: 11 +/- 4 vs. 10 +/- 2 nmol.10 g(-1).min(-1)). In agreement, the increase in muscle arginine turnover after LPS was also absent in NOS3-/- mice. In conclusion, disruption of the NOS2 gene compromises muscle glutamine release and muscle blood flow in control mice, but had only minor effects after LPS. NOS3 activity is crucial for the increase in muscle arginine and protein turnover during early endotoxemia.     

Nitric oxide synthase from cerebellum catalyzes the formation of equimolar quantities of nitric oxide and citrulline from L-arginine.

This study examined whether constitutive nitric oxide (NO) synthase from rat cerebellum catalyzes the formation of equimolar amounts of NO plus citrulline from L-arginine under various conditions. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NOx (NO+nitrite [NO2-] + nitrate [NO3-]) by chemiluminescence after reduction of NOx to NO by acidic vanadium (III). Equal quantities of NO plus citrulline were generated from L-arginine and the formation of both products was linear for about 20 min at 37 degrees C provided L-arginine was present in excess to maintain a zero order reaction rate. Deletion of NADPH, addition of the calmodulin antagonist calmidazolium, or addition of NO synthase inhibitors (NG-methyl-L-arginine, NG-amino-L-arginine) abolished or markedly inhibited the formation of both NO and citrulline. The Km for L-arginine (14 microM; 18 microM) and the Vmax of the reaction (0.74 nmol/min/mg protein; 0.67 nmol/min/mg protein) were the same whether NO or citrulline formation, respectively, was monitored. These observations indicate clearly that NO and citrulline are formed in equimolar quantities from L-arginine by the constitutive isoform of NO synthase from rat cerebellum.     

Palmitate increases nitric oxide synthase activity that is involved in palmitate-induced cell death in cardiomyocytes.

The objective of this study was to test the hypothesis that nitric oxide synthase (NOS) is subjected to regulatory control by palmitate, and that nitric oxide (NO) is operative in palmitate-induced cell death. Palmitate induced a significant ( p<0.05 ) concentration-dependent increase in NOS activity measured by the conversion of [(3)H]arginine to [3H]citrulline in embryonic chick cardiomyocytes. Cellular eNOS and iNOS, determined by immunocytochemistry, were increased by palmitate. Western blotting also showed that palmitate, 500 microM for 4h, significantly increased the amount of cellular of eNOS and iNOS by 36.2+/-6.5% ( p<0.001 ) and 38.4+/-14.4% ( p<0.05 ), respectively. The NOS inhibitor L-NAME significantly ( p<0.05 ) accentuated palmitate-induced cell death These data suggest that palmitate has a bifunctional effect on cell viability--in addition to loss of cell viability, palmitate stimulates NOS activity by inducing an increase in cellular eNOS and iNOS with the resultant NO production serving to protect cardiomyocytes from palmitate-induced cell death.     

Mitochondria: Role of citrulline and arginine supplementation in MELAS syndrome

Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.     

Manipulation of citrulline availability in humans

To determine whether circulating citrulline can be manipulated in vivo in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate (0.36 g.kg(-1).day(-1)), a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g.kg(-1).day(-1)), in randomized order. Plasma, erythrocyte (RBC), and urinary citrulline concentrations were determined by gas chromatography-mass spectrometry at 3-h intervals between 1100 and 2000 on each study day. Regardless of treatment, RBC citrulline was lower than plasma citrulline, with an RBC-to-plasma ratio of 0.60 +/- 0.04, and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an approximately 7% drop in plasma glutamine (P = 0.013), and 18 +/- 14% (P < 0.0001) and 19 +/- 17% (P < 0.01) declines in plasma and urine citrulline, respectively, with no alteration in RBC citrulline. Oral L-citrulline administration was associated with 1) a rise in plasma, urine, and RBC citrulline (39 +/- 4 vs. 225 +/- 44 micromol/l, 0.9 +/- 0.3 vs. 6.2 +/- 3.8 micromol/mmol creatinine, and 23 +/- 1 vs. 52 +/- 9 micromol/l, respectively); and 2) a doubling in plasma arginine level, without altering blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, because citrulline is bioavailable and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.     

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.     

A role for PPARα in the regulation of arginine metabolism and nitric oxide synthesis

The pleiotropic effects of PPARα may include the regulation of amino acid metabolism. Nitric oxide (NO) is a key player in vascular homeostasis. NO synthesis may be jeopardized by a differential channeling of arginine toward urea (via arginase) versus NO (via NO synthase, NOS). This was studied in wild-type (WT) and PPARα-null (KO) mice fed diets containing either saturated fatty acids (COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers of arginine metabolism were assayed in urine and plasma. mRNA levels of arginases and NOS were determined in liver. Whole-body NO synthesis and the conversion of systemic arginine into urea were assessed by using ¹⁵N₂-guanido-arginine and measuring urinary ¹⁵NO₃ and [¹⁵N]-urea. PPARα deficiency resulted in a markedly lower whole-body NO synthesis, whereas the conversion of systemic arginine into urea remained unaffected. PPARα deficiency also increased plasma arginine and decreased citrulline concentration in plasma. These changes could not be ascribed to a direct effect on hepatic target genes, since NOS mRNA levels were unaffected, and arginase mRNA levels decreased in KO mice. Despite the low level in the diet, the nature of the fatty acids modulated some effects of PPARα deficiency, including plasma arginine and urea, which increased more in KO mice fed the LIN diet than in those fed the COCO diet. In conclusion, PPARα is largely involved in normal whole-body NO synthesis. This warrants further study on the potential of PPARα activation to maintain NO synthesis in the initiation of the metabolic syndrome.     

Enhanced utilization and altered metabolism of arginine in inflammatory macrophages caused by raised nitric oxide synthesis

Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells).We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important.     

Influence of pegylated interferon-α therapy on plasma levels of citrulline and arginine in melanoma patients

Summary. The aim of this study was to evaluate the effect of pegylated interferon-alpha (PEG-IFN-α) on the plasma citrulline/arginine ratio, regarded as an index of nitric oxide (NO) synthesis, in patients with high-risk melanoma. Forty patients were randomly assigned to either PEG-IFN-α treatment (n = 22) or to observation only (control group, n = 18). The treatment group received 6 μg PEG-IFN-α/kg once a week during 8 weeks, followed by a maintenance dose of 3 μg/kg/wk. Blood was collected at different time points, plasma concentrations of citrulline and arginine were measured and the ratio of citrulline/arginine was calculated. Patients treated with PEG-IFN-α showed a significant decrease in the concentrations of citrulline and in the citrulline/arginine ratio during the whole study period, both compared to baseline values and to the control group. The data suggest that therapy with PEG-IFN-α results in a marked decrease in the synthesis of NO in melanoma patients.     

Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.     

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.     

Ethyl carbamate precursor citrulline formation from arginine degradation by malolactic wine lactic acid bacteria

Major commercially available strains for induction of malolactic fermentation in wine were examined for arginine metabolism in a resting cell system at wine pH with the aim of evaluating their ability to excrete and utilize citrulline, a precursor of carcinogenic ethyl carbamate (urethane). All strains tested excreted citrulline from arginine degradation. Citrulline was stored intracellularly during growth in arginine rich medium and was released upon lysis of the cells. All strains were found to degrade citrulline as a sole amino acid and some of them were able to reutilize previously excreted citrulline.     

Serum amino acid profile in patients with acute pancreatitis

Patients in the early phase of acute pancreatitis (AP) have reduced serum levels of arginine and citrulline. This may be of patho-biological importance, since arginine is the substrate for nitric oxide, which in turn is involved in normal pancreatic physiology and in the inflammatory process. Serum amino acid spectrum was measured daily for five days and after recovery six weeks later in 19 patients admitted to the hospital for acute pancreatitis. These patients had abnormal levels of most amino acids including arginine, citrulline, glutamine and glutamate. Phenylalanine and glutamate were increased, while arginine, citrulline, ornithine and glutamine were decreased compared to levels after recovery. NO(2)/NO(3) concentration in the urine, but not serum arginase activity, was significantly increased day 1 compared to day 5 after admission. Acute pancreatitis causes a disturbance of the serum amino acid spectrum, with possible implications for the inflammatory process and organ function both in the pancreas and the gut. Supplementation of selected amino acids could possibly be of value in this severe condition.     

Radiochemical HPLC detection of arginine metabolism: measurement of nitric oxide synthesis and arginase activity in vascular tissue.

Nitric oxide (NO) plays a key role in vascular homeostasis. Accurate measurement of NO production by endothelial nitric oxide synthase (eNOS) is critical for the investigation of vascular disease mechanisms using genetically modified animal models. Previous assays of NO production measuring the conversion of arginine to citrulline have required homogenisation of tissue and reconstitution with cofactors including NADPH and tetrahydrobiopterin. However, the activity and regulation of NOS in vivo is critically dependant on tissue levels of these cofactors. Therefore, understanding eNOS regulation requires assays of NO production in intact vascular tissue that do not depend on the addition of exogenous cofactors and have sufficient sensitivity and specificity. We describe a novel technique, using radiochemical detection of arginine to citrulline conversion, to measure NO production within intact mouse aortas, without exogenous cofactors. We demonstrate the presence of arginase activity in mouse aortas which has the potential to confound this assay. Furthermore, we describe the use of N-hydroxy-nor-L-arginine (nor-NOHA) to inhibit arginase and permit specific detection of NO production in intact mouse tissue. Using this technique we demonstrate a 2.4-fold increase in NO production in aortas of transgenic mice overexpressing eNOS in the endothelium, and show that this technique has high specificity and high sensitivity for detection of in situ NO synthesis by eNOS in mouse vascular tissue. These results have important implications for the investigation of NOS regulation in cells and tissues.     

Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor

A new metabolic pathway characterized recently that is expressed in activated macrophages involves the formation of nitric oxide ('N = O) as an intermediate. The 'N = O formed decomposes to nitrite (NO2-) and nitrate (NO3-). The substrate for the reaction is the amino acid arginine which is oxidized at the guanido nitrogen to yield citrulline as the other product of the reaction. The studies reported here show that the activity for this unusual oxidation reaction which is contained in the 100,000 x g supernatant was lost after desalting on a Sephadex G-25 column. A small molecule cofactor was found to be required for the restoration of activity. The addition of (6R)-tetrahydrobiopterin (BH4) and NADPH led to complete recovery of activity in this desalted protein. Analysis of macrophage cell extracts, using high performance liquid chromatography with electrochemical detection, showed that BH4 was present at 17 pmol/10(6) cells or 2.1 microM in macrophage supernatant. Only the (6R)-isomer was present. With the addition of BH4 and NADPH, there was loss of arginine that was equal to the NO2-, NO3-, and citrulline formed. With substoichiometric levels of NADPH relative to BH4, the loss of arginine was greater than the formation of the end products of the reaction. A scheme for the reaction pathway consistent with the results involves N-hydroxylation of arginine as the initial step. The participation of BH4 in this type of oxidative chemistry is consistent with previous characterizations of this co-factor.     

Effect of Magnesium tanshinoate B on the production of nitric oxide in endothelial cells

Nitric oxide (NO) is a potent vasodilator which plays an important role in regulating vascular tones. Danshen, a Chinese herbal medicine has been widely used for the treatment of cardiovascular diseases. The objective of this study was to investigate the effect of magnesium tanshinoate B (MTB), a compound purified from Danshen, on the production of NO in human endothelial cell line (ECV304). After cells were incubated with MTB (1-10 µM) for 1 or 4 h, amounts of NO metabolites released by cells were quantified and cellular NOS activities were determined following the conversion of [³H]arginine to [³H]citrulline. The NOS protein expression was determined by Western immunoblotting analysis. MTB (1-10 µM) stimulated the release of NO and its metabolites from endothelial cells. Following MTB treatment, the cellular NOS activities were significantly enhanced with a concomitant increase in the levels of constitutive NOS (cNOS) protein mass (110-178%). Selective activation of cNOS by MTB may be employed therapeutically in modulating NO production in endothelial cells.     

Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium

Dundas, Ian E. D. (University of Illinois, Urbana), and H. Orin Halvorson. Arginine metabolism in Halobacterium salinarium, an obligately halophilic bacterium. J. Bacteriol. 91:113-119. 1966.-Arginine was shown to be essential for growth of Halobacterium salinarium strain 1 in a chemically defined medium. Citrulline was the only compound which could substitute for arginine without affecting growth. Resting cells of H. salinarium converted arginine to citrulline and citrulline to ornithine. Cells grown in an arginine-free medium with C(14)-ureido-labeled citrulline incorporated the isotope mainly into the arginine of their proteins. The enzymes arginine desimidase and ornithine transcarbamylase were found and studied in cell-free extracts of H. salinarium. Experiments indicated that arginine was degraded in H. salinarium by arginine desimidase to citrulline, and that citrulline was further degraded by ornithine transcarbamylase to carbamyl phosphate and ornithine. Synthesis of arginine from citrulline seems to occur via the formation of argininosuccinic acid.