Increased nitric oxide formation followed by increased arginase activity induces relative lack of arginine at the wound site and alters whole nutritional status in rats almost within the early healing period

Nitric oxide (NO) production and free amino acid fluxes at the wound side during the first 3 days following cutaneous wound were investigated. Experiments were performed on Albino Oxford rats (n = 18) underwent cutaneous implantation of polyvinyl sponges. Intact animals (n = 6) were controls. Nitrites, nitrates, free amino acids and urea were measured both in plasma and wound fluids. Inducible nitric oxide synthase (iNOS) gene expressions at wound site were analyzed, too. The highest levels of both iNOS gene expression and its activity (increased wound fluid citrulline and nitrites) were at the first day. Wound fluid nitrates were significantly above plasma levels throughout the whole period, while molar nitrate to nitrite ratio steadily increased. It was associated with gradual increase of both ornithine and urea as well as steadily decreases of arginine and increases of phenylalanine at the wound site. Gradual decrease in glycine to branched-chain molar ratio was observed both in plasma and wound fluids. In conclusion, an early locally induced alterations in Arg metabolism, due to increased NO formation followed by increased arginase activity, produces relative lack of Arg at the wound site and disturbs nutritional status of the whole body almost within early healing period following cutaneous wound in rats. It is likely that NO autoxidation at the wound side is influenced by substrate availability.     

Arginine catabolism in the cyanobacterium Synechocystis sp. Strain PCC 6803 involves the urea cycle and arginase pathway

Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of L-[(14)C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of L-[(14)C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [(14)C]ornithine preceded that of [(14)C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with L-[(14)C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Delta(1)pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [(14)C]citrulline from [(14)C]arginine; (ii) a proC (Delta(1)pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [(14)C]proline, [(14)C]glutamate, and [(14)C]glutamine from [(14)C]arginine or [(14)C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [(14)C]glutamate from L-[(14)C]arginine, L-[(14)C]ornithine, or L-[(14)C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Delta(1)pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [(14)C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-L-arginyl-poly(L-aspartic acid)], a reserve material unique to cyanobacteria.     

Regulation of Arginase Activity by Intermediates of the Arginine Biosynthetic Pathway in Neurospora crassa

It has been found that, in Neurospora crassa, arginine synthesized from exogenous citrulline was not as effectively hydrolyzed as exogenous arginine. This was explained by the observed inhibition of arginase in vitro and in vivo by citrulline. The high arginine pool formed from exogenous citrulline feedback inhibits the arginine pathway. These two factors allow exogenous citrulline to be used adventitiously and efficiently as an arginine source. Finally, it was found that ornithine was a strong inhibitor of arginase. This suggests that the characteristically high ornithine pool of minimal cultures of Neurospora may act to control a potentially wasteful catabolism of endogenous arginine by arginase.     

Early effects of exogenous arginine after the implantation of prosthetic material into the rat abdominal wall

We have investigated the effects of high arginine (Arg) levels (7.5 mg/100 g body weight per hour) on the early integration of biocompatible mesh grafts into the rat abdominal wall. Studies were performed over implantation intervals of 6, 12, 24 or 48 hours (n=12, each). Arginine and related compounds were quantified in plasma, wound fluids and multiple tissues. Plasma nitric oxide (NO) production was studied. Strips were taken from the polypropylene fiber-host tissue interfaces (PTIs) for optical microscopic analysis and for immunohistochemical analysis using rat-specific antibodies against type I and type III collagens. Exogenous Arg was metabolized at the peripheral tissues but reliably reached the wound space. High amounts of Arg and ornithine (Orn) were detected in the specimens considered. No changes on citrulline (Ctr) or NO concentrations were observed, overall suggesting that, during the period studied, the arginase pathway predominated. The acute scarring response differed significantly in the two placements considered. The P-SS interface evidenced more extensive new tissue growth than the P-DS interface. Forty-eight hours after mesh implantation cellular infiltration, fibroblast proliferation, and mesh-surrounding angiogenesis were higher in the arginine-treated rats. Type III collagen staining was related to arginine treatment, being higher (++) in the study group. In conclusion, and independently of the site of mesh placement, supplemental Arg seemed to favorably affect early local collagen deposition. This could be potentially helpful to ameliorate the integration of biomaterials into the tissues and, consequently, to allow for the design of more selective therapeutic strategies to prevent hernia recurrence rates.     

Enteral arginase II provides ornithine for citrulline synthesis

The synthesis of citrulline from arginine in the small intestine depends on the provision of ornithine. To test the hypothesis that arginase II plays a central role in the supply of ornithine for citrulline synthesis, the contribution of dietary arginine, glutamine, and proline was determined by utilizing multitracer stable isotope protocols in arginase II knockout (AII(-/-)) and wild-type (WT) mice. The lack of arginase II resulted in a lower citrulline rate of appearance (121 vs. 137 μmol·kg(-1)·h(-1)) due to a reduced availability of ornithine; ornithine supplementation was able to restore the rate of citrulline production in AII(-/-) to levels comparable with WT mice. There were significant differences in the utilization of dietary citrulline precursors. The contribution of dietary arginine to the synthesis of citrulline was reduced from 45 to 10 μmol·kg(-1)·h(-1) due to the lack of arginase II. No enteral utilization of arginine was observed in AII(-/-) mice (WT = 25 μmol·kg(-1)·h(-1)), and the contribution of dietary arginine through plasma ornithine was reduced in the transgenic mice (20 vs. 13 μmol·kg(-1)·h(-1)). Dietary glutamine and proline utilization were greater in AII(-/-) than in WT mice (20 vs. 13 and 1.4 vs. 3.7 μmol·kg(-1)·h(-1), respectively). Most of the contribution of glutamine and proline was enteral rather than through plasma ornithine. The arginase isoform present in the small intestinal mucosa has the role of providing ornithine for citrulline synthesis. The lack of arginase II results in a greater contribution of plasma ornithine and dietary glutamine and proline to the synthesis of citrulline.     

Arginine catabolism in wine lactic acid bacteria: is it via the arginine deiminase pathway or the arginase-urease pathway?

The wine lactic acid bacteria Leuconostoc oenos OENO and Lactobacillus buchneri CUC-3 catabolize L-arginine to ornithine and ammonia as major end-products, with 1 mole of arginine converted into 2 moles of ammonia and 1 mole of ornithine. Some citrulline was also excreted into the medium. The excreted citrulline was reassimilated and catabolized by the lactobacillus strain, though not by the leuconostoc. Urea was not detected during arginine degradation. The activities of all three enzymes of the arginine deiminase pathway (arginine deiminase, ornithine transcarbamylase and carbamate kinase) increased significantly over time in the presence of arginine. On the other hand, arginase and urease activities were undetectable in cell extracts of cultures grown in the presence of arginine. The results show that the arginine deiminase pathway, and not the arginase-urease pathway, is the route for arginine degradation in wine lactic acid bacteria.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Oxygen and nitrate in utilization by Bacillus licheniformis of the arginase and arginine deiminase routes of arginine catabolism and other factors affecting their syntheses.

Bacillus licheniformis has two pathways of arginine catabolism. In well-aerated cultures, the arginase route is present, and levels of catabolic ornithine carbamoyltransferase were low. An arginase pathway-deficient mutant, BL196, failed to grow on arginine as a nitrogen source under these conditions. In anaerobiosis, the wild type contained very low levels of arginase and ornithine transaminase. BL196 grew normally on glucose plus arginine in anaerobiosis and, like the wild type, had appreciable levels of catabolic transferase. Nitrate, like oxygen, repressed ornithine carbamoyltransferase and stimulated arginase synthesis. In aerobic cultures, arginase was repressed by glutamine in the presence of glucose, but not when the carbon-energy source was poor. In anaerobic cultures, ammonia repressed catabolic ornithine carbamoyltransferase, but glutamate and glutamine stimulated its synthesis. A second mutant, derived from BL196, retained the low arginase and ornithine transaminase levels of BL196 but produced high levels of deiminase pathway enzymes in the presence of oxygen.     

The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.     

Submitochondrial localization of arginase and other enzymes associated with urea synthesis and nitrogen metabolism, in liver of Squalus acanthias

The submitochondrial localization of the four mitochondrial enzymes associated with urea synthesis in liver of Squalus acanthias (spiny dogfish), a representative elasmobranch, was determined. Glutamine- and acetylglutamate-dependent carbamoyl-phosphate synthetase, ornithine carbamoyltransferase, glutamine synthetase, and arginase were all localized within the matrix of liver mitochondria. The subcellular and submitochondrial localization and activities of several related enzymes involved in nitrogen metabolism and gluconeogenesis in liver and dogfish are also reported. Pyruvate carboxylase and phosphoenolpyruvate carboxykinase were localized in the mitochondrial matrix. Synthesis of citrulline by isolated mitochondria from ornithine proceeds at a near optimal rate at ornithine concentrations as low as 0.08 mM. The same stoichiometry and rates of citrulline synthesis are observed when ornithine is replaced by arginine. The mitochondrial location of arginase does not appear to reflect a mechanism for regulating ornithine availability.     

STUDIES ON FUNCTIONAL AND METABOLIC ROLE OF UREA CYCLE INTERMEDIATES IN BRAIN

Abstract— The distribution of argininosuccinate synthetase, argininosuccinase and arginase, and the synthesis of urea in cerebullum. cerebral cortex and brain stem have been studied. Cerebral cortex had high levels of argininosuccinate synthetase and argininosuccinase. and a high ability to synthesize urea from aspartic acid and citrulline. Of the three regions, cerebullum had the highest arginase activity. The activities of the enzymes transamidinase and ornithine aminotransferase in the metabolism of arginine and ornithine in pathways other than urea formation have been studied in the three regions of the rat brain. The activity of creatine phosphokinase in all regions was the same: carbamylphosphatase activity was highest in cerebullum. Cerebral cortex had a high activity of aspartic acid transcarba-mylase. The brain stem, among the three regions, had the lowest activities of glutamine synthetase and glutaminase. The activities of these enzymes in the different regions are discussed in relation to urea production and the utilization of the urea cycle intermediates.
Intraperitoneal injection of high amounts of citrulline brought about a rise in the glutamine synthetase activity of cerebellum and brain stem and a rise in ornithine aminotransferase in cerebral cortex and liver. These results are discussed in relation to the mechanism of action of citrulline in alleviating the toxicity in hyperammonaemic states.     

Expression and function of arginine-producing and consuming-enzymes in the kidney

The kidney plays a key role in arginine metabolism. Arginine production is controlled by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) which metabolize citrulline and aspartate to arginine and fumarate whereas arginine consumption is dependent on arginine:glycine amidinotransferase (GAT), which mediates creatine and ornithine synthesis. Histological and biochemical techniques have been used to study the distribution and activity of these enzymes in anatomically dissected segments, in isolated fragments of tubules and in whole tissues. ASS and ASL mRNAs and proteins are expressed in the proximal tubule. Within this nephron segment, the proximal convoluted tubule has a higher arginine synthesis capacity than the proximal straight tubules. Furthermore, this arginine-synthesizing portion of the nephron matches perfectly with the site of citrulline reabsorption from the glomerular filtrate. The kidney itself can produce citrulline from methylated arginine, but this capacity is limited. Therefore, intestinal citrulline synthesis is required for renal arginine production. Although the proximal convoluted tubule also expresses a significant amount of GAT, only 10% of renal arginine synthesis is metabolized to guanidinoacetic acid, possibly because GAT has a mitochondrial localization. Kidney arginase (AII) is expressed in the cortical and outer medullary proximal straight tubules and does not degrade significant amounts of newly synthesized arginine. The data presented in this review identify the proximal convoluted tubule as the main site of endogenous arginine biosynthesis.     

Metabolism of arginine in lactating rat mammary gland.

Significant activities of the four enzymes needed to convert arginine into proline and glutamate (arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate dehydrogenase) develop co-ordinately in lactating rat mammary glands in proportion to the increased production of milk. No enzymes were detected to carry out the reactions of proline oxidation or reduction of glutamate to pyrroline-5-carboxylate. Minces of the gland converted ornithine into proline and into glutamate plus glutamine. These conversions increased during the cycle of lactation in proportion to the increased milk production and to the content of the necessary enzymes. The minced gland did not convert labelled ornithine into citrulline, confirming the absence from the gland of a functioning urea cycle, and did not convert labelled proline or glutamate into ornithine. A metabolic flow of labelled arginine to proline and glutamate in mammary gland was confirmed in intact animals with experiments during which the specific radioactivity of proline in plasma remained below that of the proline being formed from labelled arginine within the gland. It was concluded that arginase in this tissue had a metabolic role in the biosynthesis of extra proline and glutamate needed for synthesis of milk proteins.     

Role of arginine in superficial wound healing in man

Arginine supplementation has been identified as advantageous in experimental wound healing. However, the mechanisms underlying this beneficial effect in tissue repair remain unresolved. Animal studies suggest that the beneficial role of arginine supplementation is mediated, at least in part through NO. The latter component mediates processes involved in tissue repair, including angiogenesis, epithelialization and collagen formation. This prospective study is performed to investigate arginine metabolism in acute surgical wounds in man. Expression of enzymes, known to be involved in arginine metabolism, was studied in donor sites of skin grafts of 10 hospitalized patients undergoing skin transplantation. Plasma and wound fluid levels of arginine metabolites (ornithine, citrulline, nitrate and nitrite = NOx) were measured using High Performance Liquid Chromatography. Expression of iNOS, eNOS, arginase-1 and arginase-2 was studied by immunohistochemistry in paraffin sections of skin tissue. Arginase-1 concentration was measured in plasma and wound fluid using ELISA. Arginase-2 was determined using Western blot analysis. We observed increased levels of citrulline, ornithine, NOx and arginase-1 in wound fluid when compared with plasma. Arginase-2 was expressed in both plasma and wound fluid and seemed higher in plasma. iNOS was expressed by neutrophils, macrophages, fibroblasts, keratinocytes and endothelial cells upon wounding, whereas eNOS reactivity was observed in endothelial cells and fibroblasts. Arginase-1 was expressed in neutrophils post-wounding, while arginase-2 staining was observed in endothelial cells, keratinocytes, fibroblasts, macrophages and neutrophils. For the first time, human data support previous animal studies suggesting arginine metabolism for an NO- as well as arginase-mediated reparation of injured skin.     

Metabolism of [14C]citrulline in the perfused sheep and goat udder

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-(14)C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-(14)C,-2-(14)C]citrulline or l-[carbamoyl-(14)C,1-(14)C]citrulline and l-[4-(3)H]arginine. 2. In these experiments, [(14)C]citrulline was substantially oxidized to CO(2) and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [(14)C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [(14)C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.     

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.     

Arginine hydroxamate-resistant mutants of Bacillus subtilis with altered control of arginine metabolism.

Arginine hydroxamate inhibits the growth of Bacillus subtilis. From a large number of mutants isolated as resistant to this arginine analogue, 29 were chosen for further investigation. Most of these shared diminished ability to utilize arginine, citrulline and/or ornithine as sole nitrogen source. All 29 had reduced levels of the catabolic enzymes arginase and ornithine aminotransferase under various conditions in which these enzymes are induced in the parent. In some circumstances, five of the mutants also showed elevated levels of the biosynthetic enzyme ornithine carbamoyltransferase. On the basis of these data, the 29 mutants were divided into six phenotypic classes; in four of these, control of ornithine carbamoyltransferase was the same as in the wild type, while in the other two it was altered. It is suggested that the isolates carry regulatory mutations, and that certain of these may affect simultaneously the formation of arginine catabolic and biosynthetic enzymes. The implication of the latter is that in B. subtilis, as in yeast, controls of the catabolic and biosynthetic pathways are connected. Single representatives of five of the phenotypic classes carry mutations conferring arginine hydroxamate resistance linked to cysA by transduction with phage PBSI; this did not appear to be true for a representative of the sixth class.