Valproic acid up-regulates the whole NO-citrulline cycle for potent iNOS-NO signaling to promote neuronal differentiation of adipose tissue-derived stem cells.

Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.

Argininosuccinate synthase 1 suppresses cancer cell invasion by inhibiting STAT3 pathway in hepatocellular carcinoma.

Metastasis is the main reason for high recurrence and poor survival of hepatocellular carcinoma (HCC). The molecular mechanism underlying HCC metastasis remains unclear. In this study, we found that argininosuccinate synthase 1 (ASS1) expression was significantly decreased and down-regulation of ASS1 was closely correlated with poor prognosis in HCC patients. DNA methylation led to the down-regulation of ASS1 in HCC. Stable silencing of ASS1 promoted migration and invasion of HCC cells, whereas overexpression of ASS1-inhibited metastasis of HCC cells in vivo and in vitro. We also revealed that ASS1-knockdown increased the phosphorylation level of S727STAT3, which contributed to HCC metastasis by up-regulation of inhibitor of differentiation 1 (ID1). These findings indicate that ASS1 inhibits HCC metastasis and may serve as a target for HCC diagnosis and treatment.

Human recombinant arginase I (Co)-PEG5000 [HuArgI (Co)-PEG5000]-induced arginine depletion is selectively cytotoxic to human glioblastoma cells.

In this study, we attempt to target Arginine auxotrophy in glioblastoma multiforme (GBM) cells using a pegylated recombinant human Arginase I cobalt [HuArgI (Co)-PEG5000]. We tested and characterized the activity of HuArgI (Co)-PEG5000 on a panel of 9 GBM cell lines and on human fetal glial cells (SVG-p12). HuArgI (Co)-PEG5000 was cytotoxic to all GBM cells tested. SVG-p12 cells were not sensitive demonstrating the selective cytotoxicity of HuArgI (Co)-PEG5000-induced arginine deprivation. Addition of L-citrulline led to the rescue of 6 GBM cell lines but only at concentrations of 11.4 mM, reflecting the extent of arginine auxotrophy in GBM. The ability of L-citrulline to rescue cells was dependent on the expression of argininosuccinate synthetase-1 (ASS1) with the cells that were not rescued by L-citrulline being negative for ASS1 expression. Knocking-down ASS1 reversed the ability of L-citrulline to rescue GBM cells, further illustrating the dependence of arginine auxotrophy on ASS1 expression. Inhibition of autophagy increased cell sensitivity to HuArgI (Co)-PEG5000 indicating that, following arginine deprivation, autophagy plays a protective role in GBM cells. Analysis of the type of cell death revealed a lack of AnnexinV staining and caspase activation in HuArgI (Co)-PEG5000-treated cells, indicating that arginine deprivation induces caspase-independent, non-apoptotic cell death in GBM. We have shown that GBM cells are auxotrophic for arginine and can be selectively targeted using HuArgI (Co)-PEG5000-induced arginine depletion, thus demonstrating that L-Arginine deprivation is a potent and selective potential treatment for GBM.

L-Citrulline dilates rat retinal arterioles via nitric oxide- and prostaglandin-dependent pathways in vivo.

L-Citrulline is an effective precursor of L-arginine produced by the L-citrulline/L-arginine cycle, and it exerts beneficial effects on the cardiovascular system by supporting enhanced nitric oxide (NO) production. NO dilates retinal blood vessels via the cyclooxygenase-mediated pathway. The purpose of this study was to examine the effects of L-citrulline on retinal circulation and to investigate the potential involvement of NO and prostaglandins in L-citrulline-induced responses in rats. L-Citrulline (10-300 µg kg(-1) min(-1), i.v.) increased the diameter of retinal arterioles without significantly changing mean blood pressure, heart rate, and fundus blood flow. The vasodilator response of retinal arterioles to l-citrulline was significantly diminished following treatment with N(G)-nitro-L-arginine methyl ester (30 mg/kg, i.v.), an NO synthase inhibitor, or indomethacin (5 mg/kg, i.v.), a cyclooxygenase inhibitor. In addition, α-methyl-dl-aspartic acid (147 mg/kg, i.v.), an inhibitor of argininosuccinate synthase, the rate-limiting enzyme for the recycling of l-citrulline to l-arginine, diminished the L-citrulline-induced retinal vasodilation. These results suggest that both NO- and prostaglandin-dependent pathways contribute to the L-citrulline-induced vasodilation of rat retinal arterioles. The L-citrulline/L-arginine recycling pathway may have more importance in regulating vascular tone in retinal blood vessels than in peripheral resistance vessels.

Endothelial argininosuccinate synthetase 1 regulates nitric oxide production and monocyte adhesion under static and laminar shear stress conditions.

Laminar shear stress (LSS) is known to increase endothelial nitric oxide (NO) production, which is essential for vascular health, through expression and activation of nitric oxide synthase 3 (NOS3). Recent studies demonstrated that LSS also increases the expression of argininosuccinate synthetase 1 (ASS1) that regulates the provision of L-arginine, the substrate of NOS3. It was thus hypothesized that ASS1 might contribute to vascular health by enhancing NO production in response to LSS. This hypothesis was pursued in the present study by modulating NOS3 and ASS1 levels in cultured endothelial cells. Exogenous expression of either NOS3 or ASS1 in human umbilical vein endothelial cells increased NO production and decreased monocyte adhesion stimulated by tumor necrosis factor-α (TNF-α). The latter effect of overexpressed ASS1 was reduced when human umbilical vein endothelial cells were co-treated with small interfering RNAs (siRNAs) for ASS1 or NOS3. SiRNAs of NOS3 and ASS1 attenuated the increase of NO production in human aortic endothelial cells stimulated by LSS (12 dynes·cm(-2)) for 24 h. LSS inhibited monocyte adhesion to human aortic endothelial cells stimulated by TNF-α, but this effect of LSS was abrogated by siRNAs of NOS3 and ASS1 that recovered the expression of vascular cell adhesion molecule-1. The current study suggests that the expression of ASS1 harmonized with that of NOS3 may be important for the optimized endothelial NO production and the prevention of the inflammatory monocyte adhesion to endothelial cells.

Epigenetic silencing of argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotrophy in ovarian cancer.

Evidence indicates that acquired resistance of cancers to chemotherapeutic agents can occur via epigenetic mechanisms. Down-regulation of expression of argininosuccinate synthetase (ASS1), the rate-limiting enzyme in the biosynthesis of arginine, has been associated with the development of platinum resistance in ovarian cancer treated with platinum-based chemotherapy. The aim of the present study was to analyse epigenetic regulation of ASS1 in ovarian cancer tissue taken at diagnosis and relapse and determine its significance as a predictor of clinical outcome in patients treated with platinum-based chemotherapy. In addition, expression and epigenetic regulation of ASS1 were analysed in human ovarian cancer cell lines, and ASS1 expression correlated with the ability of the lines to grow in media containing cisplatin, carboplatin or taxol or in arginine-depleted media. Our results show that aberrant methylation in the ASS1 promoter correlated with transcriptional silencing in ovarian cancer cell lines. ASS1 silencing conferred selective resistance to platinum-based drugs and conferred arginine auxotrophy and sensitivity to arginine deprivation. In ovarian cancer, ASS1 methylation at diagnosis was associated with significantly reduced overall survival (p = 0.01) and relapse-free survival (p = 0.01). In patients who relapse, ASS1 methylation was significantly more frequent at relapse (p = 0.008). These data establish epigenetic inactivation of ASS1 as a determinant of response to platinum chemotherapy and imply that transcriptional silencing of ASS1 contributes to treatment failure and clinical relapse in ovarian cancer. The collateral sensitivity of cells lacking endogenous ASS1 to arginine depletion suggests novel therapeutic strategies for the management of relapsed ovarian cancer.

Androgen receptor regulates ASS1P3/miR-34a-5p/ASS1 signaling to promote renal cell carcinoma cell growth.

Recent studies have demonstrated that the androgen receptor (AR) could play important roles to promote renal cell carcinoma (RCC) cell proliferation, and other studies have also indicated that suppressing the argininosuccinate synthase 1 (ASS1) could promote proliferation of various tumors. The potential of AR promoting cell proliferation in RCC via altering ASS1, however, remains unclear. Here we found that the expression of ASS1 was lower in RCC tissues than in adjacent normal renal tissues, and a lower ASS1 expression was linked to a worse prognosis in RCC patients. Mechanism dissection showed that AR could decrease ASS1 expression to promote RCC cell proliferation via ASS1P3, a pseudogene of ASS1. The results of RIP assay and AGO2 assay revealed that AR could bind ASS1P3 to increase RCC cell proliferation via altering miR-34a-5p function, which could bind to the 3'UTR of ASS1 to suppress its protein expression. ASS1P3 could function as a miRNA decoy for miR-34a-5p to regulate ASS1 in RCC. Preclinical study also supports the in vitro data. Together, these results demonstrated that ASS1P3 could function as a competing endogenous RNA to suppress RCC cell progression, and targeting this newly identified AR-mediated ASS1P3/miR-34a-5p/ASS1 signaling might help in blocking proliferation.

Argininosuccinate Synthase 1 is a Metabolic Regulator of Colorectal Cancer Pathogenicity.

Like many cancer types, colorectal cancers have dysregulated metabolism that promotes their pathogenic features. In this study, we used the activity-based protein profiling chemoproteomic platform to profile cysteine-reactive metabolic enzymes that are upregulated in primary human colorectal tumors. We identified argininosuccinate synthase 1 (ASS1) as an upregulated target in primary human colorectal tumors and show that pharmacological inhibition or genetic ablation of ASS1 impairs colorectal cancer pathogenicity. Using metabolomic profiling, we show that ASS1 inhibition leads to reductions in the levels of oncogenic metabolite fumarate, leading to impairments in glycolytic metabolism that supports colorectal cancer cell pathogenicity. We show here that ASS1 inhibitors may represent a novel therapeutic approach for attenuating colorectal cancer through compromising critical metabolic and metabolite signaling pathways and demonstrate the utility of coupling chemoproteomic and metabolomic strategies to map novel metabolic regulators of cancer.

Role of the L-citrulline/L-arginine cycle in iNANC nerve-mediated nitric oxide production and airway smooth muscle relaxation in allergic asthma.

Nitric oxide synthase (NOS) converts L-arginine into nitric oxide (NO) and L-citrulline. In NO-producing cells, L-citrulline can be recycled to L-arginine in a two-step reaction involving argininosuccinate synthase (ASS) and -lyase (ASL). In guinea pig trachea, L-arginine is a limiting factor in neuronal nNOS-mediated airway smooth muscle relaxation upon inhibitory nonadrenergic noncholinergic (iNANC) nerve stimulation. Moreover, in a guinea pig model of asthma iNANC nerve-induced NO production and airway smooth muscle relaxation are impaired after the allergen-induced early asthmatic reaction, due to limitation of L-arginine. Using guinea pig tracheal preparations, we now investigated whether (i) the L-citrulline/L-arginine cycle is active in airway iNANC nerves and (ii) the NO deficiency after the early asthmatic reaction involves impaired L-citrulline recycling. Electrical field stimulation-induced relaxation was measured in tracheal open-rings precontracted with histamine. L-citrulline as well as the ASL inhibitor succinate did not affect electrical field stimulation-induced relaxation under basal conditions. However, reduced relaxation induced by a submaximal concentration of the NOS inhibitor N(omega)-nitro-L-arginine was restored by L-citrulline, which was prevented by the additional presence of succinate or the ASS inhibitor alpha-methyl-D,L-aspartate. Remarkably, the impaired iNANC relaxation after the early asthmatic reaction was restored by L-citrulline. In conclusion, the L-citrulline/L-arginine cycle is operative in guinea pig iNANC nerves in the airways and may be effective under conditions of low L-arginine utilization by nNOS (caused by NOS inhibitors), and during reduced L-arginine availability after allergen challenge. Enzymatic dysfunction in the L-citrulline/L-arginine cycle appears not to be involved in the L-arginine limitation and reduced iNANC activity after the early asthmatic reaction.

Hypoxia-induced nitric oxide production and tumour perfusion is inhibited by pegylated arginine deiminase (ADI-PEG20).

The hypoxic tumour microenvironment represents an aggressive, therapy-resistant compartment. As arginine is required for specific hypoxia-induced processes, we hypothesised that arginine-deprivation therapy may be useful in targeting hypoxic cancer cells. We explored the effects of the arginine-degrading agent ADI-PEG20 on hypoxia-inducible factor (HIF) activation, the hypoxia-induced nitric oxide (NO) pathway and proliferation using HCT116 and UMUC3 cells and xenografts. The latter lack argininosuccinate synthetase (ASS1) making them auxotrophic for arginine. In HCT116 cells, ADI-PEG20 inhibited hypoxic-activation of HIF-1α and HIF-2α, leading to decreased inducible-nitric oxide synthase (iNOS), NO-production, and VEGF. Interestingly, combining hypoxia and ADI-PEG20 synergistically inhibited ASS1. ADI-PEG20 inhibited mTORC1 and activated the unfolded protein response providing a mechanism for inhibition of HIF and ASS1. ADI-PEG20 inhibited tumour growth, impaired hypoxia-associated NO-production, and decreased vascular perfusion. Expression of HIF-1α/HIF-2α/iNOS and VEGF were reduced, despite an increased hypoxic tumour fraction. Similar effects were observed in UMUC3 xenografts. In summary, ADI-PEG20 inhibits HIF-activated processes in two tumour models with widely different arginine biology. Thus, ADI-PEG20 may be useful in the clinic to target therapy-resistant hypoxic cells in ASS1-proficient tumours and ASS1-deficient tumours.

L-citrulline recycling by argininosuccinate synthetase and lyase in rat gastric fundus.

The aim of this study was to investigate in rat gastric fundus whether L-citrulline, the co-product in the nitric oxide (NO) biosynthesis catalyzed by neuronal nitric oxide synthase (nNOS), can be converted back to the nNOS substrate L-arginine. Immunohistochemistry showed that argininosuccinate synthetase and argininosuccinate lyase, that mediate transformation of L-citrulline to L-arginine in the ureum cycle in hepatocytes, co-localize with nNOS. In longitudinal smooth muscle strips, L-arginine as well as L-citrulline (10(-3) M) was capable of completely respectively partially preventing the N(G)-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M)-induced inhibition of electrically induced nitrergic relaxations, whereas D-citrulline (10(-3) M) was not. The L-citrulline-mediated prevention of the L-NAME-induced inhibition was reduced by L-glutamine (3 x 10(-3) M), the putative L-citrulline uptake inhibitor, and by succinate, an argininosuccinate lyase inhibitor. The results demonstrate that the L-citrulline recycling mechanism is active in rat gastric fundus. Recycling of L-citrulline might play a role in providing sufficient amounts of nNOS substrate during long-lasting relaxations in gastric fundus after food intake.

Hepatic ATP content and hyperammonemia induced by CCl4 in rats.

An investigation of the mechanism of development of hyperammonemia observed in CCl4-induced hepatic encephalopathy was performed in rats. CCl4 (1.0 ml/kg 3 times per week for over 10 weeks) caused a severe hyperammonemia and depletion of hepatic ATP contents in only those rats with hepatic encephalopathy. However, CCl4 (1.0 ml/kg 3 times per week for 7 weeks) did not cause hepatic encephalopathy and did not change in blood ammonia levels. Administration of 2,4-dinitrophenol (2,4-DNP) in these CCl4-treated rats caused hepatic encephalopathy within 30 min after injection and then the increase of 140 micrograms/dl in blood ammonia levels and the decrease of 80% in hepatic ATP contents were observed. However, the administration of 2,4-DNP in CCl4-untreated rats did not cause hepatic encephalopathy within 30 min after injection although the increase of 70 micrograms/dl in blood ammonia levels and the decrease of 80% in hepatic ATP contents were observed. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by 85% and 60% respectively, in rats treated with CCl4 plus 2,4-DNP. However, in rats treated with 2,4-DNP and without CCl4, the hepatic activities of CPS and ASS were inhibited only 25% and 0%, respectively. These findings suggest that the severe hyperammonemia, which may be produced by the decrease of hepatic ATP content and the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy.

Epigenetic status of argininosuccinate synthetase and argininosuccinate lyase modulates autophagy and cell death in glioblastoma.

Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated ASS1 or ASL CpG islands, ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes occur in both of the two key genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine deprivation. Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood/brain barrier.

Endothelial nitric oxide production is tightly coupled to the citrulline-NO cycle.

Nitric oxide (NO) is an important vasorelaxant produced along with L-citrulline from L-arginine in a reaction catalyzed by endothelial nitric oxide synthase (eNOS). Previous studies suggested that the recycling of L-citrulline to L-arginine is essential for NO production in endothelial cells. However, there is no direct evidence demonstrating the degree to which the recycling of L-citrulline to L-arginine is coupled to NO production. We hypothesized that the amount of NO formed would be significantly higher than the amount of L-citrulline formed due to the efficiency of L-citrulline recycling via the citrulline-NO cycle. To test this hypothesis, endothelial cells were incubated with [14C]-L-arginine and stimulated by various agents to produce NO. The extent of NO and [14C]-L-citrulline formation were simultaneously determined. NO production exceeded apparent L-citrulline formation of the order of 8 to 1, under both basal and stimulated conditions. As further support, alpha-methyl-DL-aspartate, an inhibitor of argininosuccinate synthase (AS), a component of the citrulline-NO cycle, inhibited NO production in a dose-dependent manner. The results of this study provide evidence for the essential and efficient coupling of L-citrulline recycling, via the citrulline-NO cycle, to endothelial NO production.

Yeast argininosuccinate synthetase. Purification; structural and kinetic properties.

Yeast argininosuccinate synthetase has been purified to homogeneity. The enzyme was found to have a molecular weight of 228,000 as determined by gel sieving. It is composed of identical subunits of Mr 49,000 as shown by gel electrophoresis. The quaternary structure as determined by cross-linking of the subunits with glutaraldehyde, followed by gel electrophoresis with dodecylsulfate, is tetrameric. The saturation functions by citrulline and aspartate are hyperbolic; with MgATP as the variable substrate a sigmoid character, dependent on the concentration of citrulline, aspartate, argininosuccinate and arginine, was observed. The positive cooperativity is reduced by increasing concentrations of citrulline and aspartate; it is increased by argininosuccinate and arginine. Kinetic analysis provided evidence for a random addition of substrates. Initial velocity studies as well as product and dead-end inhibition studies comply with a rapid-equilibrium random model, except for the interconversion of the central quaternary complexes; the different kinetic constants have been established on the basis. Yeast argininosuccinate synthetase has a double metabolic function: anabolic in the biosynthesis of arginine, catabolic as the first enzyme of citrulline utilization as nitrogen source. The kinetic properties of the enzyme point to a physiologically well-adjusted activity for both roles and to an economic and efficient utilization of ATP.

Citrulline as a diagnostic parameter and a major amino acid constituent of cerebrospinal fluid in hepatic coma.

Free amino acids of cerebrospinal fluid and serum in hepatic coma have been studied. Citrulline was found to form about 80% of the total free amino acids in cerebrospinal fluid, whereas serum contained slightly higher levels of tyrosine, methionine, phenylalanine and glutamine. The higher level of citrulline in cerebrospinal fluid may be attributed to the inhibition of argininosuccinic acid synthetase in this disease.

[Physico-chemical properties of guinea pig liver argininosuccinate synthetase (author's transl)]. / Propiedades físico-químicas de la arginosuccinato-sintetasa de hígado de cobaya.

The behaviour of guinea pig liver argininosuccinate synthetase with respect to pH, temperature and kinetic parameters of substrates and inhibitors has been investigated. The enzyme shows the maximum activity at pH 8 and maximum stability (15 min at 30 degrees C) at pH in the range of 6.5 and 8. With respect to temperature it remains stable (15 min at pH 8) up to 40 degrees C. The results found for the Km values of the enzyme substrates L-citrulline, L-aspartate and ATP are 0.038 mM, 0.045 mM and 0.090 mM respectively. L-ornithine, diaminopimelic acid, pyrophosphate and ATP acted as inhibitors of the enzyme (competitive or not, according to the case). These kinetic studies of substrates and inhibitors were carried out with a partially purified fraction of the enzyme which had been purified 7.3 fold, which practically suppressed the ATP-ase activity, present in crude extracts.

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies.

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.

Inhibition of urea-cycle activity by high concentrations of alanine.

1. Conditions are described in which high intracellular alanine concentrations inhibit urea-cycle flux in isolated hepatocytes. 2. Inhibition of urea-cycle flux by added alanine is DL-cycloserine-insensitive and is accompanied by an increase in intracellular citrulline and a decrease in ornithine. 3. Argininosuccinate synthetase (EC 6.3.4.5) activity in rat liver cytosol is inhibited by alanine in a competitive manner with respect to citrulline. It is concluded that this effects is the primary cause of inhibition of urea-cycle flux by alanine.