Generation of an ASS1 heterozygous knockout human embryonic stem cell line, WAe001-A-13, using CRISPR/Cas9.

The ASS1 gene encodes argininosuccinate synthetase-1, a cytosolic enzyme with a critical role in the urea cycle. Mutations are found in all ASS1 exons and cause the autosomal recessive disorder citrullinemia. Using CRISPR/Cas9-editing, we established the WAe001-A-13 cell line, which was heterozygous for an ASS1 mutation, from the human embryonic stem cell line H1. The WAe001-A-13 cell line maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.

Silencing of argininosuccinate lyase inhibits colorectal cancer formation.

Arginine and nitric oxide (NO) are important mediators of tumorigenesis in various types of cancer. Dysregulation of NO content by argininosuccinate lyase (ASL) has been previously demonstrated to inhibit the proliferation of liver and breast cancer cells. However, the function of ASL in colon cancer is not well defined. The present study aimed to determine the effect of ASL on colon cancer. Western blot analysis indicated that ASL expression was induced by endoplasmic reticulum stress in HCT116 and SW480 colon cancer cells. Additionally, the expression of ASL in colon cancer tissues was enhanced compared with that in the adjacent normal tissues, and the patients with colon cancer with higher ASL expression exhibited poorer survival rates. Transfection of ASL-targeting short hairpin RNA (shRNA) into HCT116 cells inhibited cell proliferation and decreased anchorage-independent growth in a soft agar assay. In addition, when injected subcutaneously into NOD/SCID mice, stable transfectant ASL-downregulated HCT116 cells exhibited decreased in vivo tumorigenic ability. Flow cytometric analysis of cell cycle progression indicated that ASL-targeting shRNA induced G2/M arrest, and western blot analysis showed that the inhibition of ASL was accompanied by cyclin A2 degradation. Furthermore, ASL-targeting shRNA resulted in increased autophagosomes and decreased NO levels. Inhibition of NO by the NO synthase inhibitor L-NMMA significantly reduced cell proliferation and colony formation. In summary, the results of the present study indicated that ASL-targeting shRNA-induced growth inhibition is associated with decreased cyclin A2 expression and NO content in colon cancer.

Argininosuccinate lyase is a potential therapeutic target in breast cancer.

Arginine is a non-essential amino acid that modulates nitric oxide production and cancer homeostasis. In our previous study, we observed that blocking argininosuccinate lyase (ASL) attenuates tumor progression in liver cancer. However, the role of ASL in human breast cancer has been studied to a lesser degree. In the present study, we investigated the effect of targeting ASL in breast cancer. We found that ASL was induced by ER stress and was significantly upregulated in breast cancer tissues compared to that in the corresponding normal tissues. Downregulation of ASL inhibited the growth of breast cancer in vitro and in vivo. The level of cell cycle-related gene, cyclin A2, was reduced and was accompanied by a delay in G2/M transition. ASL shRNA-induced cell inhibition was rescued by exogenous cyclin A2. Furthermore, autophagy was observed in the cells expressing ASL shRNA, and inhibition of autophagy reduced cell growth, indicating that autophagy played a cell survival role in the ASL knockdown cells. Moreover, inhibition of ASL reduced NO content. Introduction of the NO donor partially restored the growth inhibition by ASL shRNA. Thus, the mechanism induced by ASL shRNA which occurred in human breast cancer may be attributed to a decrease in cyclin A2 and NO.

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.

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.

Inactivation of the endogenous argininosuccinate lyase activity of duck delta-crystallin by modification of an essential histidine residue with diethyl pyrocarbonate.

The argininosuccinate lyase activity of duck delta-crystallin was inactivated by diethyl pyrocarbonate at 0 degrees C and pH 7.5. The inactivation followed pseudo-first-order kinetics after appropriate correction for the decomposition of the reagent during the modification period. The plot of the observed pseudo-first-order rate constant versus diethyl pyrocarbonate concentration in the range of 0.17-1.7 mM was linear and went through the origin with a second-order rate constant of 1.45 +/- 0.1 M-1.s-1. The double-logarithmic plot was also linear, with slope of 1.13, which suggested a 1:1 stoichiometry for the reaction between diethyl pyrocarbonate and delta-crystallin. L-Arginine, L-norvaline or L-citrulline protected the argininosuccinate lyase activity of delta-crystallin from diethyl pyrocarbonate inactivation. The dissociation constants for the delta-crystallin-L-arginine and delta-crystallin-L-citrulline binary complexes, determined by the protection experiments, were 4.2 +/- 0.2 and 0.12 +/- 0.04 mM respectively. Fumarate alone had no protective effect. However, fumarate plus L-arginine gave synergistic protection with a ligand binding interacting factor of 0.12 +/- 0.02. The double-protection data conformed to a random Uni Bi kinetic mechanism. Fluorescence-quenching studies indicated that the modified delta-crystallin had minimum, if any, conformational changes as compared with the native delta-crystallin. Inactivation of the enzyme activity was accompanied by an increasing absorbance at 240 nm of the protein. The absorption near 280 nm did not change. Treatment of the modified protein with hydroxylamine regenerated the enzyme activity to the original level. These results strongly indicated the modification of an essential histidine residue. Calculation from the 240 nm absorption changes indicated that only one histidine residue per subunit was modified by the reagent. This super-active histidine residue has a pKa value of approximately 6.8 and acts as a general acid-base catalyst in the enzyme reaction mechanism. Our experimental data are compatible with an E1cB mechanism [Raushel (1984) Arch. Biochem. Biophys. 232, 520-525] for the argininosuccinate lyase with the essential histidine residue close to the arginine-binding domain of delta-crystallin. L-Citrulline, after binding to this domain, might form an extra hydrogen bond with the essential histidine residue.

Screening and kinetic analysis of delta-crystallins with endogenous argininosuccinate lyase activity in the lenses of vertebrates.

Screening of lens homogenates from the representative species of five major classes of vertebrates was undertaken to search for delta-crystallin with argininosuccinate lyase activity. Purification and biochemical characterization of delta-crystallins from the avian and reptilian species revealed differences in their electrophoretic and kinetic properties in spite of their similar tetrameric structure of about 200 kDa in the native forms. Chicken delta-crystallin, in contrast to those obtained from duck, goose and caiman, is almost devoid of the enzymatic activity. Two-dimensional gel electrophoresis of lens homogenates indicated that in the chicken lens delta-crystallin is composed of a subunit with an isoelectric point of 5.9 and a subunit mass of 50 kDa whereas that of goose lenses possesses heterogeneous subunits with isoelectric points spreading in a range of 5.9 to 6.8. Immunological comparison of inactive and active delta-crystallins from the chicken, duck and caiman lenses established the apparent structural similarity of all delta-crystallins to the authentic enzyme regarding some of common surface epitopes, yet they are not completely identical. Kinetic constants for two of the active delta-crystallins, i.e. those from the duck and goose of the Anatidae family, were also determined and their catalyzed reaction was shown to conform to a random Uni-Bi kinetic mechanism similar to that of the argininosuccinate lyase from the bovine liver.

Inhibition of urea cycle enzymes by lysine and saccharopine.

Crude and purified preparations of argininosuccinate synthetase, argininosuccinate lyase and arginase were subjected to inhibition studies with L-lysine and saccharopine. Saccharopine proved to be the more potent inhibitor of argininosuccinate synthetase and lyase, whereas lysine had more effect on arginase. Similar results were found with pure enzyme and crude preparations. Computer analysis of the results suggested that inhibition of urea cycle enzymes by saccharopine and lysine might have contributed to the high levels of citrulline found in a human patient with saccharopinuria, a defect of saccharopine metabolism, but that this was unlikely to be the sole explanation.

Nitro analogs of substrates for argininosuccinate synthetase and argininosuccinate lyase.

The nitro analogs of aspartate and argininosuccinate were synthesized and tested as substrates and inhibitors of argininosuccinate synthetase and argininosuccinate lyase, respectively. The Vmax for 3-nitro-2-aminopropionic acid was found to be 60% of the maximal rate of aspartate utilization in the reaction catalyzed by argininosuccinate synthetase. Only the nitronate form of this substrate, in which the C-3 hydrogen is ionized, was substrate active, indicating a requirement for a negatively charged group at the beta carbon. The V/K of the nitro analog of aspartate was 85% of the value of aspartate after correcting for the percentage of the active nitronate species. The nitro analog of argininosuccinate, N3-(L-1-carboxy-2-nitroethyl)-L-arginine, was a strong competitive inhibitor of argininosuccinate lyase but was not a substrate. The pH dependence of the observed pKi was consistent with the ionized carbon acid (pK = 8.2) in the nitronate configuration as the inhibitory material. The pH-independent pKi of 2.7 microM is 20 times smaller than the Km of argininosuccinate at pH 7.5. These results suggest that the tighter binding of the nitro analog relative to the substrate is due to the similarity in structure to a carbanionic intermediate in the reaction pathway.

Substrate-induced inactivation of argininosuccinate lyase by monofluorofumarate and difluorofumarate.

Monofluorofumarate and difluorofumarate were tested as alternate substrates and inhibitors of the reverse reaction of bovine liver argininosuccinate lyase. Km and Vmax values relative to fumarate at pH 7.5, 25 degrees C, and 10 mM arginine are (monofluorofumarate) 1.4 mM and 5% and (difluorofumarate) 46 microM and 0.5%. As inhibitors, both of these compounds were shown to inactivate the enzyme activity in a pseudo-first-order process that is dependent on the presence of arginine. The rate of inactivation at saturating monofluorofumarate and difluorofumarate is 13 and 1.3 min-1, respectively. After removal of excess inhibitor, the inactivated enzyme can be restored to greater than 75% of its original activity with half-lives of 6 and 24 min for the monofluorofumarate- and difluorofumarate-inhibited enzyme. Evidence is presented to suggest that the time-dependent inactivation is caused by covalent addition of an enzyme nucleophile with an electrophilic reaction intermediate. In the inhibition by monofluorofumarate, the postulated intermediate is proposed to occur by the spontaneous loss of HF from 2-fluoroargininosuccinate.

Kinetic mechanism of bovine liver argininosuccinate lyase.

The kinetic mechanism of bovine liver argininosuccinate lyase has been determined at pH 7.5, 25 degrees C. Fumarate and arginine are both noncompetitive inhibitors versus argininosuccinate. The dead-end inhibitor, succinate, is competitive versus fumarate and argininosuccinate, but noncompetitive versus arginine. Citrulline is competitive versus arginine and noncompetitive versus argininosuccinate and fumarate. The results are consistent with a random mechanism with the formation of two dead-end complexes: E . argininosuccinate . fumarate and E . argininosuccinate . arginine. No evidence was obtained for nonlinear reciprocal plots. The equilibrium constant was found to be 3.7 mM.

Studies on rat liver argininosuccinate synthetase. Inhibition by various amino acids.

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.

Urea as a selective inhibitor of argininosuccinate lyase.

The effect of urea on various ornithine cycle enzymes has been investigated. It was demonstrated that argininosuccinate lyase was the only ornithine cycle enzyme inhibited by urea in a competitive manner. Based on the data presented the possible role of urea in maintaining a physiological range of intracellular and extracellular urea concentration by controlling hepatic ureogenesis was discussed.

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor.

Phi80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR+ strain but not by corresponding preparations from an argR- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on beta-galactosidase synthesis.