Properties of asparagine synthetase in asparagine-independent variants of Jensen rat sarcoma cells induced by 5-azacytidine.

Jensen rat sarcoma cells in culture require L-asparagine for growth and lack detectable levels of asparagine synthetase. Cultures exposed for 24 h to graded concentrations of 5-azacytidine give rise to asparagine-independent variants in high frequency. These prototrophs are stable phenotypically whether maintained in the presence or absence of L-asparagine. Asparagine synthetase activity in several variant clones was uniform in thermolability and several kinetic parameters, as well as in immunological properties. Parental Jensen rat sarcoma cells contained no detectable immunologically cross-reacting material. Our data suggest that transitions between asparagine dependence and independence in these cells are mediated by stable shifts in gene expression rather than by structural gene mutations.     

Ultraviolet-induced mutations to asparagine independence in Jensen sarcoma cells in vitro

UV-irradiation induces an exponential increase in the frequency of mutation from asparagine requirement to asparagine non-requirement in Jensen sarcoma cells grown in vitro. The corrected mutation frequency increases from the spontaneous rate of 5.1·10^?6 per cell to 1248·10^?6 per cell with a dose of 180 erg/mm² of 254 nm UV A substantial increase was oberved even without correction for survivors, and no significant difference was observed in the UV sensitivity of asparagine-requiring and non-requiring Jensen clones. When Jensen cells were plated at low densities in a feeder layer of LMTK-cells inactivated by HAT medium, an increase in the cloning ability of the former was observed as compared to appropriate controls without the feeder layer, but the increase was constant over all doses of UV tested. Revertants are stable and possess measurable asparagine synthetase.It is concluded that UV is an extremely effective mutagen in this system.     

Isolation of two cDNAs encoding functional human cytoplasmic cysteinyl-tRNA synthetase

Cysteinyl-tRNA synthetase catalyzes the addition of cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. To gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyl-tRNA synthetase. In this study, we sought to clone and express the full-length gene for the human cytoplasmic cysteinyl-tRNA synthetase. Although a gene encoding the human enzyme has been described, the predicted protein sequence, consisting of 638 amino acids, lacks homology with other eukaryotic enzymes in the carboxyl-terminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a full-length cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyl-tRNA synthetases in the carboxyl-terminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyl-tRNA synthetase contained an insertion of 8 bases in the 3' coding region of the mRNA. This insertion arises from an alternative splicing between the last two exons of the gene. The alternative splicing alters the reading frame and results in the replacement of the carboxy-terminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the full-length and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA(Cys) with cysteine.     

Immunochemical characterization of rat testicular asparagine synthetase.

We studied immunochemical properties of rat testicular asparagine synthetase. Western blot analysis of testis extract with polyclonal antibody raised against purified asparagine synthetase revealed an immunoreactive band at 62 kDa. The pancreas, brain, thymus, and spleen also showed 62-kDa bands. The intensities of these bands were roughly proportional to the specific activities of the enzyme in these tissues. The antibody showed some degree of cross-reactivity to asparagine synthetases from human, beef, pig, mouse, guinea pig, chicken, and frog, but not carp. But the enzyme from human HL-60 cells and lower vertebrates reacted with the antibody less strongly than enzyme from rats. The N-terminal amino acid sequence of the enzyme, determined by the Edman degradation method, in 10 recovered residues was identical to that of human asparagine synthetase deduced from corresponding cDNA (I.L. Andrulis et al., 1987, Mol. Cell. Biol. 7, 2435-2443). Immunohistochemical staining of the testis showed the presence of asparagine synthetase mainly in Sertoli cells in the seminiferous tubules.     

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells.

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.     

Purification and properties of asparagine synthetase from rat liver.

Asparagine synthetase (L-aspartate:ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation. DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4-7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110,000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent Km values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl- were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by L-asparagine, nucleoside triphosphates except ATP, and sulfhydryl reagents. It has been observed that the properties of asparagine synthetase from rat liver are not so different from those of tumors such as Novikoff hepatoma and RADA 1.     

Isolation and characterization of CHO cell mutants with altered asparagine synthetase.

Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat- strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10(-6) per cell per generation. Three particular revertants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase.     

A sulfoximine-based inhibitor of human asparagine synthetase kills l-asparaginase-resistant leukemia cells

An adenylated sulfoximine transition-state analogue 1, which inhibits human asparagine synthetase (hASNS) with nanomolar potency, has been reported to suppress the proliferation of an l-asparagine amidohydrolase (ASNase)-resistant MOLT-4 leukemia cell line (MOLT-4R) when l-asparagine is depleted in the medium. We now report the synthesis and biological activity of two new sulfoximine analogues of 1 that have been studied as part of systematic efforts to identify compounds with improved cell permeability and/or metabolic stability. One of these new analogues, an amino sulfoximine 5 having no net charge at cellular pH, is a better hASNS inhibitor (K_I¹ = 8 nM) than 1 and suppresses proliferation of MOLT-4R cells at 10-fold lower concentration (IC₅₀ = 0.1 mM). More importantly, and in contrast to the lead compound 1, the presence of sulfoximine 5 at concentrations above 0.25 mM causes the death of MOLT-4R cells even when ASNase is absent in the culture medium. The amino sulfoximine 5 exhibits different dose-response behavior when incubated with an ASNase-sensitive MOLT-4 cell line (MOLT-4S), supporting the hypothesis that sulfoximine 5 exerts its effect by inhibiting hASNS in the cell. Our work provides further evidence for the idea that hASNS represents a chemotherapeutic target for the treatment of leukemia, and perhaps other cancers, including those of the prostate.     

Glutamine-dependent asparagine synthetase in fetal, adult and neoplastic rat tissues.

Three enzyme reactions related to asparagine synthesis were studied in rat tissues: formation of aspartylhydroxamate, either from aspartate or by transfer from asparagine, and actual synthesis of asparagine from aspartate. Actual asparagine synthesis occurred at one-thousandth the rate of the other two reactions. Optimal conditions for quantitative assay of asparagine synthesis were determined in fetal liver extract, which is a rich source of the enzyme. Demonstrable activity in liver fell 6 days after birth to 20% of the fetal value and decreased slowly thereafter to the low adult value. Adult pancreas was the most active tissue found. The asparagine synthetase of fetal liver extracts was significantly inhibited when combined with adult liver or tumor extracts. The inhibitor fractionated with ammonium sulfate in close association with the asparagine synthetase. Therefore, demonstrable activities of asparagine synthetase in tissue extracts, measured in the presence of this inhibitor, do not necessarily parallel the concentrations of the enzyme present.     

Expression of human asparagine synthetase in Escherichia coli

Human asparagine synthetase was expressed in Escherichia coli. Synthesis of the enzyme was demonstrated by immunoblotting and by complementation of asparagine auxotrophy in E. coli. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. Compared to asparagine synthetase isolated from beef pancreas, the one expressed in E. coli migrated at a slightly slower rate on a denaturing protein gel. In contrast with previous reports, the data obtained here strongly suggest that the active enzyme is a homodimer. The production of soluble and active enzyme was shown to be highly temperature-dependent. Expression at 37 degrees C yielded no soluble enzyme, whereas growth at 30 and 21 degrees C favored the production of soluble asparagine synthetase. The incubation temperature was also important for complementation of asparagine auxotrophy in E. coli, as growth in the absence of asparagine occurred at 30 degrees C and not at 37 degrees C.     

Expression of human asparagine synthetase in Saccharomyces cerevisiae

Human asparagine synthetase was expressed in the yeast Saccharomyces cerevisiae. The identity of the expressed protein was confirmed by immunoblotting and in vitro enzymatic activity. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. In contrast to overproduction in Escherichia coli, the expressed protein was found to be soluble in the yeast cell. Furthermore, expression in yeast made it possible to isolate non-degraded human asparagine synthetase which had also the N-terminal methionine correctly processed. The yeast expression plasmid was constructed for optimal production of the recombinant enzyme. In addition, unique restriction enzyme sites that bracket the first five codons of the human asparagine synthetase gene were introduced. This will allow the use of oligonucleotide cassette mutagenesis to investigate the role of the N-terminal amino acids in asparagine synthetase enzymatic activity.     

Methotrexate stimulation of asparagine synthetase activity in rat liver

Methotrexate was found to stimulate asparagine synthetase activity in vivo by approximately six-fold in rat liver. The maximum effect of methotrexate on hepatic asparagine synthetase activity was observed sixteen hours after intraperitoneal injection of the drug. Cycloheximide, like methotrexate, is a protein synthesis inhibitor and was used to determine that asparagine synthetase activity was not preferentially stimulated under stress. As expected, hepatic asparagine synthetase activity falls markedly with the decreased protein synthesis caused by injection of cycloheximide. It is proposed that methotrexate inhibits serine-dependent glycine biosyn-thesis by decreasing the concentration of tetrahydrofolate for serine hydroxymethyltransferase. This leads to a stimulation of asparagine synthetase to provide nitrogen for asparagine-dependent glycine synthesis. This may provide an explanation of the observed chemotherapeutic synergism between asparaginase and methotrexate treatment.     

Purification and properties of asparagine synthetase from rat liver

Asparagine synthetase (L-aspartate: ammonia ligase (AMP-forming, EC 6.3.1.1) activity in rat liver increased when the animals were put on a low casein diet. The enzyme was purified about 280-fold from the supernatant of rat liver homogenate by a procedure comprising ammonium sulfate fractionation, DEAE-Sepharose column chromatography, and Sephadex G-100 gel filtration. The optimal pH of the enzyme was in the range 7.4–7.6 with glutamine as an amide donor. The molecular weight was estimated to be approximately 110 000 by gel filtration. Chloride ion was required for the enzyme activity. The apparent K_m values for L-aspartate, L-glutamine, ammonium chloride, ATP, and Cl⁻ were calculated to be 0.76, 4.3, 10, 0.14, and 1.7 mM, respectively. The activity was inhibited by l-asparagine, nucleoside triphosphates except ATP, and sulflhdryl reagents.It has been observed that the properties of asparagine synthetase from rat liver are not different from those of tumors such as Novikoff hepatoma and RADA 1.     

Dark-induced and organ-specific expression of two asparagine synthetase genes in Pisum sativum.

Nucleotide sequence analysis of cDNAs for asparagine synthetase (AS) of Pisum sativum has uncovered two distinct AS mRNAs (AS1 and AS2) encoding polypeptides that are highly homologous to the human AS enzyme. The amino-terminal residues of both AS1 and AS2 polypeptides are identical to the glutamine-binding domain of the human AS enzyme, indicating that the full-length AS1 and AS2 cDNAs encode glutamine-dependent AS enzymes. Analysis of nuclear DNA shows that AS1 and AS2 are each encoded by single genes in P.sativum. Gene-specific Northern blot analysis reveals that dark treatment induces high-level accumulation of AS1 mRNA in leaves, while light treatment represses this effect as much as 30-fold. Moreover, the dark-induced accumulation of AS1 mRNA was shown to be a phytochrome-mediated response. Both AS1 and AS2 mRNAs also accumulate to high levels in cotyledons of germinating seedlings and in nitrogen-fixing root nodules. These patterns of AS gene expression correlate well with the physiological role of asparagine as a nitrogen transport amino acid during plant development.     

Processing and expression of rat and human clotting factor-X-encoding cDNAs

The cDNA encoding clotting factor X, which participates in the middle stage of the blood coagulation cascade was cloned from a rat liver cDNA library. Sequencing of the rat factor-X-encoding cDNA revealed that this vitamin-K-dependent protein has a dibasic Arg-Arg sequence at the propeptide cleavage site, as occurs in other vitamin-K-dependent proteins. Although the human and rat deduced amino acid sequences are remarkably similar (76% identical), they do significantly differ in that human factor-X contains a unique Thr-Arg sequence at the propeptide cleavage site [Fung et al., Proc. Natl. Acad. Sci. USA 82 (1985) 3591–3595], where a dibasic sequence would normally be expected. This specific site is the recognition motif for the endoprotease, furin, which is located in the Golgi apparatus. Both rat and human cDNAs expressed in Cos-1 cells resulted in secretion of a mixture of single- and two-chain forms of factor X. The two-chain forms were devoid of the propeptide and were produced at similar rates by the transfected cells. The efficient processing of human factor X, when compared to rat factor X, may indicate that an additional protease(s), which recognizes the Thr-Arg motif, may be involved in proteolytic processing of the human enzyme