Effect of hepatocarcinogens on the adenine purine nucleotide cycle during the initiation phase of carcinogenesis.
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Activities of the adenine purine nucleotide cycle enzymes, i.e., adenylosuccinate (SAMP) synthetase, SAMP lyase, and adenosine 5'-monophosphate deaminse, were determined in hepatic tissue of rats fed and/or given injections of 3'-methyl-4-dimethylaminoazobenzene, 4'-methyl-4-dimethylaminoazobenzene, thioacetamide, ethionine, or 2-acetylaminofluorene. SAMP lyase activity showed an early increase in all regimens containing hepatocarcinogens. Adenosine 5'-monophosphate deaminse showed increases with 3'-methyl-4-dimethylaminoazobenzene and thioacetamide but not with ethionine or 2-acetylaminofluorene. SAMP synthetase either was nonresponsive or else showed inhibition to the carcinogens. Increase in SAMP lyase activity was noted as early as 48 to 72 hr following i.p. injections of these carcinogens. The response of SAMP lyase was not duplicated by analogs of carcinogens such as 4'-methyl-4-dimethylaminoazobenzene or methionine. These data imply interaction of active carcinogens with SAMP lyase and to some extent adenosine 5'-monophosphate deaminse or to some mechanism responsible for their synthesis and/or release. This interaction may be a significant component of the initiation phase of carcinogenesis. (+info)
Effect of hadacidin on growth and adenylosuccinate synthetase activity of Dictyostelium discoideum.
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The growth of the eucaryotic microorganism Dictyostelium discoideum in liquid culture was completely inhibited by the aspartic acid analog hadacidin (N-formylhydroxyamino-acetic acid). Growth arrest occurred both in chemically defined medium and in complex growth medium containing aspartic acid and AMP precursors such as adenine and adenosine. Although these compounds could not overcome the effect of hadacidin, growth was restored if cells were washed and resuspended in fresh growth medium. Additional experiments showed that D. discoideum contains adenylosuccinate synthetase, the enzyme which catalyzes the synthesis of adenylosuccinate from IMP, aspartic acid, and GTP in the de novo biosynthesis of purines. A partially purified preparation of this enzyme was obtained, and the effect of hadacidin on its activity was studied. We found that maximum inhibition of the D. discoideum activity occurs at a ratio of aspartic acid to hadacidin of 5:1, suggesting that the affinity of the drug for this enzyme is less than for the enzyme from rabbit muscle and plants but greater than for that from Escherichia coli. The effect of the drug can be overcome by a 10-fold excess of aspartic acid, suggesting that the drug acts as a competitive inhibitor. A comparison of the adenylosuccinate synthetase activity levels at various stages of growth showed that its specific activity decreases about 60% as cells enter the stationary growth phase, and decreases about 75% after starvation for 2 h. Further studies showed that in cells treated with hadacidin the rate of uptake of exogenous nutrients is reduced about 75% and that these cells are more resistant to rupture by osmotic shock. While the results of this study are consistent with the proposal that growth arrest is contingent upon inhibition of adenylosuccinate synthetase activity, they also suggest that, as a consequence of this inhibition, some physiological properties of the cell have been altered. (+info)
Computer simulation of purine metabolism.
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A computer model of purine metabolism, including catabolism, salvage pathways and interconversion among nucleotides, is given. Steady-state rate equations corresponding to metabolic enzymes are written based on information from the literature about their kinetic behaviour. Numerical integration of this set of equations is performed employing selected parameters taken from the literature. After stabilization of purine compound concentrations is reached, simulation of enzyme deficit and enzyme overproduction is carried out. The latter is calculated by varying specified maximum velocities in the numerical integration. A pattern of intermediate metabolite concentrations is found. These results form a basis for the comparison of normal patterns or patterns reflecting the effects of inborn errors of metabolism. The aim of this paper is to demonstrate the usefulness of this computer simulation method in complex metabolism pathways. (+info)
The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion.
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The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate. (+info)
The mechanism of the adenylosuccinate synthetase reaction as studied by positional isotope exchange.
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In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of various ligands. Positional isotope exchange occurred, as measured by nuclear magnetic resonance spectroscopy, when IMP was present. In the absence of IMP, with or without aspartate or succinate, the [gamma-18O]GTP did not exhibit scrambling. These results suggest that the adenylosuccinate synthetase reaction involves the participation of 6-phosphoryl-IMP as an obligatory intermediate. On the basis of experiments carried out in our laboratory as well as in others, we believe the GDP remains bound to the enzyme until the product, adenylosuccinate, is formed. All products may then dissociate randomly from the enzyme. The positional isotope exchange experiments, along with initial-rate experiments carried out in our laboratory, serve to explain the lack of partial exchange reactions associated with the synthetase (Fromm, H. J. (1958) Biochim. Biophys. Acta 29, 255-262), as well as the net inversion of configuration when chiral thio-GTP is converted to thiophosphate (Webb, M. R., Reed, G. H., Cooper, B. F., and Rudolph, F. B. (1984) J. Biol. Chem. 259, 3044-3046). (+info)
Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis.
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A mutant clone (AU-100) which is 90% deficient in adenylosuccinate synthetase activity was characterized from wild-type murine S49 T-lymphoma cells. This AU-100 cell line and its hypoxanthine-guanine phosphoribosyltransferase-deficient derivative, AUTG-50B, overproduce purines severalfold and excrete massive amounts of inosine into the culture medium (Ullman et al., Proc. Natl. Acad. Sci. U.S.A. 79:5127-5131, 1982). We introduced a mutation into both of these cell lines which make them incapable of taking up nucleosides from the culture medium. The genetic deficiency in nucleoside transport prevents the adenylosuccinate synthetase-deficient AU-100 cells from excreting inosine. Because of an extremely efficient intracellular inosine salvage system, the nucleoside transport-deficient AU-100 cells also no longer overproduce purines. AUTG-50B cells which have been made genetically deficient in nucleoside transport still overproduce purines but excrete hypoxanthine rather than inosine. These studies demonstrate genetically that nucleoside transport and nucleoside efflux share a common component and that nucleoside transport has an important regulatory function which profoundly affects the rates of purine biosynthesis and purine salvage. (+info)
Purine oversecretion in cultured murine lymphoma cells deficient in adenylosuccinate synthetase: genetic model for inherited hyperuricemia and gout.
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Alterations in several specific enzymes have been associated with increased rates of purine synthesis de novo in human and other mammalian cells. However, these recognized abnormalities in humans account for only a few percent of the clinical cases of hyperuricemia and gout. We have examined in detail the rates of purine production de novo and purine excretion by normal and by mutant (AU-100) murine lymphoma T cells (S49) 80% deficient in adenylosuccinate synthetase [IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4]. The intracellular ATP concentration of the mutant cells is slightly diminished, but their GTP is increased 50% and their IMP, four-fold. Compared to wild-type cells, the AU-100 cells excrete into the culture medium 30- to 50-fold greater amounts of purine metabolites consisting mainly of inosine. Moreover, the AU-100 cell line overproduces total purines. In an AU-100-derived cell line, AU-TG50B, deficient in adenylosuccinate synthetase and hypoxanthine/guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), purine nucleoside excretion is increased 50- to 100-fold, and de novo synthesis is even greater than that for AU-100 cells. The overexcretion of purine metabolites by the AU-100 cells seems to be due to the primary genetic deficiency of adenylosuccinate synthetase, a deficiency that requires the cell to increase intracellular IMP in an attempt to maintain ATP levels. As a consequence of elevated IMP pools, large amounts of inosine are secreted into the culture medium. We propose that a similar primary genetic defect may account for the excessive purine excretion in some patients with dominantly inherited hyperuricemia and gout. (+info)
Molecular transformation of tumor adenylosuccinate synthetase and characteristics of its converting factor.
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The properties of adenylosuccinate synthetase [inosine monophosphate:L-aspartate ligase (guanosine 5'-diphosphate-forming) EC 6.3.4.4]) of rat Yoshida sarcoma ascites tumor cells changed during purification. The isoelectric points (pI) of the crude and purified enzymes were 5.0 and 5.9, respectively. The Km values of the crude enzyme for inosine monophosphate, aspartate, and guanosine 5'-triphosphate were calculated to be 99 +/- 1 (S.E.), 870 +/- 40, and 27 +/- 2 microM, respectively, while those of the purified enzyme were 410 +/- 10, 980 +/- 50, and 69 +/- 5 microM, respectively. These data indicate that the crude enzyme should be more effective for activity than the purified one. It was found that the change in pI occurred during diethylaminoethyl cellulose column chromatography and that, during this step a compound, named pI-converting factor (ICF), was separated from the enzyme molecule. On addition of ICF, the pI of the purified enzyme changed from 5.9 to 5.0, indicating that the pI conversion was dependent on ICF and was reversible. ICF was nondialyzable, heat stable, and partly precipitated with 1 N perchloric acid but was not affected by 1 N KOH. It was partially degraded by DNAse I. These results suggest that ICF is a DNA-like compound. (+info)