Utilization Of Hypoxanthine Research Articles

Genetic characterization of the Neurospora crassa molybdenum cofactor biosynthesis

Molybdenum (Mo) is a trace element that is essential for important cellular processes. To gain biological activity, Mo must be complexed in the molybdenum cofactor (Moco), a pterin derivative of low molecular weight. Moco synthesis is a multi-step pathway that involves a variable number of genes in eukaryotes, which are assigned to four steps of eukaryotic Moco biosynthesis. Moco biosynthesis mutants lack any Moco-dependent enzymatic activities, including assimilation of nitrate (plants and fungi), detoxification of sulfite (humans and plants) and utilization of hypoxanthine as sole N-source (fungi). We report the first comprehensive genetic characterization of the Neurospora crassa (N. crassa) Moco biosynthesis pathway, annotating five genes which encode all pathway enzymes, and compare it with the characterized Aspergillus nidulans pathway. Biochemical characterization of the corresponding knock-out mutants confirms our annotation model, documenting the N. crassa/A. nidulans (fungal) Moco biosynthesis as unique, combining the organizational structure of both plant and human Moco biosynthesis genes.

Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G-3H] hypoxanthine (Hy) into [3H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [3H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

Mode of action of 2-amino-6-chloro-1-deazapurine

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5′-phosphate which accumulated to concentrations as high as 1000 nmoles/10 9 cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibited the synthesis of 5′-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides. IMP dehydrogenase from L1210 cells was not inhibited by the nucleotides of 2-amino-6-chloropurine or 2-amino-6-chloro-1-deazapurine at 1 mM comcentrations. The nucleotide of 2-amino-6-chloropurine was dechlorinated by AMP deaminase; the apparent K m was about the same as that of AMP and the V max was 21% that of AMP. The nucleotide of 2-amino-6-chloro-1-deazapurine was not a substrate. Conversion of the nucleotide of 2-amino-6-chloropurine to GMP by the action of AMP deaminase explains both the lower toxicity of 2-amino-6-chloropurine as compared to its 1-deaza analog and its capacity to increase pools of guanine nucleotides. Thus, of the two compounds the 1-deaza analog may be the more interesting as an antitumor agent because of the greater metabolic stability of its nucleotide.

HYPOXANTHINE AND ALLANTOIN AS NITROGEN SOURCES FOR THE GROWTH OF SOME FRESHWATER GREEN ALGAE

SummarySix freshwater green algae, viz. Scenedesmus obliquus, Scenedesmus pannonicus, Chlorella glucotropa, Crucigenia tetrapedia, Tetradesmus cumbricus and Stichococcus mirabilis, were examined for their ability to utilize hypoxanthine and allantoin as sole nitrogen sources for growth in light. All the six algae could utilize both hypoxanthine and allantoin for growth. None of the algae needed any significant period of adaptation and growth was often as rapid on hypoxanthine/allantoin as it was on nitrate. Higher concentrations (3 mat) of the purines inhibited the growth of Stichococcus mirabilis and Crucigenia tetrapedia, possibly because of the formation of intracellular pools of the purines. Activity of the enzyme nitrate reductase was not markedly affected by growth on either hypoxanthine or allantoin. It is concluded that the ready utilization of hypoxanthine and allantoin indicates the operation of the standard pathway of purine catabolism, i.e. hypoxanthine → xanthine → uric acid → allantoin → end products, in these algae.

Guanosine metabolism in Neurospora crassa.

Two aspects of guanosine metabolism in Neurospora have been investigated. (a) The inability of adenine mutants (blocked prior to IMP synthesis) to use guanosine as a nutritional supplement; and (b) the inhibitory effect of guanosine on the utilization of hypoxanthine as a purine source for growth by these mutants. Studies on the utilization of guanosine indicated that the proportion of adenine derived from guanosine may be limiting for the growth of adenine mutants. In wild type, adenine is produced through the biosynthetic pathway when grown in the presence of guanosine. The amount of adenine produced through the de novo biosynthesis in wild type increases with increasing concentrations of guanosine in the medium. However, the total purine synthesis does not increase. Guanosine inhibits the uptake of hypoxanthine severely. In addition, guanosine and its nucleotide derivatives also inhibit the hypoxanthine phosphoribosyltransferase activity, at the same time stimulating the adenine phosphoribosyltransferase activity. Guanosine's effects on the uptake of hypoxanthine and its conversion to the nucleotide form may be the reasons why guanosine inhibits the utilization of hypoxanthine but not adenine by these mutants.

Inhibition of hypoxanthine-guanine phosphoribosyl transferase

The affinities of eighteen purines or purine analogs for human erythrocytic hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase) were compared to assess the feasibility of obtaining active inhibitors of the enzyme. Three compounds appeared to inhibit the utilization of hypoxanthine by L5178Y cells in vitro due to inhibition of the enzyme rather than depletion of the intracellular 5-phosphoribosyl-1-pyrophosphate pool. The three competitive inhibitors and their affinity constants ( K i ) using 6-mercaptopurine as substrate were: 6-mercapto-9-(tetrahydro-2-furyl)-purine, 37 μM; 2,6-bis-(hydroxyamino)-9-β- d-ribofuranosyl-purine, 12 μM; and 6-iodo-9-(tetrahydro-2-furyl)-purine, 108 μM. The KIn m for 6-mercaptopurine was 9 μM. Thus, the enzyme tolerates bulky substitution at N 9. 6-Mercapto-9-(tetrahydro-2-furyl)-purine also potentiated the chemotherapeutic effect of azaserine, an inhibitor of de novo purine biosynthesis, in L5178Y ascites tumor-bearing mice. Four 2-substituted, oxazolo-[5, 4- d]-pyrimidine-7-ones and 2-methylthiazolo-[5, 4- d]-pyrimidine-7-one had K i values in the range of 84–173 μM. Consequently, isosteric substitution at N 9 may also be a fruitful and logical course to pursue in the design and synthesis of more potent inhibitors of this important enzyme.

Effects of diazoacetyl-glycine amide on purine nucleotide metabolism in ehrlich ascites tumour cells

On the basis of the known inhibitory activity of diazoacetyl-glycine amide (DGA) on DNA and purine nucleotide synthesis, a series of experiments was performed to study, in greater detail, the effects of the drug on purine nucleotide metabolism. DGA produces a marked inhibition of de novo utilization of glycine and formate and a less pronounced inhibition in the “salvage” utilization of hypoxanthine for IMP synthesis. The synthesis of adenine and guanine nucleotides from hypoxanthine is also depressed by DGA, and the drug also causes unbalance in the ratio of the concentration of adenine and guanine nucleotides and a drop in the energy status of nucleotides. These findings and those already reported are related to a rather unspecific interaction of the drug with cellular components.

Differential induction of chromosome deletions by natural and synthetic macromolecules in Drosophila melanogaster.

HE genetic phenomenon usually associated with macromolecules, is the process of transformation in certain bacteria in the presence of deoxyribonucleic acid (DNA). This involves the replacement of specific gene loci in the bacterial cell by their alleles when supplied in the form of physiologically competent exogenous DNA. In recent years, “somatic transformation” of metazoan cells was frequently attempted in various biological systems, but on the whole without success (PERRY and WALKER 1958; BEARN and KIRBY 1959; BILLET, HAMILTON and NEWTH 1964). In contrast, SZYBALSKA and SZYBALSKI (1962) reported the successful transformation of human sternal cells in tissue culture. Cells incompetent in the utilization of hypoxanthine (deficient in the enzyme inosinic acid pyrophosphorylase) were transformed into competent cells by their exposure to DNA extracted from the normal strain. In these experiments the transformation frequency was considerably lower than in bacteria and the reverse process (from competent to incompetent cells) has not, so far, been demonstrated. It would thus appear that metazoan cells might, indeed, be capable of genetic transformation, although the conditions for its occurrence are probably very critical and, as yet, largely unknown. Doubtless, this is due to the much higher complexity of biological organisation of metazoan genetic systems. The mode of action of DNA on metazoan cells was extensively analysed on the germ line of Drosophila (FAHMY and FAHMY 1961, 1962, 1963, 1964, 1965). The injection of DNA extracted from the wild-type flies around the testes of a stock carrying seven mutant loci (both autosomal and sex-linked) did not result in any transformants. More recently, the reverse experiment was undertaken: the DNA was extracted from the 7-mutant stock and injected into the wild-type males, but again no specific locus transformation occurred (FAHMY and FAHMY 1965). In contrast to-the mtive transformation results, mutagenicity tests revealed the activity uf DNA in the induction of small chromosome deficiencies (resulting in the M,inute phenotype) and-to a much lesser extent-sex-linked recessive visibles (FAHMY and FAHMY 1961). It was later shown that the DNA type of mutagenicity was also shared by a diversity of macromolecules of natural and synthetic origin (FAHMY and FAHMY 1962, 1964). This indicated that the mutagenicity of DNA is a function of its polymeric chemical structure, and is

Biochemical effects of duazomycin A in the mouse plasma cell neoplasm 70429

Sublines of the 70429 mouse plasma cell neoplasm exhibited cross-resistance between duazomycin A and the other glutamine antagonists, DON and azaserine. Like these compounds, duazomycin A inhibited incorporation of formate into soluble purine nucleotides and into nucleic acids in growing tumor cells in vivo, and produced large accumulations of formylglycinamide ribotide in the soluble fraction of these cells. The utilization of hypoxanthine for purine nucleotide and nucleic acid synthesis was correspondingly increased. Tnese findings are in agreement with a major site of duazomycin inhibition, as for the other two antagonists, at the glutamine-mediated amidination of formylglycinamide ribotide to formylglycinamidine ribotide. The accumulations of formylglycinamide ribotide were much reduced at higher levels of duazomycin, although purine nucleotide synthesis continued to be blocked; this would be consistent with an effective secondary inhibition earlier in the de-novo pathway of purine biosynthesis, in the glutamine-requiring synthesis of phosphoribosylamine. In this respect duazomycin resembled DON, but azaserine did not show this effect. Duazomycin was also an effective inhibitor of the conversion of uridine to cytidine nucleotides in these cells, although it was not so potent as DON in this respect. At the level tested duazomycin did not exert apparent inhibition of the synthesis of RNA guanylic acid from hypoxanthine, although such inhibition was evident with DON. Duazomycin was readily deacetylated, evidently to DON, by mammalian acylase I purified from hog kidney. In addition, acylase activity in mouse kidney deacetylated some duazomycin; acylase activity was weak in extracts of 70429 cells and had no detectable effect on duazomycin.