Purine and pyridine nucleotide production in human erythrocytes

Human erythrocyte adenyl and pyridine nucleotide production has been tested in cell-free lysates and in intact cells. The main products obtained in cells incubated with adenine and nicotinic acid are adenosine triphosphate and nicotinate mononucleotide, respectively, under any experimental condition used (incubation time, base concentration). Adenine-phosphoribosyltransferase activity determined in crude lysates is about 100 times higher than nicotinate-phosphoribosyltransferase activity, while cellular adenyl nucleotide production is only three times higher than that of pyridine nucleotide. A strong intracellular regulation for the former, but not latter, synthetic process is thus suggested. Intact erythrocyte nicotinate nucleotide production is inhibited by adenine, while nicotinate-phosphoribosyltransferase activity is not. The possible regulation by adenyl nucleotides is discussed in light of the modulating action of ATP on nicotinate-phosphoribosyltransferase activity. The kinetic characteristics of both adenine- and nicotinate-phosphoribosyltransferases, determined on crude lysates, are reported.     

Purine nucleotide cycle enzymes in dystrophic and normal mouse muscle

A comparative study of the operation of the purine nucleotide cycle and of the activity of adenylosuccinase in extracts of muscle from the two strains of dystrophic mouse shows that the cycle is defective in both cases in the conversion of adenylosuccinate to AMP. However, adenylosuccinase activity is only slightly reduced in the standard conditions for its direct assay.     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.     

Purine nucleotide content of developing Ascaris lumbricoides eggs

Farland W. H. &; Macinnis A. J. 1978. Purine nucleotide content cf developing Ascaris lumbricoides eggs. International Journal for Parasitology8: 177–186. Populations of Ascaris lumbricoides eggs obtained from the terminal portion of the uteri of mature females were shown to develop synchronously, allowing the detection of quantitative and qualitative changes in nucleotide content during development. Application of a method for the preparation of perchloric acid-soluble fractions from Ascaris eggs utilizing Alamine 336S in chloroform is described. This method was useful in neutralizing the extract and for removal of interfering lipids.Guanine nucleotides were found in high concentration in ovarian tissue of adult female worms, and comprise more than 80% of the total acid-soluble nucleotides in 0-day eggs. Muscle tissue contained a predominance of adenine nucleotides.To determine the fate of large concentrations of guanine nucleotides present in 0-day eggs, perchloric acid-soluble fractions were prepared from embryonating eggs on various days of development. Nucleotide content was determined after fractionation on DEAE-cellulose columns,A dramatic decrease (6·2 fold) in guanine nucleotides between 5 and 8 days' embryonation was seen; [GMP] and [GDP] showed the greatest change. ATP concentration increases 3·3 fold through 21 days' embryonation. The total acid-soluble nucleotide content decreased 60% during this time. Uric acid, an end product of purine metabolism, was detectable in 5-day eggs and accumulated through the course of embryonation. The decrease of guanine nucleotides correlates temporally with the increase in DNA content during embryonation. The methodology and results of this study provide a basis for additional study of nucleic acid metabolism during development of Ascaris eggs.     

Purine nucleotide metabolism of germinating soybean embryonic axes

Isolated soybean (Glycine max L. cv. Kent) embyronic axes metabolized [¹⁴C]glycine to ATP within the 1 hour of imbibition. Radioactivity was not detected in GTP until the 3rd hour. Throughout most of the first 24 hours of germination about 10 to 26 times as much label from [¹⁴C]glycine appears in ATP as GTP. About five times as much [¹⁴C]hypoxanthine and [¹⁴C]inosine was converted into GTP as into ATP in embryonic axes. Two independent pools of IMP appear to be used in purine nucleotide synthesis of soybean axes.     

Purine base transport in normal and mutant erythrocytes.

The uptake of adenine and hypoxanthine in HGPRT-deficient and normal human erythrocytes was measured using a rapid filtering centrifugation technique. The transport of hypoxanthine as well as of adenine is impaired in the mutant cells. The transport of hypoxanthine into HGPRT-deficient erythrocytes differs from that into normal cells with respect to a higher accumulation capacity, to lower initial velocities and to the kinetic properties of the translocator. In addition, a higher accumulation capacity and lower initial velocities of adenine uptake could be demonstrated in mutant cells. A linkage of the purine translocator with purine phosphoribosyltransferases associated with the erythrocyte membrane is discussed.     

Induction of HPRT- mutants in Chinese hamster V79 cells after heavy ion exposure

The induction of resistance to 6-thioguanine by heavy ion exposure was investigated with various accelerated ions (oxygen-uranium) up to linear energy transfer (LET) values of about 15000 keV/µm.31 y Survival curves are exponential with fluence; mutation induction shows a linear dependence. Cross-sections (_i: inactivation, _m: mutation) were derived from the respective slopes. Generally, _i rises over the whole LET range, but separateas into different declining curves for single ions with LET values above 200 keV/µm. Similar behaviour is seen for _m. The new SIS facility at GSI, Darmstadt, makes it possible to study the effects of ions with the same LET but very different energies and track structures. Experiments using nickel and oxygen ions (up to 400 MeV/u) showed that inactivation cross-sections do not depend very much on track structure, i.e. similar values are found with different ions at the same LET. This is not the case for mutation induction, where very energetic ions display considerably smaller induction cross-sections compared with low-energy ions of identical LET. Preliminary analyses using the polymerase chain reaction (PCR) demonstrate that even heavy ions cause small alterations (small deletions or base changes). The proportion of the total deletions seems to increase with LET.Submitted paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Purine salvage enzyme activities in normal and neoplastic human tissues

The enzymatic pattern of five enzymes involved in the purine salvage pathway, namely purine nucleoside phosphorylase (EC 2.4.2.1), adenosine deaminase (EC 3.5.4.4), 5'-nucleotidase (EC 3.1.3.5), alkaline phosphatase (EC 3.1.3.1), and hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been evaluated both in human intestinal and breast carcinomas and compared to that of normal tissues. A higher level of hypoxanthine-guanine phosphoribosyltransferase was associated with tumor tissues. This metabolic alteration should lead to an elevated synthesis of nucleotides in cancer cells, might confer selective growth advantages to neoplastic tissues, and account, at least in part, for the difficulties encountered in the chemotherapy of human tumors, by using compounds affecting only the purine de novo biosynthesis.     

Purine metabolism in thioguanine-resistant glioma cells

6-Thioguanine resistant strains of rat glioma cells were selected spontaneously and after mutagen treatment. Both mutant lines exhibited a severe deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase, increased intracellular concentrations of 5-phosphoribosyl-1-pyrophosphate and rate of the early steps of purine biosynthesis, and an inability to incorporate guanine, but not adenine, into soluble purine nucleotides.     

Purine and oxypurine production in mitochondria of ischemic and reperfused myocardium

The present study was undertaken to determine whether significant breakdown of adenine nucleotides to purine bases and oxypurines occurred in mitochondria following myocardial ischemia and ischemia followed by reperfusion, and whether allopurinol prevented this effect. The adenine nucleotides adenosine, hypoxanthine, xanthine and uric acid were measured in the mitochondria and the results suggest that breakdown did occur. Malondialdehyde concentration was determined to gauge lipid peroxidation. This substance did not increase during ischemia or reperfusion, but did so in the presence of allopurinol. Xanthine dehydrogenase was converted to xanthine oxidase during reperfusion and the activity of both enzymes were inhibited by allopurinol. The results also suggested the presence of a mitochondrial 5'-nucleotidase. We conclude that significant breakdown of adenine nucleotide took place in myocardial mitochondria during ischemia and ischemia followed by reperfusion and that allopurinol may have a protective effect.     

Purine base composition analysis of normal and tumor rat DNA

Chemical and viral induced rat tumors were analyzed for their purine base composition and compared to normal tissue DNA'S. The tumors were induced by 7,12-dimethylbenz[a]anthracene (DMBA), 20-methylcholanthrene (MC), 3,4-benzopyrene (BP), 1,2-dimethylhydrazine (DMH) and Rous sarcoma virus (RSV). Normal DNAs were extracted from colon, caecum, liver, spleen and embryo and used as reference standards for base composition of normal rat DNA. The composition of purines was obtained by spectrophotometric estimation of the total adenine and guanine (A/G) contents after depurination of the DNA with 66% formic acid at 30 degrees C for 18 h and passage over a cationic exchange resin. Statistical comparison of the A/G molar ratios in normal rat DNAs (1.271) to those of chemical-induced primary tumors (1.342) has shown a highly significant increase. No significant differences could be detected when the base composition of the normals were compared to transplanted tumors, whether chemically or virally induced. Possible explanations from a mutational point of view are discussed.