Hypoxanthine Phosphoribosyltransferase Activity Research Articles

Segregation of human hypoxanthine phosphoribosyltransferase activity from somatic cell hybrids isolated from fusion of mouse gene transfer cells with Chinese hamster cells.

Four independent mouse gene transfer clones, i.e. mouse cells which had taken up a human chromosomal fragment carrying the human gene for hypoxanthine phosphoribosyltransferase (HPRT), were characterized with the following results: 1. None of the clones expressed human α-galactosidase activity whose structural gene has been previously located on the human X-chromosome in the neighbourhood of the gene coding for HPRT. Thus the human chromosomal fragment in these mouse cells is presumably so small that it does not carry the gene for human α-galactosidase A. 2. All the gene transfer clones when maintained in logarithmic growth for at least 100 generations in selective medium became phenotypically stable, i.e. they no longer segregated the transferred human HPRT activity after being shifted to non-selective growth conditions. 3. Somatic cell hybrids were isolated after fusion of phenotypically stable mouse gene transfer cells, expressing human HPRT, with chinese hamster cells, mouse cells which were derived from the same parental line as the gene transfer cells but which expressed reverted mouse HPRT were also fused with these chinese hamster cells. All isolated hybrid cell clones were counter-selected for the absence of functional HPRT.

Levels and Significance of Erythrocyte Purine Enzymes in Hyperuricemia

SummaryTo examine possible relationships among purine enzyme patterns in hy-peruricemia and gout, purine enzyme activities were measured in erythrocytes from young adults who were either normouricemic, hyperuricemic, or had a family history of gout. The purine enzymes whose activities were measured included adenine phospho-ribosyltransferase (APRT), hypoxanthine phosphoribosyltransferase (HPRT), adeno-sine deaminase (AD), adenosine kinase (AK), and 5'-nucleotidase. AD and AK activities were not different among the normouricemic, hyperuricemic, or family history of gout groups. The activity of APRT was significantly higher in the group with a family history of gout compared with the normouricemics, and the activity of HPRT was significantly lower in the hyperuricemic and family history of gout groups compared with the normouricemics. The ratio of APRT/HPRT was significantly greater in 66% of those subjects who had a family history of gout and were also hyperuricemic compared with normouricemics without a f...

Regulation of hypoxanthine transport in Neurospora crassa.

Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'monophosphate cyclohydrolase (EC 3.5.1.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found to the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyl-transferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH+/4, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH+/4, none of the strains excreted any detectable purine compounds.

Mutant alleles for hypoxanthine phosphoriboxyltransferase: codominant expression, complementation, and segregation in hybrid Chinese hamster cells.

Chinese hamster ovary cell mutants resistant to the purine analogs 6-thioguanine or 8-azaguanine have been isolated following mutagenesis with ethyl methane sulfonate. The activities of hypoxanthine phosphoribosyltransferase (HPRT) in three such mutants have been found to exhibit an increased Km for the substrate 5-phosphoribosyl-1-pyrophosphate. The isoelectric point of the mutant enzyme activity has also changed in two mutants. Hybrid cells containing one mutant and one wild-type allele express both genes. Segregants that have lost only the wild-type allele can be selected on the basis of drug resistance. Two mutants exhibiting different alterations in HPRT activity can complement in a hybrid cell to yield a wild-type growth pattern and enzyme activity with intermediate electrophoretic and kinetic properties. The results suggest intracistronic complementation between structural gene mutants of HPRT.

Studies on adenine and adenosine metabolism by intact human erythrocytes using high performance liquid chromatography

Adenine and adenosine metabolism has been studied in intact human erythrocytes in vitro using high performance liquid chromatography, isotopic labelling and electrophoresis. Their metabolism to nucleotides was controlled by phosphoribose diphosphate synthesis which was phosphate dependent. Adenosine formed hypoxanthine or IMP depending upon P i concentration, but adenosine kinase and deaminase activities were not affected by P i levels. Free [ 14C]adenine and [ 14C]hypoxanthine were found in cellular extracts. Rapid interconversions occurred to give a distribution for ATP : ADP : AMP of 10 : 1 : 0.1. Marked decomposition of ATP to ADP and AMP occurred during incubations in plasma and Earle's media in air on nitrogen, but ATP levels remained stable in phosphate buffers and in the presence of oxygen. At physiological P i (1 mM) adenosine kinase activity grossly exceeded adenine phosphoribosyltransferase activity. The latter was approximately 7 fold that of hypoxanthine phosphoribosyltransferase activity. These differences decreased with increasing P i levels. No significant increase in corresponding nucleotides was obtained by incubation with high levels (0.5 mM) of adenine, guanine or guanosine at physiological P i, ATP increased by 10% independently of the substrate employed and significant amounts of IMP and GTP were formed from adenosine and guanosine, respectively. The existence of a bound intracellular pool of ATP is suggested.

Monoamine oxidase activity decreased in cells lacking hypoxanthine phosphoribosyltransferase activity.

The Lesch-Nyhan syndrome in humans is characterized by lack of hypoxanthine phosphoribosyltransferase activity and neurologic abnormalities that suggest changes in catecholamine metabolism. Monoamine oxidase, which degrades biogenic amines, has decreased activity in noradrenergic murine neuroblastoma cell lines lacking hypoxanthine phosphoribosyltransferase activity and in skin fibroblasts from patients with the Lesch-Nyhan syndrome.

Hypoxanthine phosphoribosyltransferase activity in intact fibroblasts from patients with X-linked hyperuricemia.

Discordance between clinical phenotype and the level of a mutant enzyme activity may reflect differences between enzyme function in vivo and that measured by the customary enzyme assays on cell extracts. In the present study, the conversion of hypoxanthine to phosphorylated products was measured in intact skin fibroblasts and in cell extracts from seven patients with mutant hypoxanthine-guanine phosphoribosyltransferase (HPRT) and six control subjects. The patient's phenotypes ranged from asymptomatic hyperuricemia to the Lesch-Nyhan syndrome. Although there was a general correlation between the HPRT activity in cell extracts assayed by the usual methods and the function of the purine salvage pathway in patients, as reflected by urinary oxypurine excretion, there were notable exceptions. A more accurate appraisal of the functioning of the pathway at the cellular level is achieved by measuring the conversion of substrate to product in the intact cell at physiological concentrations of substrates, activators, and product and metabolite inhibitors, and in a physiological ionic environment. In one of the seven patients, the standard enzyme assay indicated normal function, whereas measurements in the intact cell exposed severe dysfunction of the salvage system. In another, the standard assay suggested a severe deficiency not evident in the intact cell or in the patient.

Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.

Expression of human hypoxanthine phosphoribosyl transferase in Chinese hamster cells treated with isolated human chromosomes

Chinese hamster cells deficient for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) were incubated with isolated human metaphase chromosomes and 21 colonies were isolated in HAT medium. Three different types of cell lines were established from these clones. First, 4 cell lines had 10-30% of normal Chinese hamster HPRT activity with the same electrophoretic mobility as human HPRT. This HPRT activity remains detectable during at least 8 weeks of growth of the cells in nonselective medium. Second, 3 cell lines also had human-like HPRT with the same activity as the first type. This HPRT persists only if the cells are grown in HAT medium and disappears during 8 weeks of growth in nonselective medium. Third, other clones survived in HAT medium as well as in medium with 8-azaguanine. These cells had no detectable HPRT activity. Using differential chromosome staining techniques no recognizable human chromosome fragments were found in any of the cell lines.

A comparative study of hypoxanthine phosphoribosyltransferase activity in birds and mammals

1. 1. Hypoxanthine phosphoribosyltransferase activities in birds were compared with those in mammals. 2. 2. In the livers of chicken, quail and pigeon, the activities were much lower than that in rat liver, though the activities in the avian kidney were on almost the same level as that of rat kidney. 3. 3. The incorporation of [ 14C]hypoxanthine into acid soluble nucleotides and RNA was also very low in the avian liver.

CHANGES IN THE METABOLIC PATHWAYS OF HYPOXANTHINE IN STREPTOMYCES

Changes in the metabolic pathways of hypoxanthine in a strain of Streptomyces sp. has been studied. Hypoxanthine was converted to inosine 5′-monophosphate by the cells of the early period of cultivation, in which a high activity of hypoxanthine phosphoribosyltransferase but none of xanthine dehydrogenase was found, and was oxidized to xanthine and 6, 8-dihydroxypurine by those of the middle and the late periods, in which both activities were observed. If the cells of the early growth period were starved of the nitrogen source, the activity of xanthine dehydrogenase increased and hypoxanthine stimulated the increase, whereas that of hypoxanthine phosphoribosyltransferase remained constant. Synthesis of the nucleotide but not the oxidation was inhibited under these conditions of nitrogen limitation. If the cells were then supplied with the nitrogen source, however, the purine base was metabolized to both the synthetic and oxidative directions at the same time. These results suggest that the nitrogen source in the medium plays an important role in the control of hypoxanthine metabolism; repressing the formation of xanthine dehydrogenase on the one hand and promoting the reaction of hypoxanthine phosphoribosyltransferase on the other.

Hypoxanthine phosphoribosyltransferase deficiency: association of reduced catalytic activity with reduced levels of immunologically detectable enzyme protein.

In the present study hemolysates from fourteen patients with a genetically determined deficiency of hypoxanthine phosphoribosyltransferase (EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase) activity were examined immunologically for the presence of material that crossreacts with the normal enzyme. A quantitative assay for crossreacting material in enzyme-deficient hemolysates was based on the inhibition of the immunoprecipitation of the normal enzyme. As little as 3% of normal crossreacting material could be detected. One patient in this series was found to have a normal amount of crossreacting material, whereas the remainder had no detectable crossreacting protein. The lack of detectable crossreacting material in these patients raises the possibility that a defect in synthesis or degradation of enzyme protein may be present in many patients deficient in hypoxanthine phosphoribosyltransferase.

Hypoxanthine phosphoribosyltransferase activity in normal, developing, and neoplastic tissues of the rat

The activity of hypoxanthine phosphoribosyltransferase, an enzyme of purine salvage, has been measured in ten normal tissues of the rat, in regenerating liver, in developing liver, in starved and glucocorticoid-treated rats, and in several Morris hepatomas. A new method was employed, making a multiple-time-point assay practicable. Liver contained the highest activity of those tissues assayed; activity was maximum in this tissue at 32 days after birth. No correlation of enzyme activity with tissue growth rate was apparent. Kinetic studies on the enzymes from normal liver and Morris hepatoma 3924A indicated half-maximal velocities were attained at concentrations of about 4 μM hypoxanthine, and about 5 μM phosphoribosylpyrophosphate.

Isolation of mammalian cell mutants deficient in glucose-6-phosphate dehydrogenase activity: linkage to hypoxanthine phosphoribosyl transferase.

Mutants of Chinese hamster ovary cells deficient in glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP 1-oxidoreducatse, EC 1.1.1.49) activity were isolated after mutagenesis with ethyl methane sulfonate. The mutants were induced at frequencies of about 10-4 and do not differ in growth properties from wild-type cells. They were isolated by means of a sib selection technique coupled with a histochemical stain of colonies for enzyme activity. The lack of enzyme activity is not due to a dissociable inhibitor, and is recessive in hybrid cells. Multiple mutants that lack hypoxanthine phosphoribosyltransferase activity (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and adenine phosphoribosyltransferase activity (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7) were isolated by further mutagenesis. By following segregation of wild-type phenotypes from heterozygous multiply marked hybrid cells, it was shown that the genes responsible for glucose-6-phosphate dehydrogenase activity and hypoxanthine phosphoribosyltransferase activity are linked in Chinese hamster cells, in agreement with the location of both on the X chromosome in humans. No linkage to adenosine phosphoribosyltransferase was found. The isolation of mutant cells carrying linked markers should prove useful for studying chromosomal events such as segregation, breakage, recombination, and X-chromosome reactivation.

Hypoxanthine phosphoribosyltransferase and hypoxanthine uptake in human erythrocytes.

A system of hypoxanthine uptake and IMP retention was studied and characterized in human erythrocytes. It follows closely the system already described for rabbit erythrocytes[7]. IMP formation and retention are dependent on the activity of hypoxanthine phosphoribosyl-transferase and on intracellular availability of phosphoribosyl pyrophosphate (P-Rib-PP), which is one of the substrates. In the extrecellular medium, neither P-Rib-PP nor GMP -- a potent inhibitor of the enzyme in vitro -- has any influence on IMP retention. The amount of residual hypoxanthine phosphoribosyltransferase in erythrocyte ghost preparations is directly related to the residual hemoglobin content. Thus the enzyme is characterized as typically soluble and "loosely bound" to membranes. There is a slight difference in the kinetic properties of the ghost-bound and the free soluble enzyme. The possible importance of these results for purine uptake and utilization in human red cells is discussed.

Cycloheximide inhibitation on hypoxanthine transport in cultured cells

Cycloheximide preincubation inhibits hypoxanthine uptake into the acid-soluble fractions of cultured rat hepatoma cells (MH 1C 1) and human skin epithelial cells (NCTC 2544, HE cells) in a time- and dose-dependent manner 50% inhibition is seen after 4 h preincubation with 10 −4 M cycloheximide of MH 1C 1 cells and after 2.5 h of HE cells. Adenine uptake is much less affected, after 10 h preincubation with 10 −4 M cycloheximide it was reduced to 83% and 67% of controls in MH 1C 1 cells and HE cells respectively. Cycloheximide inhibits hypoxanthine uptake in a dose-dependent manner above 10 −7 M, with 50% inhibition in MH 1C 1 cells at 4 · 10 −7 M after 12 h preincubation and at 10 -6 M in HE cells after 6 h preincubation. Puromycin mimics the action of cycloheximide. The inhibition of hypoxanthine uptke is not caused by reduction of the activity of hypoxanthine phosphoribosyltransferase in the two cell lines. 10 −4 M cycloheximide preincubation for 10 h does not significantly reduce the uptake of the two non-metabolizable amino acids α-aminoisobutyric acid or 1-aminocyclopentane-1-carboxylic acid (cycloleucine). It is suggested that cycloheximide inhibits the synthesis of a rapidly turning over the protein involved in hypoxanthine transport.

Incorporation of hypoxanthine by PHA-stimulated HPRT-deficient lymphocytes

Phytohemagglutinin (PHA) markedly stimulates 3H-hypoxanthine incorporation by lymphocytes of normal subjects as revealed by radioautography. There is no corresponding increase in activity of hypoxanthine phosphoribosyltransferase (HPRT) in lysates but the level of phosphoribosylpyrophosphate (PRPP), the cosubstrate for the reaction, is higher. Lymphocytes from a patient with partial HPRT deficiency responded to PHA as did the normals, whereas the response in Lesch-Nyhan (LN) subjects was variable. PHA-stimulated lymphocytes from two LN patients showed some increase in 3H-hypoxanthine incorporation, while two others failed to respond. The observations provide further evidence of genetic heterogeneity among LN patients.

Changes in purine phosphoribosyltransferase activities in mouse brain, liver and muscle with age

Hypoxanthine (or guanine) phosphoribosyltransferase (EC 2.4.2.8) and adenine phosphoribosyltransferase (EC 2.4.2.7) activities have been evaluated in mouse brain, liver and muscle as a function of age. In these tissues adenine phosphoribosyltransferase activity (per g of tissue) decreased from birth until about 30 days of age, at which time it attained a plateau in the brain and increased slightly in liver and muscle. The pattern of hypoxanthine (or guanine) phosphoribosyltransferase activity differed more with the tissue. In muscle it decreased continuously; in liver it decreased until 30 days and then increased significantly: in brain it increased considerably and attained a plateau at 30 days. Until 20 days of age the total phosphoribosyltransferase activity per organ was greatest in the brain; subsequently the total liver activity became largest. Simultaneous determinations of xanthine oxidase and guanine deaminase indicated that, shortly after birth, hypoxanthine (or guanine) phosphoribosyltransferase in liver and muscle must compete for its substrates with high levels of these two enzymes. In brain from birth, the guanine deaminase activity was superior to the phosphoribosyltransferase activity with guanine; however, phosphoribosyltransferase activity with hypoxanthine as substrate was always considerably greater than the xanthine oxidase activity.

Modulation of the activity of an avian gene transferred into a mammalian cell by cell fusion.

Mouse A9 cells, deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8), were fused with normal chick erythrocytes and selected in hypoxanthine-aminopterin-thymidine medium for cells with hypoxanthine phosphoribosyltransferase activity. Recovered hybrid cells produced the chick hypoxanthine phosphoribosyltransferase exclusively, as demonstrated by electrophoretic mobility and immunoprecipitation tests, even though no chick chromosomes or chick cell-surface antigens could be identified in the hybrids. Surprisingly, the expression of the chick hypoxanthine phosphoribosyl-transferase activity in the mouse/chick hybrids required the presence of aminopterin in the growth medium; in its absence, enzyme synthesis decreased markedly. Because of the rapid and reversible modulation of hypoxanthine phosphoribosyltransferase activity, the hybrid cells could proliferate equally well in media containing hypoxanthine-aminopterin-thymidine or 8-azaguanine. Cellular selection was definitely ruled out as a possible cause. These results confirm previous reports that specific genetic information can be selectively transferred from one cell to another of a distant species. Furthermore, they demonstrate that an avian gene, whose activity is normally expressed constitutively, can become facultative when integrated into a mammalian cell. This seems to be the first instance where heterologous gene activity has been shown to be reversibly modulated in hybrid cells.

Purine metabolism in normal and thioguanine-resistant neuroblastoma.

Purine metabolism has been examined in a clonal line of mouse neuroblastoma cells resistant to the cytotoxic effects of 6-thioguanine. Comparative studies in the resistant and parental lines indicate that the former cells have less than 1% of normal hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity. The activities of other enzymes important in the de novo and salvage pathways of purine biosynthesis were not significantly different in the two lines. Hypoxanthine phosphoribosyltransferase deficiency in this neuroblastoma line was associated with increased intracellular concentrations of 5-phosphoribosyl-1-pyrophosphate, an increased rate of purine biosynthesis de novo, and failure to incorporate hypoxanthine, but not adenine, into nucleotides. There are essentially the same alterations in purine metabolism that occur in hypoxanthine phosphoribosyltransferase-deficient fibroblasts or lymphoblasts derived from individuals with the Lesch-Nyhan syndrome. Clonal lines of hypoxanthine phosphoribosyltransferase-deficient neuroblastoma cells may therefore be of use in elucidating the mechanisms by which the enzyme defect leads to the neurologic dysfunction seen in children with this disease.