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PubMed1983

[Chinese hamster cells mutant for the hypoxanthine-guanine phosphoribosyltransferase locus. IV. The biochemical characteristics of the hypoxanthine-guanine phosphoribosyltransferase of hybrid clones obtained by intragenic complementation].

A biochemical study of hypoxanthine-guanine-phosphoribosyltransferase (HPRT) has been carried out in hybrid clones of Chinese hamster cells obtained in complementation experiments. A wide range of biochemical characteristics made it possible to identify a hybrid form of HPRT differing from the enzyme of parental clones in virtually every hybrid tested. The presence of hybrid HPRT was detected by the changed kinetic properties of temperature sensitivity and electrophoretic mobility compared to the enzyme in mutant cells. Since HPRT consists of identical subunits, the hybrid nature of the enzyme in cells obtained through hybridization of HPRT-mutant clones may be regarded as evidence for intragenic complementation. None of the hybrid clones contained an enzyme with the normal properties. Groups of hybrids with similar biochemical characteristics of HPRT can be obtained, if one of the mutant partners involved in hybridization belongs to one and the same complementation group; the major characteristics of hybrid HPRT are then determined by the partner having the higher level of enzyme activity. The series of studies of intragenic complementation in the HPRT gene is summarized.

Animals↗
PubMed1982

[Chinese hamster cells with mutation in the hypoxanthine-guanine phosphoribosyltransferase locus. III. The biochemical characteristics of the hypoxanthine-guanine phosphoribosyltransferase of complementing mutants].

The paper presents a biochemical study of hypoxanthine-guanine phosphoribozyltransferase (HRPT) in mutant clones of Chinese hamster cells showing an ability for complementation. In order to characterize HPRT, its kinetic properties, temperature sensitivity and electrophoretic mobility in polyacrylamide gel were assayed. According to the complementation map, the nine mutant clones studied can be divided into four complementation groups. All these clones have been shown to be mutants with respect to the HPRT structural gene, as they synthesize the structurally and functionally altered enzyme. A comparative biochemical analysis of HPRT in the four complementation groups revealed substantial differences in mutant enzymes from different groups; hence, the possibility of complementation on the molecular level. All biochemical characteristics of HPRT tested are similar in clones belonging to one and the same complementation group, which could indicate that they have the same structural variant of the enzyme, regardless of the manner in which the mutants were obtained. Having revealed the similarity and the distinctive features of mutant enzymes within complementation groups, the biochemical analysis confirmed the results of complementation analysis and added the structural information concerning mutant variants of the enzyme. Thus, the complementation map of the HPRT gene yielded by hybridological analysis has been tested and confirmed by an independent biochemical study. Complementation analysis applied to the HPRT mutants made it possible to identify qualitatively distinct groups. Each of these groups may be regarded as an allele of the gene, and the sum of the groups may be regarded as a series of multiple alleles.

Animals↗
PubMed1992

Purine nucleoside phosphorylase. Inosine hydrolysis, tight binding of the hypoxanthine intermediate, and third-the-sites reactivity.

Purine nucleoside phosphorylase from calf spleen is a trimer which catalyzes the hydrolysis of inosine to hypoxanthine and ribose in the absence of inorganic phosphate. The reaction occurs with a turnover number of 1.3 x 10(-4) s-1 per catalytic site. Hydrolysis of enzyme-bound inosine occurs at a rate of 2.0 x 10(-3) s-1 to form a stable enzyme-hypoxanthine complex and free ribose. The enzyme hydrolyzes guanosine; however, a tightly-bound guanine complex could not be isolated. The complex with hypoxanthine is stable to gel filtration but can be dissociated by acid, base, or mild denaturing agents. Following gel filtration, the E.hypoxanthine complex dissociates at a rate of 1.9 x 10(-6) s-1 at 4 degrees C and 1.3 x 10(-4) s-1 at 30 degrees C. The dissociation constant for the tightly-bound complex of enzyme-hypoxanthine is estimated to be 1.3 x 10(-12) M at 30 degrees C on the basis of the dissociation rate. The stoichiometry of the reaction is 1 mol of hypoxanthine bound per trimer. The reaction is reversible since the same complex can be formed from enzyme and hypoxanthine. Addition of ribose 1-phosphate to the complex results in the formation of inosine without release of hypoxanthine. Thus, the complex is catalytically competent. Inorganic phosphate or arsenate prevents formation of the tightly-bound E.hypoxanthine complex from inosine or hypoxanthine. Direct binding studies with hypoxanthine in the presence of phosphate result in 3 mol of hypoxanthine bound per trimer with a dissociation constant of 1.6 microM. In the absence of phosphate, three hypoxanthines are bound, but higher hypoxanthine concentrations cause the release of two of the hypoxanthines with an apparent inhibition constant of 130 microM. The results establish that enzymatic contacts with the nucleoside alone are sufficient to destabilize the N-glycosidic bond. In the absence of phosphate, water attacks slowly, causing net hydrolysis. The hydrolytic reaction leaves hypoxanthine stranded at the catalytic site, tightly bound to the enzyme with a conformation related to the transition state. In the phosphorolysis reaction, ribose 1-phosphate causes relaxation of this conformation and rapid release of hypoxanthine.

Animals↗
PubMed1986

Modulation of thiopurine cytotoxicity in the HL-60 cell line by physiological concentrations of hypoxanthine.

Thioguanine and mercaptopurine are clinically important agents in widespread use for the treatment of acute leukemia. In this study the effect of low, physiological concentrations of hypoxanthine on thiopurine cytotoxicity was examined in the HL-60 human acute promyelocytic leukemia cell line. Initially the effect of cell concentration on medium hypoxanthine levels was investigated. When 10(5) to 10(6) cells/ml were used, medium hypoxanthine concentrations fell to undetectable (less than 0.1 microM) levels by 24 h, due to cell utilization. However, when the cell density was reduced to 10(3) cells/ml, it was possible to maintain a medium hypoxanthine level as low as 0.5 microM for 24 h without significant hypoxanthine depletion. This allowed for the investigation of the extent to which physiological concentrations of hypoxanthine (1.0 to 10 microM) could modulate thiopurine cytotoxicity. HL-60 cells were incubated in medium containing from 1.0 to 100 microM hypoxanthine concentrations and varying levels of thioguanine or mercaptopurine for 24 h. Cells were then washed and cloned in soft agar. Physiological concentrations of hypoxanthine (1.0 to 10 microM) provided significant protection to HL-60 cells from the cytotoxic effects of both thioguanine and mercaptopurine. An increase in medium hypoxanthine level from 1.0 to 3.0 microM resulted in a 3-fold increase in the thiopurine concentration required to kill 50% of cells. There was a linear relationship between the thiopurine concentration required to reduce clonogenic survival by 50% and medium hypoxanthine level over the hypoxanthine concentrations studied. Although thioguanine was 200-fold more potent than mercaptopurine, and an analogue of guanine rather than hypoxanthine, there was a similar degree of modulation of the cytotoxicity of both agents by hypoxanthine. These results indicate that low, physiological hypoxanthine concentrations can significantly modulate thiopurine cytotoxicity and suggest that endogenous hypoxanthine pools may have an important effect on the clinical activity of thioguanine and mercaptopurine.

Cell Count↗
PubMed1993

In vitro deleterious effect of hypoxanthine in Ham's Nutrient Mixture F-10 culture medium on human oocyte fertilization and early embryonic development.

OBJECTIVE: To determine whether hypoxanthine in Ham's Nutrient Mixture F-10 (GIBCO, Grand Island, NY) culture medium affects murine embryo development or the outcome of patients undergoing IVF-ET. DESIGN: For the two-cell embryo bioassay, embryos from each female were equally distributed and incubated in Ham's F-10 with or without hypoxanthine supplementation for up to 72 hours. To assess the effect of hypoxanthine in human IVF-ET, oocytes, sperm, and embryos were cultured in Ham's F-10 medium with or without hypoxanthine. Fertilization and embryo cleavage were assessed at 18 and 40 hours, respectively, after insemination. SETTING: University medical research laboratory. PATIENTS, PARTICIPANTS: Nine couples undergoing IVF-ET. RESULTS: Two-cell mouse embryos incubated for 24 hours without hypoxanthine developed 40% to morula, compared with 6.5% for the hypoxanthine group. At 72 hours, 99.5% of the embryos without hypoxanthine reached the expanded blastocyst stage with 65% of them exhibiting hatching, compared with 72% and 19.5%, respectively, for the group with hypoxanthine. Human oocytes cultured in Ham's F-10 without hypoxanthine showed higher fertilization rates than the group incubated in the presence of hypoxanthine (69% versus 53%). The proportion of cleaved embryos was not different between the two culture media; however, the rate of embryos cleaving without cytoplasmic fragments was significantly higher in the group without hypoxanthine (75% versus 35%). CONCLUSION: Ham's F-10 medium containing hypoxanthine significantly reduced embryo development in the two-cell mouse embryo bioassay. Hypoxanthine in culture medium for IVF-ET may have a deleterious effect on human gametes, leading to decreased fertilization and increased incidence of cytoplasmic fragments in the conceptuses.

Adult↗
PubMed1997

Uptake and salvage of hypoxanthine mediates developmental arrest in preimplantation mouse embryos.

Preimplantation mouse embryos become arrested after first or second cleavage when cultured in hypoxanthine-supplemented Whitten's medium. We present evidence that the hypoxanthine-induced arrest is dependent on uptake and salvage of hypoxanthine and depletion of phosphoribosylpyrophosphate (PRPP) levels. Hypoxanthine uptake increased during the 2-cell stage and was augmented by glucose. HPLC analysis of [14C]hypoxanthine metabolism revealed that hypoxanthine was salvaged and converted to ATP and guanosine triphosphate (GTP), with a shift to more guanyl nucleotide production at the 3- to 4-cell stage. In embryos from mice with a null mutation for the salvage enzyme hypoxanthine-guanine phosphoribosyltransferase, hypoxanthine did not block development nor was it taken up by the embryos. Glucose, which is required for the hypoxanthine-induced arrest, produced a 5.3-fold increase in PRPP levels at the 2-cell stage, which was eliminated by hypoxanthine. We conclude that metabolism of hypoxanthine to nucleotides mediates its inhibitory action on preimplantation mouse embryos via negative feedback on PRPP synthetase, ultimately resulting in decreased PRPP availability and arrest of other PRPP-dependent pathways. Finally, reversal of the block by EDTA and cAMP-elevating agents may be mediated by alterations in hypoxanthine or glucose uptake, or by changes in the relative metabolism of hypoxanthine.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Hypoxanthine: a marker for asphyxia.

It has been hypothesized that hypoxanthine concentrations in the blood of newborn infants are a marker of asphyxia. To test this hypothesis, we measured serum hypoxanthine levels in relationship to perinatal and neonatal asphyxia, and compared arterial hypoxanthine levels with arterial pH and base deficit. We also compared hypoxanthine levels of survivors with those of asphyxiated non-survivors. Forty-two newborns were classified as asphyxiated by either of two methods: 1) Infants from whom umbilical cord hypoxanthine levels were taken were classified as asphyxiated if they had an Apgar score of 6 or less at 1 or 5 minutes, fetal heart rate below 100 beats per minute, or meconium-stained amniotic fluid; and 2) infants from whom peripheral arterial hypoxanthine samples were taken were classified by clinical assessment, whereby one author, blinded to the infants' hypoxanthine levels, prospectively assessed each patient's condition for evidence of asphyxia. Hypoxanthine levels correlated with increased base deficit (P less than .001; r = 0.8) and with decreased pH (P less than .001; r = -0.5). By both of our asphyxia classification methods, hypoxanthine levels were significantly higher (P less than .002) in the asphyxiated groups. We also noted a higher hypoxanthine level in asphyxiated non-survivors as compared with all survivors (P less than .02). We propose that serum hypoxanthine levels may help define asphyxia. Because hypoxanthine, when metabolized by xanthine oxidase, generates oxygen radicals that are highly destructive to tissue, hypoxanthine levels may have important therapeutic implications for asphyxiated patients.

Asphyxia Neonatorum↗
PubMed1981

Distinct mechanisms of hypoxanthine and inosine transport in membrane vesicles isolated from Chinese hamster ovary and Balb 3T3 cells.

Both enzyme-mediated group translocation and facilitated diffusion have been proposed as mechanisms by which mammalian cells take up purine bases and nucleosides. We have investigated the mechanisms for hypoxanthine and inosine transport by using membrane vesicles from Chinese hamster ovary cells (CHO), Balb/c 3T3 and SV3T3 cells prepared by identical procedures. Uptake mechanisms were characterized by analyzing intravesicular contents, determining which substrates could exchange with the transport products, assaying for hypoxanthine phosphoribosyltransferase activity, and measuring the stimulation of uptake of hypoxanthine by phosphoribosyl pyrophosphate (PRib-PP). We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3--4-fold by PRib-PP. The intravesicular product was predominantly IMP. The hypoxanthine phosphoribosyltransferase activity copurified with the vesicle preparation. These results suggest the possible involvement of this enzyme in hypoxanthine uptake in 3T3 vesicles. In contrast to the 3T3 vesicles, CHO vesicles prepared under identical procedures did not retain hypoxanthine phosphoribosyltransferase activity and did not demonstrate PRib-PP-stimulated hypoxanthine uptake. The intravesicular product of hypoxanthine uptake in CHO vesicles was hypoxanthine. These results and data from our kinetic and exchange studies indicated that CHO vesicles transport hypoxanthine via facilitated diffusion. An analogous situation was observed for inosine uptake; CHO vesicles accumulated inosine via a facilitated diffusion mechanism, while in the same experiments SV3T3 vesicles exhibited a purine nucleoside phosphorylase-dependent translocation of the ribose moiety of inosine. Vesicles prepared from a CHO cell line temperature-sensitive for hypoxanthine uptake (Azarts) showed a temperature-sensitivity in Km for uptake parallel to that of the intact cells. This suggests that the defect in Azarts may be caused by a missense mutation in the gene coding for the hypoxanthine transport carrier.

Animals↗
PubMed1997

Effects of hypoxemia and reoxygenation with 21% or 100% oxygen in newborn piglets: extracellular hypoxanthine in cerebral cortex and femoral muscle.

OBJECTIVE: To determine whether reoxygenation with an FIO2 of 0.21 (21% oxygen) is preferable to an FIO2 of 1.0 (100% oxygen) in normalizing brain and muscle hypoxia in the newborn. DESIGN: Prospective, randomized, animal study. SETTING: Hospital surgical research laboratory. SUBJECTS: Twenty-six anesthetized, mechanically ventilated, domestic piglets, 2 to 5 days of age. INTERVENTIONS: The piglets were randomized to control or hypoxemia groups. Hypoxemia was induced by ventilating the piglets with 8% oxygen in nitrogen, which was continued until mean arterial pressure decreased to <20 mm Hg. After hypoxemia, the piglets were further randomized to receive reoxygenation with an FIO2 of 0.21 (21% oxygen group, n = 9) or an FIO2 of 1.0 for 30 mins followed by an FIO2 of 0.21 (100% oxygen group, n = 9), and followed for 5 hrs. The piglets in the control group were mechanically ventilated with 21% oxygen (n = 8). MEASUREMENTS AND MAIN RESULTS: We measured extracellular concentrations of hypoxanthine in the cerebral cortex and femoral muscle (in vivo microdialysis), plasma hypoxanthine concentrations, cerebral arterial-venous differences for hypoxanthine, acid base balances, arterial and venous (sagittal sinus) blood gases, and mean arterial pressures. The lowest pH values of 6.91 +/- 0.11 (21% oxygen group, mean +/- SD) and 6.90 +/- 0.07 (100% oxygen group) were reached at the end of hypoxemia and then normalized during the reoxygenation period. Plasma hypoxanthine increased during hypoxemia from 28.1 +/- 9.3 to 119.1 +/- 31.9 micromol/L in the 21% oxygen group (p < .001) and from 32.6 +/0- 14.5 to 135.0 +/- 31.4 micromol/L in the 100% oxygen group (p <.001). Plasma hypoxanthine concentrations then normalized over the next 2 hrs in both groups. In the cerebral cortex, extracellular concentrations of hypoxanthine increased during hypoxemia from 3.9 +/- 2.8 to 20.2 +/- 7.4 micromol/L in the 21% oxygen group (p < .001) and from 5.9 +/- 5.0 to 25.1 +/- 7.1 micromol/L in the 100% oxygen group (p < .001). In contrast to plasma hypoxanthine, extracellular hypoxanthine in the cerebral cortex increased significantly further during early reoxygenation, and, within the first 30 mins, reached maximum values of 24.9 +/- 6.3 micromol/L in the 21% oxygen group (p < .01) and 34.8 +/- 10.9 micromol/L in the 100% oxygen group (p < .001). This increase was significantly larger in the 100% oxygen group than in the 21% oxygen group (9.7 +/- 4.7 vs. 4.7 +/- 2.6 micromol/L, p < .05). There were no significant differences between the two reoxygenated groups in duration of hypoxemia, hypoxanthine concentrations in femoral muscle, plasma hypoxanthine concentrations, pH, or mean arterial pressure. The cerebral arterial-venous difference for hypoxanthine was positive both at baseline, at the end of hypoxemia, and after 30 mins and 300 mins of reoxygenation, and no differences were found between the two reoxygenated groups. CONCLUSIONS: Significantly higher extracellular concentrations of hypoxanthine were found in the cerebral cortex during the initial period of reoxygenation with 100% oxygen compared with 21% oxygen. Hypoxanthine is a marker of hypoxia, and reflects the intracellular energy status. These results therefore suggest a possibly more severe impairment of energy metabolism in the cerebral cortex or an increased blood-brain barrier damage during reoxygenation with 100% oxygen compared with 21% oxygen in this newborn piglet hypoxia model.

Animals↗
PubMed1987

The effect of hypoxanthine on mouse oocyte growth and development in vitro: maintenance of meiotic arrest and gonadotropin-induced oocyte maturation.

The concentration of hypoxanthine in mouse follicular fluid has been estimated to be 2-4 mM, and although this concentration maintains meiotic arrest in fully grown mouse oocytes in vitro, oocyte maturation in vivo is not induced by a decrease in the concentration of this purine in follicular fluid (J. J. Eppig, P. F. Ward-Bailey, and D. L. Coleman, Biol. Reprod. 33, 1041-1049, 1985). In the present study, the effect of 2 mM hypoxanthine on oocyte growth and development in vitro was assessed and the ability of gonadotropins to stimulate oocyte maturation in the continued presence of hypoxanthine was determined. Oocyte-granulosa cell complexes were isolated from 10- to 11-day-old mice and cultured in the presence or absence of 2 mM hypoxanthine. Oocytes from 10- to 11-day-old mice are in mid-growth phase and, without further development, are incompetent of undergoing meiotic maturation. During a 12-day culture period the granulosa cell-enclosed oocytes approximately doubled in size and, regardless of the presence or absence of hypoxanthine, 50-70% developed competence to undergo germinal vesicle breakdown (GVBD). Hypoxanthine promoted the continued association of oocytes with their companion granulosa cells during the 12-day culture period, and therefore had a beneficial effect on oocyte development. Most of the oocytes that acquired GVBD competence in the absence of hypoxanthine underwent spontaneous GVBD. In contrast, 95% of the GVBD-competent oocytes were maintained in meiotic arrest by hypoxanthine. Following withdrawal of the hypoxanthine after the 12-day culture, 75% of the GVBD-competent oocytes underwent GVBD. These results show that hypoxanthine, and/or its metabolites, maintains meiotic arrest in oocytes that grow and acquire GVBD competence in vitro. Follicle-stimulating hormone (FSH), but not luteinizing hormone or human chorionic gonadotropin, induced oocyte GVBD in the continued presence of hypoxanthine. FSH stimulated oocyte maturation at a significantly (P less than 0.01) higher frequency than coculture of the granulosa cell-denuded oocytes with granulosa cells in the continued presence of hypoxanthine. FSH did not induce the maturation of denuded oocytes cocultured with granulosa cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1976

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.

Cell Membrane↗
PubMed1997

Hypoxanthine uptake at the fetal side of human placenta proceeds through a nucleobase-preferring carrier and a non-saturable process.

Uptake and metabolism of hypoxanthine by human placenta were studied using the single-circulation paired-tracer technique. In isolated cotyledons perfused through the fetal (basal) circulation, at mean pressures of 31.7 +/- 4.0 mmHg and mean flow rates maintained at 5.5 +/- 0.15 ml/min, the [3H]hypoxanthine uptake was 36 +/- 2.4 per cent (16.5 +/- 1.1 pmol/g wet weight). Hypoxanthine uptake was significantly inhibited by unlabelled (mM) hypoxanthine (0.5), adenine (0.5), guanine (0.5) and papaverine (15.0), but was unaffected by nitrobenzylthioinosine (0.01). Adenosine failed to inhibit hypoxanthine uptake. The kinetic analysis of hypoxanthine uptake showed it to be partially mediated by a saturable (apparent K(m) = 12.1 +/- 1.85 microns; Jmax = 7.1 +/- 0.52 nmol/min) and Na(+)-dependent mechanism. A greater fraction of hypoxanthine influx proceeded through a non-saturable process. Thin layer chromatographic analysis of venous perfusate after the intra-arterial injection of [3H]hypoxanthine showed a negligible degradation of nucleobase. These overall results show that hypoxanthine uptake at the fetal side of human placenta occurs by a saturable plus a non-saturable process. The carrier showed specificity for nucleobases and high affinity-low capacity for hypoxanthine. Since the fetal blood concentration of hypoxanthine is normally low, its uptake would be mediated by the high affinity transport system. Because the non-saturable mechanism can be operative at high concentrations of hypoxanthine, it may have primary importance to clear the nucleobase coming from the fetus during intrauterine hypoxia.

Adenine↗