Hypoxanthine Phosphoribosyltransferase Activity Research Articles

Use of selective media for distinguishing variant forms of hypoxanthine phosphoribosyl transferase

Growth properties of cultured fibroblasts in selective media were used to characterize the HPRT enzyme of a patient with a new variant of hypoxanthine phosphoribosyl transferase (HPRT) with deficient activity. The clinical phenotype of the patient was typical of the Lesch-Nyhan syndrome. However, cells of the patient were not selected for by growth in either 8-azaguanine or 6-thioguanine. Assay of the activity of the enzyme in erythrocyte lysates revealed values of approximately zero, while in the intact fibroblast assay the level of activity was 1.4% of normal. The heterozygous mother of the patient, unlike heterozygotes for the classic Lesch-Nyhan enzyme, had a level of activity in erythrocyte lysates that was 45% of control. In the presence of selective agents in vitro the cells of the patient retained sufficient HPRT activity to permit a degree of toxicity indistinguishable from that observed in normal cells although the degree of the deficiency was so great that it led to the complete Lesch-Nyhan phenotype. These findings call into question the use of selective agents for the identification of HPRT- cells in the detection of heterozygosity.

Simultaneous Determination for Adenine-Phosphoribosyltransferase and Hypoxanthine-Phosphoribosyltransferase Activities in Human Erythrocytes by High-Performance Liquid Chromatography

Simultaneous Determination for Adenine-Phosphoribosyltransferase and Hypoxanthine-Phosphoribosyltransferase Activities in Human Erythrocytes by High-Performance Liquid Chromatography

Subcellular localisation of purinemetabolising enzymes in Leishmania mexicana mexicana

1. 1. Leishmania mexicana mexicana cultured promastigotes were fractionated by isopycnic centrifugation on linear sucrose gradients. 2. 2. Guanine, hypoxanthine and xanthine phosphoribosyltransferase activities were found to be associated with glycosomes, whereas adenine phosphoribosyltransferase was cytosolic. 3. 3. 3′- and 5′-nucleotidases and IMP dehydrogenase were shown to be particulate, the former two possibly being associated with the plasma membrane, IMP dehydrogenase with the endoplasmic reticulum. 4. 4. Nucleosidases and deaminases were found to be cytosolic. 5. 5. The results demonstrate that intracellular separation of enzymes could play a part in the regulation of the parasite's purine metabolism.

FIRST-TRIMESTER DIAGNOSIS OF LESCH-NYHAN SYNDROME

Chorionic villi were sampled at 8-9 weeks' gestation in four women whose fetuses were at risk for Lesch-Nyhan syndrome. Radiochemical assay of hypoxanthine phosphoribosyl transferase activity and fetal sexing (in two fetuses by means of a Y chromosome specific cDNA probe) showed that three fetuses were affected. The biochemical findings were confirmed after therapeutic abortion in two cases and spontaneous abortion in the third. Chorion biopsy and ultramicroscale enzymology may be a suitable alternative to recombinant-DNA-based methods for first-trimester diagnosis in selected diseases where the abnormal gene product is an enzyme expressed in the chorion by the 8th week of pregnancy.

Studies in fibroblasts of patients with the Lesch-Nyhan syndrome and HPRT variants. Correlation of HPRT activity with hypoxanthine utilization and growth in selection media.

In the present study, hypoxanthine phosphoribosyltransferase (HPRT) has been investigated in fibroblasts of 19 patients from 16 different families with HPRT deficiency, concerning activity, incorporation of 14C-hypoxanthine, and growth in 8-azaguanine and HAT (hypoxanthine, azaserine, thymidine containing) selection media. According to these data we could classify the patients into 5 groups (patients with classical Lesch-Nyhan syndrome and patients with HPRT variants of types A, B, C, D). In 3 groups (patients with classical Lesch-Nyhan syndrome, HPRT variants C and D), a correlation of residual HPRT activity with the incorporation of 14C-hypoxanthine as well as growth in 8-azaguanine and HAT selection was observed. The variant A, from a patient with the classical Lesch-Nyhan syndrome, exhibited higher HPRT activity than that from all the other patients with the Lesch-Nyhan syndrome. However, the values of hypoxanthine incorporation and growth in selection media were as in the classical syndrome. The cells of variant B were resistant to azaguanine and grew in HAT selection media in the range of control cells, but had HPRT residual activities similar to those of variants A and C. For the characterization of the genetic heterogeneity of HPRT, it seems necessary to study the enzymatic properties in cell extracts as well as the purine uptake and proliferation of cells in different selection media.

Purine synthesis and salvage in brain and liver.

Previous work in which we measured the specific activities of amidophosphoribosyltransferase (PRPP-At; EC 2.4.2.14) and hypo-xanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) in the brains of rats at different ages suggested that the HPRT-catalysed purine salvage pathway became more important relative to the purine de novo synthesis pathway after the main bursts of neuroblast and neuroglial proliferation.1 This interpretation involves the assumption that changes in the specific activity of PRPP-At, which catalyses the first and presumably ratelimiting reaction on the purine de novo synthesis pathway, are a valid measure of changes in the flow of metabolites along the whole pathway. The evidence2 that the cyclic nucleotides (cAMP and cGMP) have important second messenger functions in the central nervous system, some of which are related to post-synaptic neurotransmission, and which presumably become more important as neuronal function increases, suggested to us that HPRT might have a specific function in maintaining the supply of a neuropharmacologically important low molecular weight purine derivative which could be cGMP. We also suggested that a failure of this mechanism might be responsible for the functional disorders of the Lesch-Nyhan syndrome3.

Purine metabolism in myeloid precursor cells during maturation. Studies with the HL-60 cell line.

In studies with the human promyelocytic leukemia cell line HL-60, we defined changes in intermediary purine metabolism that appear to contribute to the regulation of terminal maturation in myeloid cells. When HL-60 cells were exposed to compounds that induce maturation, consistent alterations in purine metabolism were found to occur within 24 h of culture. Perturbation of guanosine nucleotide synthesis and decreases of up to 50% in intracellular guanylate pool sizes were associated with the induced maturation of these cells in response to diverse inducing agents. While immature HL-60 cells were observed to synthesize purine nucleotides by both de novo and salvage pathways, the activity of both pathways decreased in cells induced to mature, although the relative contribution of purine salvage increased. Moreover, incorporation of the salvage pathway precursor, [14C]hypoxanthine from the intermediate, inosine monophosphate (IMP), into guanylates was reduced by approximately 65% in induced HL-60 cells, reflecting decreased activity of both hypoxanthine phosphoribosyltransferase and IMP dehydrogenase. When various inhibitors of IMP dehydrogenase (mycophenolic acid, 3-deazaguanosine, and 2-beta-D-ribofuranosylthiazole-4-carboxamide) were evaluated for their effects upon HL-60 cells, each agent was found to induce the cells to mature morphologically and functionally. Like other inducers, these agents decreased HL-60 cell proliferation and caused the cells to acquire an ability to phagocytose opsonized yeast and reduce nitroblue tetrazolium. Each agent reduced intracellular guanosine nucleotide pool sizes and induced HL-60 cell maturation at micromolar concentrations. These observations suggest that the size of intracellular guanosine nucleotide pools, the biosynthesis of guanosine nucleotides, and the activity of IMP dehydrogenase may be central to the regulation of terminal maturation in myeloid cells.

Expression of maternally and embryonically derived hypoxanthine phosphoribosyl transferase (HPRT) activity in mouse eggs and early embryos.

X-chromosome activity in early mouse development has been studied by a gene dosage method that involves measuring the activity level of the X-linked enzyme hypoxanthine phosphoribosyl transferase (HPRT) in single eggs and embryos from XO females and from females heterozygous for In(X)1H, a paracentric inversion of the X chromosome. The HPRT activity in oocytes increased threefold over a 24-hr period beginning after ovulation. Afterward, the activity plateaued in unfertilized eggs but continued to increase for at least 66 hr in presumed OY embryos. Both before and after ovulation, the level of activity in unfertilized eggs from In(X)/X females was twice that from XO females, and the distributions of activity in eggs for both sets of females remained unimodal. Beginning with the two-cell stage, distributions of activity for embryos from In(X)/X females were trimodal, which is evidence for embryonic activity. It is proposed that activation of a maternal mRNA or proenzyme is responsible for the HPRT activity increase in oocytes and early embryos and is supplemented by dosage-dependent activity of the embryonic Hprt gene as early as the two-cell stage.

Some regulatory and integrative aspects of purine nucleotide biosynthesis and its control: An overview

The regulation and integration of purine nucleotide biosynthesis is considered from the viewpoint of the main groups of reaction sequences involved and with respect to some specific organs and tissues. Inhibiting either IMP dehydrogenase or adenylosuccinate synthetase in rat liver in vitro reduced the rate of purine do novo synthesis with respect to the purine remaining in the tissue and did not materially affect the rate with respect to the purines extruded into the incubation medium. These results are considered in contrast to the results of previous studies in cultured lymphoblasts. The relative activities of purine de novo synthesis and of purine salvage have been assessed in different tissues by the activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase (HPRT), respectively. Changes in purine de novo synthesis as measured by [ 14C]formate incorporation into cellular purines were reflected in the amidophosphoribosyltransferase activities. The capacity of different tissues to synthesize purines de novo is widespread and the role of the liver as the main site of purine de novo synthesis in vivo and exporting purines to other tissues appears questionable. Regulatory mechanisms may well be tissue specific. The age-related changes in the activity of the purine de novo synthesis and purine salvage pathways, respectively, in the brain suggest that it is physiological or neuropharmacological functions of the developed brain rather than cell division and organogenesis which require a high level of purine salvage relative to purine de novo synthesis. This is compatible with the observation that purine de novo synthesis alone can meet the needs for additional purine nucleotides which lectin induced lymphocyte transformation involves. The mechanism whereby purine de novo synthesis is initiated during lectin induced lymphoblast transformation remains obscure.

Clinical, Post-mortem, Biochemical and Therapeutic Observations on the Lesch-Nyhan Syndrome with Particular Reference to the Neurological Manifestations

SUMMARY This paper reports our 10 years experience in the management of patients with the Lesch-Nyhan syndrome. It is based on the detailed study of eight patients, the longest period of follow-up being about eight years. The usual clinical descriptions have emphasized spasticity with pyramidal tract signs, choreoathetosis, compulsive self-injurious behaviour and mental handicap as the outstanding features of the syndrome. We report the development of a cervical cord lesion in two cases and the modification of the clinical phenotype which this complication produces. We have not encountered evidence of a pyramidal tract lesion except in association with this complication. In our experience, a generalized muscle hypotonia which is present from infancy is the basic motor abnormality on which torsion dystonia with its two components of abnormal posturing and episodic rigidity are superimposed. The apparent degree of mental handicap may be affected by the extreme disorder of expressive motor function, which exceeds the comprehension defect, by lack of basic social and educational opportunities and by lack of intelligence tests suitable for older children who have lacked these opportunities. Detailed histopathological and electron microscopic examination of the brain in one case showed no abnormalities. None of the 13 regions of the brain examined showed residual hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) activity but phosphoribosylpyrophosphate amidotransferase (PRPP-At; EC 2.4.2.14) activity was consistently present and of similar activity to that observed in a non HPRT-deficient control brain. A wide range of other tissues showed a similar enzymological picture. Computerized cranial tomography was normal in the three patients examined. The electroencephalographic findings were also normal in the three patients studied in this way. Neither haloperidol, pimozide, diazepam, nor a combination of 5-hydroxytryptophan with clomipramine and carbidopa influenced significantly the severity of either the torsion dystonia or the compulsive self injurious behaviour. Levodopa with carbidopa ( Sinemet ) increased the torsion dystonia in the one patient to whom it was given. The bearing of these observations on the practical management of the patients and on our understanding of the pathogenesis of the neurological manifestations of HPRT deficiency are discussed.

X-chromosome activity in the germ cells of sex-reversed mouse embryos.

Germ cells were isolated from XX ovaries and XY and XX Sex-reversed (Sxr/+) testes of mouse embryos 14-16 days post coitum, and the activity of an X-chromosome-coded enzyme, hypoxanthine phosphoribosyl transferase (HPRT), relative to an autosomal one, adenine phosphoribosyl transferase was determined. The ratio of enzyme activities in XX Sxr/+ prospermatogonia was significantly higher than that in XY prospermatogonia, up to 2-fold, suggesting that the silent X chromosome is reactivated in XX male germ cells before birth, as it is in female germ cells. The ratio was several times higher still in XX oocytes than in XX prospermatogonia, confirming that the increase in HPRT activity reported in oocytes is only partly due to an X-chromosome dosage effect.

Protein variations associated with Lesch-Nyhan syndrome.

Patients having Lesch--Nyhan syndrome were studied by using enzymatic, immunologic, and two-dimensional electrophoretic techniques. Four hundred proteins were analyzed on each two-dimensional electrophoretogram for positional or quantitative variation. In autoradiograms of lymphocytes stimulated with phytohemagglutinin, there were 11 quantitative differences found in all patients that were significant at the 2P less than 0.01 level. A significant quantitative difference was also found in an analysis of silver-stained gels of unstimulated lymphocytes. Patients had trace amounts of erythrocyte hypoxanthine phosphoribosyl transferase (HPRT) activity and trace or no immunoprecipitable HPRT. However, HPRT was observed in silver-stained erythrocyte electrophoretograms and in autoradiograms from phytohemagglutinin-stimulated lymphocytes. Unstimulated lymphocytes contained 65% of the control HPRT concentration. Currently, the technology of two-dimensional electrophoresis detects a fraction of the total cellular proteins and defective proteins may not show electrophoretic alterations. However, specific secondary changes in other polypeptides may be observed and, when catalogued, will serve as an aid in the diagnosis and understanding of the pathophysiology of metabolic diseases.

Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in chinese hamster V79 cells by tritium suicide

Tritium suicide was shown to be highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [ 3H]hypoxanthine for 5 or 10 min, followed by storage of 3H-labelled cells at −70°C for 4–10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [ 3H]hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent K m for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine.

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 ( P Rib-PP ). We found that the uptake of hypoxanthine in Balb 3T3 vesicles was stimulated 3–4-fold by P Rib-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 P Rib-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.

Adenovirus-induced mutations at the hypoxanthine phosphoribosyltransferase locus of Chinese hamster cells.

Hpt-13 is a Chinese hamster cell line deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) and sensitive to a medium containing 10(-4) M hypoxanthine, 5.5 X 10(-6) M aminopterin, and 10(-4) M thymidine. In this cell line there is a high incidence of cells resistant to this selective medium after an incubation with either ethyl methane sulfonate or adenovirus type 2 complete virions or their incomplete particles. The rate of reversion in the presence of these agents was 34-fold higher with ethyl methane sulfonate and 2.5- to 5.6-fold higher with adenovirus particles than the spontaneous rate of reversion. The revertant phenotypes were stable for many generations without selective pressure. All of the revertants tested recovered the hypoxanthine phosphoribosyltransferase activity. Most of them, however, carried an enzyme of lower activity and faster electrophoretic mobility than that of the wild type. The preferential reversion to this type of enzyme was observed among spontaneous revertants as well as among those induced by mutagenesis with ethyl methane sulfonate or exposure to viral particles. Our results suggest that adenovirus particles and ethyl methane sulfonate induce mutations at the hpt locus of Hpt-13 cells through similar mechanisms.

Gene for monoamine oxidase type A assigned to the human X chromosome

Inherited variations in monoamine oxidase (MAO) activity are thought to affect human behavior and expression of disease. The present study has established the chromosomal location of one of the structural genes coding for this enzyme. Mapping was carried out by somatic cell hybridization between normal human skin fibroblasts and mouse neuroblastoma cells. Selective media for growth of cells with or without hypoxanthine phosphoribosyltransferase (HPRT) activity were used to obtain hybrid lines which had retained or lost the human X chromosome, respectively. Cytogenetic techniques, isozyme analysis, and limited proteolysis and peptide mapping of [3H]pargyline-labeled MAO were used to characterize hybrid lines. With one exception, only lines containing the human X chromosome and human forms of two X-linked enzymes (phosphoglycerate kinase and glucose-6-phosphate dehydrogenase) expressed the human form of the flavin polypeptide of type A MAO. The exceptional hybrid line contained a putative translocation of part of the human X chromosome, since it expressed human forms of both MAO and phosphoglycerate kinase but neither the human form of glucose-6-phosphate dehydrogenase nor HPRT activity. This evidence indicates that the structural gene for the flavin polypeptide of MAO-A is on the human X chromosome. This represents the first chromosomal assignment of a human gene coding for an enzyme of neurotransmitter metabolism. This information will help to elucidate the structure of MAO and modes of its inheritance in the human population.

Specific resistance to 8-azaguanine in cells with normal hypoxanthine phosphoribosyltransferase (HPRT) activity: the role of guanine deaminase.

The role of guanine deaminase in selective cellular resistance to 8-azaguanine was examined, using eight mammalian cell lines and their subclonal derivatives isolated on the basis of increasing resistance to this drug. 8-Azaguanine and 6-thioguanine are synthetic analogs of guanine and are lethal to cells with normal hypoxanthine phosphoribosyltransferase (HPRT) activity. In principle, however, HPRT-positive cells could become selectively resistant to 8-azaguanine if, by any mechanism, the cells expressed higher levels of guanine deaminase. This is because 8-azaguanine, but not 6-thioguanine, is converted by this enzyme to a noncytotoxic metabolite, 8-azaxanthine. Our study shows that HPRT-positive cells inherently resistant to relatively high levels of 8-azaguanine contain high levels of guanine deaminase. In general, guanine deaminase activity was higher in 8-azaguanine-resistant cells, regardless of their HPRT activity. Our results support the view that elevated guanine deaminase activity constitutes a potential mechanism of selective 8-azaguanine resistance in cells with normal HPRT activity. Guanine deaminase levels were significantly elevated in HPRT-positive cells briefly exposed to sublethal concentrations of 8-azaguanine, but this elevation was transient. Long-term exposure of cells to increasingly higher levels of the drug did not lead to high stable levels of guanine deaminase, indicating that 8-azaguanine is not an inducer of guanine deaminase in the cells examined.

Immunochemical identification of the chick HPRT gene transferred from chick erythrocytes to mammalian somatic cells.

Immunochemical methods were used to identify the genetic origin of hypoxanthine phosphoribosyltransferase (HPRT) expressed in heteroploid, HPRT-deficient mouse (A9) cells and Chinese hamster ovary (K627) cells, after these cells were fused with chick embryo erythrocytes and selected for resistance to hypoxanthine-aminopterin-thymidine (HAT) medium. All of the HAT-selected clones produced HPRT activity which was immunoprecipitable by an antiserum specific for chick HPRT, but not by an antiserum specific for mouse and hamster HPRT. Furthermore, the HPRT activity in these clones was electrophoretically indistinguishable from chick liver HPRT and clearly different from mouse liver HPRT. These data provide evidence that the HPRT activity expressed in cell hybrids produced by the fusion of HPRT-negative mammalian cells and chick erythrocytes containing genetically inactive nuclei is indeed coded by the chick HPRT gene and that an avian gene can be stably incorporated and correctly expressed in a mammalian cells.

Subcellular distribution of PRibPP synthetase activity of rat intestinal mucosa.

5-Phosphoribosyl 1-pyrophosphate synthetase (PRibPP synthetase EC 2.7.6.1) isolated from rat intestinal mucosa was found to be membrane associated. The subcellular distribution of PRibPP synthetase activity seems to parallel that of gamma-glutamyl transpeptidase, indicating it to be in the brush border. The tip cells of rat intestinal mucosa were richer in PRibPP synthetase than the crypt cells. Chromatography of a Triton-solubilized particulate fraction unmasked a peak of hypoxanthine phosphoribosyltransferase activity that was not detectable before. The activity, too, was concentrated in the brush border. The coexistence of these two activities in the fraction of the bowl involved in absorption has led to the suggestin that the synthetase and phosphoribosyl-transferase are part of a coupled transport system.

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.