Isoenzymes of hypoxanthine-guanine-phosphoribosyl transferase in a family with partial deficiency of the enzyme

The isoenzymes of hypoxanthine-guanine-phosphoribosyl transferase (HGPRT; E. C. 2.4.2.8) were studied by polyacrylamide gel disc electrophoresis in the erythrocytes of a family in which there was a partial deficiency of this X-linked enzyme. Hyperuricemic males, in whom HGPRT activity was 4% of normal, were found to have a variant enzyme which had altered kinetic and electrophoretic properties. In acrylamide gel, this variant migrated about 15% faster than the normal enzyme, and its K _m for hypoxanthine was twice that of the normal. The sister of two patients had 34% of normal activity in her erythrocytes and was thought to be a heterozygote. Electrophoresis of her hemolysate yielded profiles in which there were two zones of HGPRT activity. The more slowly migrating isoenzyme behaved electrophoretically like the normal isoenzyme. The faster-migrating isoenzyme had a mobility identical to that of the variant enzyme found in hemolysates from her hyperuricemic siblings. However, in her profile the activity of the variant enzyme was three times greater than that of the HGPRT found in the boys. This increased activity appears to be due to an interaction of the variant enzyme with the normal enzyme. Electrophoresis of a mixture of normal enzyme and the variant from a hyperuricemic male yielded a profile similar to that observed in this girl and a dramatic increase in the amount of activity in the variant zone.Aided by U.S. Public Health Service Grants No. HD04608 and GM 17702 from the National Institute of Child Health and Human Development and from the National Institute of General Medical Sciences, respectively, National Institutes of Health. Presented in part at the 1971 Annual Meeting of the Western Society for Pediatric Research, Carmel, California.     

Electrophoretic properties of hypoxanthine-guanine phosphoribosyl transferase in erythrocytes of subjects with Lesch-Nyhan syndrome

Hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C. 2.4.2.8) has been studied in erythrocytes of patients with Lesch-Nyhan syndrome by polyacrylamide gel electrophoresis. The location of this enzyme in gel was determined by radiochemical assay. Inosine monophosphate (the reaction product of HGPRT with radioactive hypoxanthine and 5-phosphorylribose-1-pyrophosphate) was precipitated in the gel at the site of its formation with lanthanum chloride. The zone containing radioactive inosine monophosphate was located by continuous monitoring of mechanically fractionated gels in a scintillation spectrometer. The sensitivity of this method has permitted the detection of the very low HGPRT activity in the electropherograms of hemolysates of patients with Lesch-Nyhan syndrome. Among six patients, four had a mutant enzyme which migrated 15% faster than the normal; the other two had a mutant enzyme with about 12% faster migration. These mutants were designated HGPRT-LN and HGPRT-LN slow, respectively. These observations indicate that the mutant gene on the X chromosome codes for a protein of altered structure.Aided by U.S. Public Health Service grants Nos. HD 04608 and HD 03015 from the National Institute of Child Health and Human Development and GM 17702 from the National Institute of General Medical Sciences, National Institutes of Health.Presented in part at the 1971 meeting of the Western Society for Pediatric Research, Carmel, California.     

Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase with reduced affinity for PP-ribose-P in four related males with gout

Summary A family is described in which four affected males, spanning two generations, have hyperuricemia and gout accompanied by hematuria but are without severe neurologic involvement. The affected males were found to have markedly reduced levels of erythrocytic hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity; these were 5–12% with hypoxanthine and 0.5–3% with guanine as compared to controls. Erythrocytic adenine phosphoribosyltransferase (APRT) was approximately three-fold elevated in the affected individuals.The residual HGPRT activity in affected males enabled characterization of some of the properties of this mutation. The apparent Michaelis constants (km) for both hypoxanthine and guanine were essentially unchanged, whereas the km for PP-ribose-P was approximately 10–20-fold elevated for all four affected males. The enzyme was more sensitive to product inhibition by IMP and GMP than controls, and exhibited greater thermal lability at 65°C than found with control lysates.     

Profiles of hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase activities measured in single preimplantation human embryos by high-performance liquid chromatography

The profiles of hypoxanthine guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyl transferase (APRT) activities were examined in normally fertilized human embryos developing at the normal rate in vitro between the 2-4-cell stage on Day 2 and the blastocyst stage on Day 6 after insemination. The activities of both enzymes were assayed simultaneously in extracts of single embryos by measuring the rate of production of the reaction products, inosine monophosphate (IMP) and adenine monophosphate (AMP), separated by high-performance liquid chromatography (HPLC). The activity profiles of the two enzymes over this period showed marked differences. The activity of HGPRT, coded by the X chromosome, increased between Days 2 and 4 (P less than 0.01) but declined sharply by Day 6 (P less than 0.001), whereas autosome-coded APRT activity remained low between Days 2 and 5, but increased on Day 6 (P less than 0.05). The profile of HGPRT activity may reflect a combination of decreasing levels of maternal enzyme inherited from the oocyte and the initiation of embryonic gene expression followed by X inactivation at the blastocyst stage on Day 6.     

Adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-hamster cell hybrids.

Male New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no reduction of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.     

Heterogeneity of hypoxanthine guanine phosphoribosyl transferase from human erythrocytes.

Hypoxanthine guanine phosphoribosyl transferase (E.C.: 2.4.2.8) has been purified 4000- to 4500-fold from normal human erythrocytes by three different schemes of protein fractionation. In one scheme, the enzyme was separated by preparative polyacrylamide gel electrophoresis in an LKB Uniphor system and purified by affinity column chromatography employing Sepharose/phosphoribosyl/pyrophosphate. In the second, the enzyme was isolated by isotachophoresis in the presence of Amphiline carrier ampholytes employing a Tris/phosphate/β-alanine ion system. The enzyme was then purified by isotachophoresis in the presence of carrier ampholytes using a Tris/acetate/glycine ion system. The hypoxanthine guanine phosphoribosyl transferase purified by affinity chromatography and isotachophoresis consisted, on immunoelectrophoresis, mainly of one component and had less than 5% impurities. When subjected to analytical polyacrylamide gel electrophoresis, such preparations were resolved into four isoenzymes. In the third scheme, the enzyme was isolated by isoelectric focusing. In this system, the enzyme was also resolved into four isoenzymes. Their isoelectric points were: 5.47, 5.63, 5.74, and 5.84. When subjected to analytical polyacrylamide gel electrophoresis each isoenzyme migrated at a different rate. In sodium dodecyl sulfate gel electrophoresis each isoenzyme yielded one major and one minor band. Protein appearing in the major and minor bands migrated at rates consistent with a molecular size of 33,500 and 26,500, respectively.     

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells.

A micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of 14C hypoxanthine and 14C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines. Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.     

Transfer of purine metabolites between cells through the medium and via cell contacts in cocultures of HGPRT+ and HGPRT- cells

Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT- Chinese hamster and human fibroblasts were able to incorporate 3H-labeled purine metabolite(s) from medium in which mouse HGPRT+ B82 cells had been grown for 24 h with [3H]hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to [3H]MTA, which has been shown previously to be the major 3H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to [3H]adenine, which is phosphoribosylated by APRT to [3H]AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with [3H]hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. This is termed proximity-dependent metabolite transfer (PDMT). Both CIMT and PDMT interfered with the assessment of nucleotide exchange between HGPRT+ and HGPRT- cells through cell contacts, which is referred to as contact-dependent metabolite transfer (CDMT). These problems were unique to HGPRT+ mouse L cells. However, HGPRT- mouse L cells, A9, could be used as potential recipients. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators. CDMT could not be studied with [3H]guanine because the nuclei of HGPRT- cells became labeled.     

Developmental changes in purine phosphoribosyltransferases in human and rat tissues.

1. The hypoxanthine/guanine and adenine phosphoribosyltransferase activities in a wide variety of human tissues were studied during their growth and development from foetal life onward. A wide range of activities develop after birth, with especially high values in the central nervous system and testes. 2. Postnatal development of hypoxanthine/guanine phosphoribosyltransferase was also defined in the rat. Although there were increases in the central nervous system and testes, there was also a rise in activity in the liver, which was less marked in man. 3. A sensitive radiochemical assay method, using dTTP to inhibit 5'-nucleotidase activity, suitable for tissue extracts, was developed. 4. No definite evidence of the existence of tissue-specific isoenzymes of hypoxanthine/guanine or adenine phosphoribosyltransferase was found. Hypoxanthine/guanine phosphoribosyltransferase in testes, however, had a significantly different thermal-denaturation rate constant. 5. The findings are discussed in an attempt to relate activity of hypoxanthine/guanine phosphoribosyltransferase to biological function. Growth as well as some developmental changes appear to be related to increase in the activity of this enzyme.     

Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method

The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in a group of man-mouse somatic cell hybrids, produced by Sendai virus-mediated cell fusion and HAT selection, has been analyzed by a new electrophoretic technique. Evidence is presented which shows that the hybrid lines derived from fusion of a mouse fibroblast deficient in HGPRT with various human cell strains have an HGPRT activity that is characteristic of the human enzyme, whereas a hybrid line derived from a mouse fibroblast which is deficient in thymidine kinase has an HGPRT activity characteristic of the mouse. This new technique involves electrophoresis of cell extracts on cellulose acetate gel, followed by the localization of the enzyme activity by autoradiography.This research was supported in part by a research grant from the U.S. National Institutes of Health (No. GM-13415).     

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells

Summary A micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of ¹⁴C hypoxanthine and ¹⁴C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10 000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines.Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Enzymes of the purine interconversion system in chronic lymphatic leukemia: decreased purine nucleoside phosphorylase and adenosine deaminase activity.

Activities of adenosine deaminase (ADA), adenosine kinase (AK), adenine phosphoribosyltransferase (APRT), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP), all enzymes of the purine interconversion system, were determined in lymphocytes of 25 patients with chronic lymphatic leukemia (CLL) and in 23 controls. A statistically significant decrease of PNP activities and a reduction of ADA activities at borderline levels were found in the patients, whereas for the other enzymes assayed no deviation from normal values was observed.     

Hypoxanthine phosphoribosyltransferase activity in bovine embryos during the early embryonic development

The activity of hypoxanthine phosphoribosyltransferase (HPRT) was determined in the bovine embryo during early embryonic development. Microassay, using [(3)H] hypoxanthine, was improved to measure enzyme activity in the embryonic extract. This activity depended on the reaction time and the concentration of phosphorybosyl pyrophosphate (PRPP) in a reaction. mixture. Maximum activity was obtained at 4 hours of reaction time and at a concentration of 1 mM PRPP, but was much lower than the activity recorded in the mouse embryo. During early embryonic development, HPRT activity rapidly increased beyond the 8-cell stage. When distributions and activities of HPRT, adenine phosphorybosyltransferase (APRT), and the ratio of HPRT: APRT were examined in individual blastocysts, HPRT activity was broadly distributed, but it did not clearly show the bimodal distribution expected. Six of demi-embryos with high or low HPRT:APRT ratios were transferred to recipient cows from which 2 calves were obtained. Both offspring were of the sex predicted by the HPRT: APRT ratio. These results indicate that HPRT activity of bovine preimplantation embryos can be microassayed using radiolabeled hypoxanthine, and this assay could provide an alternative method for embryo sexing.     

Rapid method for the diagnosis of partial adenine phosphoribosyltransferase deficiencies causing 2,8-dihydroxyadenine urolithiasis

Summary More than half of the Japanese patients with 2,8-dihydroxyadenine urolithiasis only partially lack adenine phosphoribosyltransferase (APRT), while all the Caucasian patients with the same disease completely lack the enzyme. APRT activities in healthy heterozygotes for the complete APRT deficiencies were at the same levels as the Japanese patients, and simple enzyme assay does not distinguish between these two conditions. We have previously shown, using viable T-cells, that the enzyme was non-functional in the cells from the Japanese patients although they contain considerable APRT activities in the cell extracts. In the present investigations, we devised a rapid method using erythrocytes for the diagnosis of partial APRT deficiencies accompanied by severe impairment in adenine metabolism causing 2,8-dihydroxyadenine lithiasis. Thus, erythrocytes from three different families with 2,8-dihydroxyadenine urolithiasis associated with partial APRT deficiencies incorporated only minimal amounts of radioactive adenine, while normal erythrocytes incorporated significant amounts. These data indicate that severe impairment in adenine metabolism is shown not only in viable T-cells but also in viable erythrocytes. The present procedures provide a rapid method suitable for routine clinical use for the diagnosis of partial APRT deficiencies causing 2,8-dihydroxyadenine lithiasis.     

Acyclovir transport into human erythrocytes

The mechanism of transport of the antiviral agent acyclovir (ACV) into human erythrocytes has been investigated. Initial velocities of ACV influx were determined with an "inhibitor-stop" assay that used papaverine to inhibit ACV influx rapidly and completely. ACV influx was nonconcentrative and appeared to be rate-saturable with a Km of 260 +/- 20 microM (n = 8). However, two lines of evidence indicate that ACV permeates the erythrocyte membrane by means other than the nucleoside transport system: 1) potent inhibitors (1.0 microM) of nucleoside transport (dipyridamole, 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and dilazep) had little (less than 8% inhibition) or no effect upon the influx of 5.0 microM ACV; and 2) a 100-fold molar excess of several purine and pyrimidine nucleosides had no inhibitory effect upon the influx of 1.0 microM ACV. However, ACV transport was inhibited competitively by adenine (Ki = 9.5 microM), guanine (Ki = 25 microM), and hypoxanthine (Ki = 180 microM). Conversely, ACV was a competitive inhibitor (Ki = 240-280 microM) of the transport of adenine (Km = 13 microM), guanine (Km = 37 microM), and hypoxanthine (Km = 180 microM). Desciclovir and ganciclovir, two compounds related structurally to ACV, were also found to be competitive inhibitors of acyclovir influx (Ki = 1.7 and 1.5 mM, respectively). These results indicate that ACV enters human erythrocytes chiefly via the same nucleobase carrier that transports adenine, guanine, and hypoxanthine.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

Characterization of the biochemical basis of a complete deficiency of the adenine phosphoribosyl transferase (APRT)

Summary In order to study the biochemical basis of a complete deficiency of adenine phosphoribosyl transferase (APRT) the enzyme was purified to homogeneity, its properties were characterized, and antibodies raised. The enzyme is indirectly involved in adenine uptake. Apparently, by forming AMP the internal concentration of adenine is kept low allowing its diffusion.The same APRT is present in various tissues as was revealed by antibody inactivations employing anti-erythrocyte APRT as well as by direct enzyme assays in cells from the APRT deficient patient. In vitro cultured fibroblasts derived from this patient had less than 0.02% enzyme activity. No cross-reacting material was found in erythrocytes obtained from an APRT deficient child.     

Purine mutants of mammalian cell lines: III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells.

Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.