Abstract
Hypoxanthine-guanine phosphoribosyltransferase is a ubiquitous human enzyme, the inherited deficiency of which leads to a specific metabolic-neurological syndrome. Native acrylamide isoelectric focusing revealed that the human enzyme consists of different numbers of isoenzymes depending on the tissue of origin. The erythrocytic enzyme has the most isoenzymes while the enzyme from cultured fibroblasts has only a single isoenzyme. The isoenzyme pattern of the erythrocytic enzyme changes on storage of the crude hemolysate at 4 C. Treatment of the stored crude hemolysate with 4.5 m urea and 0.35 mm β-mercaptoethanol results in an isoenzyme pattern similar to that of the fresh crude extract. Thus the additional isoenzymes are generated on storage not by covalent modification of the enzyme but probably by binding of small molecules to the enzyme or to association of the enzyme molecules. Hypoxanthine-guanine phosphoribosyltransferase has been purified to 80% homogeneity in three steps, DEAE Sephadex chromatography, heat treatment at 85 C for 5 min, and hydroxylapatite chromatography. Denaturing two-dimensional gel electrophoresis of the erythrocytic enzyme revealed that the erythrocytic enzyme is composed of three major types of subunits (1–3) with the same molecular weight but different isoelectric points. In contrast, the fibroblast enzyme is composed of only a single type of subunit, which comigrates with subunit 1 of the erythrocytic enzyme. Since there is a single genetic locus in humans for HGPRTase (the enzyme is X linked) (Nyhan et al., 1967), the observed subunit modification of the erythrocyte enzyme appears to be the result of posttranslational modification. These findings provide a simple explanation for the observed electrophoretic properties of human HGPRTase. A patient with 0.5% of HGPRTase activity in his erythrocytes was found to have small amounts (> 0.5% but < 5% of normal) of the erythrocytic HGPRTase subunits.
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References
Bakay, B., and Nyhan, W. L. (1971). The separation of adenine and hypoxanthine-guanine phosphoribosyl transferase isoenzymes by disc gel electrophoresis. Biochem. Genet. 581.
Chasin, L. A., and Urlaub, G. (1976). Mutant alleles for hypoxanthine phosphoribosyltransferase: Codominant expression, complementation, and segregation in hybrid Chinese hamster cells. Somat. Cell Genet. 2453.
Davies, M. R., and Dean, B. M. (1971). The heterogeneity of erythrocyte IMP: Pyrophosphate phosphoribosyl transferase and purine nucleoside phosphorylase by isoelecric focusing. FEBS Lett. 18283.
Ghangas, G. S., and Milman, G. (1975). Radioimmune determination of hypoxanthine phosphoribosyltransferase crossreacting material in erythrocytes of Lesch-Nyhan patients. Proc. Natl. Acad. Sci. USA 724147.
Ghangas, G. S., and Milman, G. (1977). Hypoxanthine phosphoribosyltransferase: Two-dimensional gels from normal and Lesch-Nyhan hemolyzates. Science 1961119.
Gudas, L. J., Zannis, V. I., Clift, S. M., Ammann, A. J., Staal, G. E. J., and Martin, D. W. Jr. (1978). Characterization of mutant subunits of human purine nucleoside phosphorylase. J. Biol. Chem. 2538916.
Hughes, S. H., Wahl, G. M., and Capecchi, M. R. (1975). Purification and characterization of mouse hypoxanthine-guanine phosphoribosyltransferase. J. Biol. Chem. 250120.
Kelley, W. N., and Wyngaarden, J. B. (1974). Enzymology of gout. C. Hypoxanthine-guanine phosphoribosyltransferase. Advan. Enzymol. 4114.
Krenitsky, T. A., Papaioannou, R., and Elion, G. B. (1969). Human hypoxanthine phosphoribosyltransferase. I. Purification, properties, and specificity. J. Biol. Chem. 2441263.
Lesch, M., and Nyhan, W. L. (1964). A familial disorder of uric acid metabolism and central nervous system function. Am. J. Med. 36561.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193265.
Martin, D. W., Jr., and Maler, B. (1976). Phosphoribosylpyrophosphate synthetase is elevated in fibroblasts from patients with the Lesch-Nyhan syndrome. Science 193408.
Migeon, B. R., Der Kaloustian, V. M., Nyhan, W. L., Young, W. J., and Childs, B. (1968). X-linked hypoxanthine-guanine phosphoribosyl transferase deficiency: heterozygote has two clonal populations. Science 160425.
Milman, G., Lee, E., Ghangas, G. S., McLaughlin, J. R., and George, M. (1976). Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: Evidence for silent gene action. Proc. Natl. Acad. Sci. USA 734589.
Nyhan, W. L., Pesek, L., Sweetman, L., Carpenter, D. G., and Carter, C. H. (1967). Genetics of an X-linked disorder of uric acid metabolism and cerebral function. Pediat. Res. 15.
O'Farrell, P. J. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 2504007.
Olsen, A. S., and Milman, G. (1974a). Chinese hamster hypoxanthine-guanine phosphoribosyltransferase. J. Biol. Chem. 2494030.
Olsen, A. S., and Milman, G. (1974b). Subunit molecular weight of human hypoxanthine-guanine phosphoribosyltransferase. J. Biol. Chem. 2494038.
Ricciutti, F., and Ruddle, F. H. (1973). Assignment of nucleoside phosphorylase to D-14 and localization of X-linked loci in man by somatic cell genetics. Nature New Biol. 241180.
Rosenbloom, F. M., Kelley, W. N., Miller, J., Henderson, J. F., and Seegmiller, J. E. (1967). Inherited disorder of purine metabolism: Correlation between central nervous system dysfunction and biochemial defects. J. Am. Med. Assoc. 202175.
Rubin, C. S., Dancis, J., Yip, L. C., Nowinski, R. C., and Balis, M. E. (1971). Purification of IMP: Pyrophosphate phosphoribosyltransferases, catalytically incompetent enzymes in Lesch-Nyhan disease. Proc. Natl. Acad. Sci. USA 681461.
Seegmiller, J. E., Rosenbloom, F. M., and Kelley, W. N. (1967). Enzyme defect associated with a sex-linked human neurological disorder and excessive purine synthesis. Science 1551682.
Shin, S., Khan, P. M., and Cook, P. R. (1971). Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method. Biochem. Genet. 591.
Steinberg, R. A., O'Farrell, P. H., Friedrich, U., and Coffino, P. (1977). Mutations causing charge alterations in regulatory subunits of the cAMP-dependent protein kinase of cultured S49 lymphoma cells. Cell 10381.
Turner, B. M., Fisher, R. A., and Harris, H. (1971). An association between the kinetic and electrophoretic properties of human purine-nucleoside-phosphorylase isozymes. Eur. J. Biochem. 24288.
Upchurch, K. S., Leyva, A., Arnold, W. J., Holmes, E. W., and Kelley, W. N. (1975). Hypoxanthine phosphoribosyltransferase deficiency: Association of reduced catalytic activity with reduced levels of immunologically detectable enzyme protein. Proc. Natl. Acad. Sci. USA 724142.
Uy, R., and Wold, F. (1977). Posttranslational covalent modification of proteins. Science 198890.
Zannis, V. I., Doyle, D., and Martin, D. W., Jr. (1978). Characterization of human erythrocyte purine nucleoside phosphorylase and its subunits. J. Biol. Chem. 253504.
Zannis, V. I., Gudas, L. J., and Martin, D. W., Jr. (1979). Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts. Biochem. Genet. 17621.
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This work was supported by a grant from NIAMDD, National Institutes of Health, United States Public Health Service. L. J. G. was supported by a fellowship from the National Institute of Child Health and Human Development. D. W. M. is an Investigator, Howard Hughes Medical Institute.
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Zannis, V.I., Gudas, L.J. & Martin, D.W. Characterization of the subunit composition of HGPRTase from human erythrocytes and cultured fibroblasts. Biochem Genet 18, 1–19 (1980). https://doi.org/10.1007/BF00504356
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DOI: https://doi.org/10.1007/BF00504356