Studies of an unusually basic hypoxanthine-guanine phosphoribosyltransferase.
Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) from beef brain has been purified 3100-fold to apparent homogeneity using a purification procedure based on GMP-Sepharose affinity chromatography. The native enzyme has a molecular weight of 84,000 as determined by gel filtration studies. A subunit molecular weight of 26,000 was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme is a trimer. Two forms of the enzyme have been separated by nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Basic pI values of 7.85 and 8.10 were obtained for the two forms. These values are much higher than have been observed with any other purified phosphoribosyltransferase. The amino acid composition of the enzyme is 18 Lys, 6 His, 9 Arg, 1 Trp, 6 Cys, 28 Asx, 12 Thr, 16 Ser, 19 Glx, 10 Pro, 23 Gly, 16 Ala, 17 Val, 5 Met, 11 Ile, 19 Leu, 9 Tyr, and 8 Phe. An unusual basic amino acid, yet to be identified, was also present. The enzyme exhibits Km values of 0.42 microM for guanine, 0.99 microM for hypoxanthine, 18.6 microM for P-Rib-PP in the presence of guanine, and 2.9 microM for P-Rib-PP in the presence of hypoxanthine.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D007041:Hypoxanthine Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight
D000596:Amino Acids; D046911:Macromolecular Substances; D007041:Hypoxanthine Phosphoribosyltransferase