Chinese Hamster Hypoxanthine-Guanine Phosphoribosyltransferase: PURIFICATION, STRUCTURAL, AND CATALYTIC PROPERTIES

Abstract

Abstract Hypoxanthine-guanine phosphoribosyltransferase from Chinese hamster brain, liver, and V79 tissue culture cells appears to have identical structural and catalytic properties. The enzyme has been purified 540-fold to apparent homogeneity from Chinese hamster brain. The native molecular weight is 78,000 to 85,000 determined by Sephadex G-100 column chromatography and acrylamide gel electrophoresis. The enzyme appears to consist of three subunits of molecular weight 25,000 determined by sodium dodecyl sulfate acrylamide gel electrophoresis. Electrofocusing and acrylamide gel electrophoresis demonstrate the presence of at least three isozymes. The enzyme is remarkably stable at 85° if first incubated in 1 mm 5-phosphoribosyl 1-pyrophosphate. The enzyme is active from pH 5.5 to 11 with maximum activity at pH 10. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for hypoxanthine, guanine, and phosphoribosylpyrophosphate of 0.52, 1.1, and 5.3 µm, respectively.