Facilitated Purification of Hypoxanthine Phosphoribosyltransferase

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Abstract
Three major approaches to the complete purification of hypoxanthine phosphoribosyltransferase [EC 2.4.2.8] from human erythrocytes and rat brain are described. Preparative isolelectric focusing which was used for the isolation of the human enzyme was not fully successful in the case of rat brain. Preparative polyacrylamide-gel electrophoresis in gel blocks yields enzyme samples of high purity as judged by analytical gel electrophoresis, but with a comparatively low specific enzyme activity. The most rapid and convenient method, a modification of the affinity chromatography on GMP agarose 1st described by Hughes gives hypoxanthine phosphoribosyltransferase which is superior to the other preparations in its homogeneity and its specific activity. All 3 methods produce an identical enzyme protein detected by polyacrylamide electrophoresis on nondenaturing and sodium dodecylsulfate gels. Molecular data of hypoxanthine phosphoribosyltransferase derived from these studies are: isoelectric points of 5.60; 5.85 and 5.90 for 3 isozyme peaks of the rat brain enzyme; and a MW of 72,000 for the native rat brain enzyme and of 25,000-27,000 for the subunit of human and rat enzyme. Guanylate kinase does not interfere with the purification of hypoxanthine phosphoribosyltransferase on GMP agarose and is itself partially purifiedl by this chromatography.