Evidence against the existence of real isozymes of hypoxanthine phosphoribosyltransferase.

A method for reducing the degree of heterogeneity in the electrophoretic enzyme activity pattern of hypoxanthine phosphoribosyltransferase preparations by incubation with a (magnesium) phosphoribosyl diphosphate substrate is described. Hypoxanthine phosphoribosyltransferase was isolated from human erythrocytes and Chinese hamster livers. A subunit molecular weight of 26000--27000 as reported by other authors was obtained for both enzymes by gel electrophoresis in the presence of dodecylsulfate. Gradient gel electrophoresis revealed that the native enzymes mainly have a molecular weight of 105000--110000 and are thus apparently tetrameric, when held in the active state by the presence of phosphoribosyl diphosphate. The dimeric enzyme with a molecular weight of 52000--55000, was also found under other conditions. The trimer occurred only in the absence of phosphoribosyl diphosphate, for instance by glycerol gradient centrifugation. The enzyme from human erythrocytes was partly degraded during purification in the absence of a protease inhibitor. The purified enzyme has a very low protease contamination level. Proteolysis is an additional cause of heterogeneity and might therefore explain earlier conflicting results. Since the heterogeneous nature of hypoxanthine phosphoribosyltransferase is caused only by the secondary processes of dissociation/association and, in the case of the human erythrocyte enzyme, degradation, we suggest that the use of the term 'isozyme' to describe the different forms should be avoided.