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 26 000–27 000 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 105 000–110 000 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 52 000–55 000, 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.
1) Efficient separation of the proteins from rat liver ribosomes can by achieved by two-dimensional polyacrylamide gel electrophoresis. Complete separation of all components, however, is not possible with one system only. Comparison of the results obtained with different systems suggests further heterogeneity of S15, L22, L28, L33 and L35 and enables identification of S15a, S15b, L22a, L22b, L28a, L28b, L33, L33a, L35a and L36b. 2) Ribosomal proteins were substituted with iodoacetamide prior to electrophoresis or handled in all steps of the procedure in the presence of reducing agents. These procedures prevent the formation of oxidation products described erroneously as ribosomal proteins S5, S6, L15, L17 and L32 in earlier papers. 3) Estimation of the molecular weights was performed by two-dimensional separation of the small and large subunit proteins using sodium dodecyl sulphate in the second dimension. The positions of the 70 basic proteins in the 2-D patterns were identified. 4) The small and large subunit proteins have molecular weights in the range of 8000 to 35,000 and 11,000 to 55,500 Dalton, respectively. The number average molecular weights for the small and large subunit proteins are 22,500 and 26,500 Dalton, respectively. The sum of the molecular weights is 0.67 x 10(6) Dalton for the proteins of the small subunit and 1.05 x 10(6) Dalton for the proteins of the large subunit.
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