An Association between the Kinetic and Electrophoretic Properties of Human Purine‐Nucleoside‐Phosphorylase Isozymes

Abstract

Human purine‐nucleoside phosphorylase has been studied in extracts of red‐blood cells, cultured fibroblasts and cultured‐lymphocytoid‐cell lines. The enzyme from red cells has been found to contain at least seven electrophoretically distinct isozymes while the enzyme from the cultured cells gives a single electrophoretic band corresponding to the slowest band of the red‐cell enzyme. Human erythrocytes have been separated into groups of different mean‐ages by densitygradient centrifugation; nucleoside phosphorylase from the youngest cells was found to give only four or five electrophoretic bands of which the slowest was the most intensely staining. There was a progressive increase in number and intensity of the faster bands as the mean age of the red‐cell fraction increased. An age‐related decline in the total activity of the enzyme was also observed. The Lineweaver‐Burk plot for human‐erythrocyte purine‐nucleoside phosphorylase assayed in a red‐cell haemolysate with inosine as the variable substrate showed a downward curvature at inosine concentrations above about 0.2 mM. When a form of the enzyme giving only a single electrophoretic band was used the Lineweaver‐Burk plots were found to be completely linear. Generation of secondary isozymes in vitro resulted in a change in this linear plot to the curved form shown by the red‐cell enzyme. It is concluded that the secondary isozymes generated as a result of ageing in vivo or in vitro differ in kinetic behaviour from the primary isozyme. This may account for the observed curvature of the Lineweaver‐Burk plot at high inosine concentrations.