Purine nucleoside phosphorylase in erythrocytes: determination of optimum reaction conditions.

Reaction conditions for determining the activity of purine nucleoside phosphorylase (PNP; E.C. 2.4.2.1) were investigated. We examined the kinetic parameters for the enzymatic reaction with respect to the substrates inosine and phosphate. We confirmed the pH optimum, established the optimal concentration of xanthine oxidase and that of calcium and magnesium. The Km values for inosine and phosphate were found to be 60 uM and 667 uM, respectively. Optimum assay conditions for PNP activity were established. This optimized method has been compared with other procedures and found to be more sensitive and to yield significantly higher activities. The experimental variation of a manual procedure using these optimum reaction conditions was less than 4.5%. The mean erythrocyte PNP activity of 28 healthy subjects was estimated to be 9.71 U/mL packed cells at 25 degrees C and 18.60 U/mL packed cells at 37 degrees C.