A SIMPLIFIED METHOD FOR THE DETERMINATION OF PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE-ACTIVITY IN HEMOLYSATES

Methods currently employed for measurement of phosphoribosylpyrophosph ate synthetase (ribophosphate pyrophosphokinase; PRPPs) activity are c umbersome and expensive, requiring an auxiliary enzyme reaction, a rad ioactive nucleobase and multiple incubations, or do not allow a comple te kinetic study. The aim of the present study is to describe a simpli fied, single step, non-isotopic method for determination of PRPPs in h emolysate, appropriate for a complete screening of PRPPs activity diso rders. Briefly, the charcoal-treated hemolysate is incubated with satu rating amounts of substrates and P1 P5 di(adenosine 5') pentaphosphate (Ap5A), an inhibitor of human adenylate kinase, to prevent conversion of AMP to ADP. AMP generated in this reaction is then measured by HPL C. Adenylate kinase activity was fully inhibited by Ap5A, allowing the accurate determination of AMP. The method was sensitive and reproduci ble and mean and variance values compared closely with those reported using other, more complicated, assay procedures. The hyperbolic curve relating Pi concentration to initial reaction velocity was shifted to sigmoidal by addition of 0.02 mmol/l GDP which inhibited PRPPs activit ies only at inorganic phosphate concentrations < 2 mmol/l. This sugges ts that this method should provide sensitive and accurate screening fo r regulatory, as well as catalytic, defects underlying PRPPs superacti vity.