39] Pi32-ATP exchange enzyme system: Adenosine triphosphate (A-R-P-P-P) + orthophosphate-P32 (Pi32) ⇄ adenosine triphosphate-P32 (A-R-P-P-P32) + orthophosphate (Pi) adenosine triphosphate (A-R-P-P-P) + adenosine diphosphate-P32 (A-R-P-P32) ⇄ adenosine triphosphate-P32 (A-R-P-P32P) + adenosine diphosphate (A-R-P-P)

Abstract

Publisher Summary This chapter explains the P i 32 –ATP exchange enzyme system. The purification of the P i 32 –ATP exchange activity is most conveniently followed by observing the incorporation of the label from orthophosphate into bound organic phosphate compounds by means of the molybdate extraction procedure. Because this method merely results in the removal of orthophosphate into the organic solvent phase, leaving other phosphate compounds in the aqueous layer, it is highly unspecific and cannot be used for the determination of the P i 32 –ATP exchange in very crude tissue preparations. The use of isolated mitochondria as the starting material for the enzyme extraction leaves behind most of the interfering enzymic activities. Even in this initial extract from mitochondrial acetone powder, radioactivity from P 32 is found in both ATP and ADP, probably because of the initial exchange reaction between P i 32 and ATP which is followed by a redistribution of label from formed ATP 32 into ADP via the adenylic kinase reaction. The exchange between P i 32 and ATP occurs specifically with the terminal phosphate group of ATP; however, this reaction is stimulated by the presence of ADP in the reaction mixture. ADP seems to be a participant in the P i 32 –ATP reaction, because purified enzyme preparations carry out an ADP–ATP exchange at the same rate as the P i 32 –ATP reaction.