Use of actin-bound adenosine 5′-diphosphate as a method to determine the specific 32P-radioactivity of the γ-phosphoryl group of adenosine 5′-triphosphate in a highly compartmentalized cell, the platelet

Abstract

Determination of the specific 32P-radioactivity of cytoplasmic ATP in 32P- P i-labeled platelets is complicated by the presence of a large pool of metabolically inactive, granule-stored nucleotides. Moreover, our data show that the specific 32P-radioactivity of cytoplasmic ATP is severely underestimated when determined in platelets after the complete secretion of granule-stored nucleotides, possibly due to isotopic dilution with granule-stored phosphate. As F-actin-bound ADP is ethanol-insoluble, this pool can be readily separated from the other nucleotide pools in platelets. Here we show that the specific 32P-radioactivity of F-actin-bound ADP accurately reflects that of the γ-phosphoryl group of cytoplasmic ATP. During uptake of 32P- P i by human platelets the specific 32P-radioactivity of F-actin-bound ADP equals that of the monoester phosphates of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, which are in metabolic equilibrium with cytoplasmic ATP. Therefore, this method enables the determination of the specific 32P-radioactivity of the γ-phosphoryl group of cytoplasmic ATP in platelets even under short-term labeling conditions.