Use of actin-bound adenosine 5'-diphosphate as a method to determine the specific 32P-radioactivity of the gamma-phosphoryl group of adenosine 5'-triphosphate in a highly compartmentalized cell, the platelet.
Determination of the specific 32P-radioactivity of cytoplasmic ATP in 32P-Pi-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 gamma-phosphoryl group of cytoplasmic ATP. During uptake of 32P-Pi 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 gamma-phosphoryl group of cytoplasmic ATP in platelets even under short-term labeling conditions.
Verhoeven
A J
AJ
Department of Biochemistry, Bergen, Norway.
Cook
C A
CA
Holmsen
H
H
D000199:Actins / Q000097:blood*; D000225:Adenine / Q000097:blood; D000244:Adenosine Diphosphate / Q000097:blood*; D000255:Adenosine Triphosphate / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism*; D011756:Diphosphates / Q000097:blood; D010710:Phosphates / Q000097:blood*; D010761:Phosphorus Radioisotopes; D014316:Tritium
D000199:Actins; D011756:Diphosphates; D010710:Phosphates; D010761:Phosphorus Radioisotopes; D014316:Tritium; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000225:Adenine