Protein phosphorylation in intact bovine epididymal spermatozoa: identification of the type II regulatory subunit of cyclic adenosine 3',5'-monophosphate-dependent protein kinase as an endogenous phosphoprotein.

An obstacle to the study of protein phosphorylation in mammalian spermatozoa has been the inability to incorporate sufficient amounts of 32P1 into cellular adenosine triphosphate (ATP) (Babcock et a!., 1975). We report conditions under which 32P1 is effectively incorporated into the ATP of intact bovine spermatozoa. In the presence of a bicarbonate-buffered medium containing glucose, spermatozoa incorporated 32P into intracellular ATP in a time-dependent manner; after 2 h of incubation, the specific activity of [y-32PJATP (2.3 x i#{252}� cpm/nmol ATP) was estimated to be 50-65% of the specific activity of the intracellular phosphate pooi. In the absence of glucose or other added substrates, the specific activity of [7�32 P]ATP was 1025% that of the specific activity observed in the presence of glucose. Washed spermatozoa incubated in carrier-free 32P1 for 2 h at 37#{176}C, and solubilized in a solution containing final concentrations of 6.8 M urea, 6% NP4 0, and 5% 13-mercaptoethanol contained in excess of 40 32P�labeled proteins as assessed by two-dimensional polyac,ylamide gel electrophoresis. Major phosphoproteins had approximate molecular weights of 93,000, 40,000, and 22,000. A different two-dimensional gel pattern was observed when cells were extracted with a solution containing 38.5mM 2[N-cyclohexylaminoJ ethanesulfonic acid (CHES), pH 9.5/1.5% sodium dodecyl sulphate (SDS) at 100#{176} C. In contrast to the urea/Nonidet P-40 (NP4O)43-mercaptoethanol extract, a 56,000 Mr phosphoprotein represented a major component while the 40,000 Mr and several of the 22,000 Mr polypeptides were markedly reduced in radioactive intensity. The 56,000 Mr species present in the CHES/SDS extract comigrated with the purified, phosphoiylated regulatory subunit (Rfl) of cyclic adenosine 3’, 5’-monophosphate-dependent protein kinase from bovine heart. Antibodies to RH immunoprecipitated a 56,000 Mr, 32P-labeled polypeptide from the CHES/SDS extract that comigrated with purified, 132 P1 Rjj after two-dimensional electrophoresis. Rjj, then, appears to represent one of the endogenous phosphoproteins of intact bovine epididymal spermatozoa.