Protein phosphorylation of plasma membranes from bovine epididymal spermatozoa.
Plasma membranes from bovine epididymal spermatozoa possess both cAMP-independent and cAMP-dependent protein kinase activity. With the synthetic peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly as substrate, the basal activity of the membrane-associated protein kinase(s) was 0.1 nmol phosphate incorporated X min X mg protein. In the presence of 5 microM cAMP, the apparent activity was increased about twofold. The addition of Nonidet P-40 (0.05%) to the assay mixture increased protein kinase activity to 0.4 and 4.0 nmol phosphate incorporated X min X mg protein in the absence or presence of 5 microM cAMP, respectively. Both isozymes of the cAMP-dependent protein kinase were detected in detergent-solubilized membranes but 95% of the activity appeared as a Type II form based on DEAE-Sephacel chromatography. Several polypeptide components of the plasma membrane served as substrates for membrane-associated cAMP-dependent protein kinases, in vitro. In the absence of detergent, two cAMP-dependent phosphoproteins of 41,000 Mr and 60,000 Mr were detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When 0.05% Nonidet P-40 was included in the assay mixture, a cAMP-dependent phosphoprotein of 43,000 Mr appeared. Two-dimensional polyacrylamide gel electrophoresis of membranes phosphorylated in the presence of 5 microM and 0.05% Nonidet P-40 revealed phosphoproteins of the following molecular weights/isoelectric points: 56,000/6.7, 56,000/6.9, 51,000/6.2, 42,000/5.9, 42,000/6.0, 38,000/6.1, 38,000/6.4, 14,000/7.2, 12,000/7.4 and a train of five polypeptides appearing at 14,000/5.4-6.0.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis; D011506:Proteins / Q000378:metabolism*; D013094:Spermatozoa / Q000378:metabolism*
D011506:Proteins; D004533:Egtazic Acid; D000242:Cyclic AMP; D011494:Protein Kinases