Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa.
Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [gamma-32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004822:Epididymis / Q000201:enzymology*; D005110:Extracellular Space / Q000201:enzymology; D006041:Goats; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011494:Protein Kinases / Q000378:metabolism*; D013010:Sonication; D013081:Sperm Motility; D013094:Spermatozoa / Q000201:enzymology*; D013997:Time Factors
D008565:Membrane Proteins; D010750:Phosphoproteins; D011092:Polyethylene Glycols; D011494:Protein Kinases; D010447:Peptide Hydrolases