Enzymic characteristics of an ecto-cyclic AMP-dependent protein kinase in rat epididymal spermatozoa.
Intact spermatozoa from rat cauda epididymides possess an ecto-(cyclic AMP-dependent protein kinase) activity that causes the transfer of the terminal phosphate group of ATP to the serine residues of all the histone fractions. The enzyme showed a high degree of substrate specificity for the phosphorylation of histones rather than protamine, casein and phosvitin. The cell-external-surface protein kinase requires Mg2+ for activity, and other bivalent cations such as Mn2+ and Co2+ can substitute partially for Mg2+, whereas Ca2+ and Zn2+ are potent inhibitors of the enzyme. The enzyme has markedly higher affinity for cyclic AMP than for other cyclic nucleotides for its activation, with an apparent Km value for cyclic AmP of 80 nM. Spermatozoal ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. There was no loss of kinase activity from the cells when washed with 2 mM-EDTA, and the histones phosphorylated by intact spermatozoa were located outside the cells. Protein kinase activity of intact cells was strongly inhibited (approx. 90%) by p-chloromercuribenzenesulphonic acid (10 microM), which is believed not to enter the cells. These data provide further support for the localization of a protein kinase on the external surface of spermatozoa.
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D000242:Cyclic AMP / Q000494:pharmacology; D004822:Epididymis; D006657:Histones / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D009712:Nucleotides, Cyclic / Q000494:pharmacology; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity
D002413:Cations, Divalent; D006657:Histones; D009712:Nucleotides, Cyclic; D047428:Protein Kinase Inhibitors; D000242:Cyclic AMP; D011494:Protein Kinases