An adenosine 3':5' monophosphate dependent protein kinase from sea urchin spermatozoa.
A cyclic AMP dependent protein kinase (EC 2.7.1.37) from sea urchin sperm as purified to near homogeneity and characterized. A 68-fold purification of the enzyme was obtained. This preparation had a specific activity of 389 000 units/mg protein with protamine as the substrate. On the basis of the purification required, it may be calculated that the protein kinase constitutes as much as 1.5% of the soluble protein in sperm. There appeared to be a single form of the enzyme in sea urchin sperm, based on the behavior of the enzyme during DEAE-cellulose and Sephadex G-200 column chromatography. Magnesium ion was required for enzyme activity. The rate of phosphorylation of protamine was stimulated 2.5-fold by an optimal concentration of 0.9 M NaCl. The Km for ATP (minus cyclic AMP) was 0.119 +/- 0.013 (S.D.) and 0.055 mM +/- 0.009 (S.D.) in the presence of cyclic AMP. The specificity of the enzyme toward protein acceptors, in decreasing order of phosphorylation, was found to be histone f1 protamine, histone f2b, histone f3 and histone f2a; casein and phosvitin were not phosphorylated. The holoenzyme was found to have an apparent molecular weight of 230 000 by Sephadex G-200 chromatography. In the presence of 5 - 10(-6) M cyclic AMP, the holoenzyme was dissociated on Sephadex G-200 to a regulatory subunit of molecular weight 165 000 and a catalytic subunit of Mr 73 000. The dissociation could also be demonstrated by disc gel electrophoresis in the presence and absence of cyclic AMP.
D000818:Animals; D002413:Cations, Divalent; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009928:Organ Specificity; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D012617:Sea Urchins; D013045:Species Specificity; D013094:Spermatozoa / Q000201:enzymology*; D013737:Testis / Q000201:enzymology
D002413:Cations, Divalent; D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases; D008274:Magnesium