Purification and characterization of two casein kinases from ejaculated bovine spermatozoa.
Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.
Ruzzene
M
M
Department of Biological Chemistry, University of Padova, Italy.
Vianello
F
F
Donella-Deana
A
A
Deana
R
R
D000818:Animals; D000906:Antibodies; D047388:Casein Kinases; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004542:Ejaculation; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D011107:Polylysine / Q000494:pharmacology; D011494:Protein Kinases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D011794:Quercetin / Q000494:pharmacology; D011994:Recombinant Proteins / Q000276:immunology; D013094:Spermatozoa / Q000201:enzymology*; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity
D000906:Antibodies; D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011107:Polylysine; D013096:Spermine; D011794:Quercetin; D011494:Protein Kinases; D047388:Casein Kinases