title: Purification and characterization of two casein kinases from ejaculated bovine spermatozoa.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, University of Padova, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Ruzzene
authors.forename: F
authors.initials: F
authors.lastname: Vianello
authors.forename: A
authors.initials: A
authors.lastname: Donella-Deana
authors.forename: R
authors.initials: R
authors.lastname: Deana
mesh_terms: 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
chemical_list: D000906:Antibodies; D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011107:Polylysine; D013096:Spermine; D011794:Quercetin; D011494:Protein Kinases; D047388:Casein Kinases