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J. Biochem, 1992, Vol. 112, No. 6 768-774
© 1992 Japanese Biochemical Society

research-article

Purification and Characterization of Two Casein Kinases from Ejaculated Bovige Spermatozoa1

Maria Ruzzene, Fabio Vianello, Arianna Donella-Deana and Renuzo Deana2

Department of Biological Chemistry and C.N.R Unit for the Study of the Mitochondrial Physiology, University of Padova Padova, Italy

2To whom correspondence should be addressed at: Dipartimento di Chimica Biologia, Via Trieste 75, I 30121 Padova, Italy

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 µM). 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 ß, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 µM) 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, MgC12 (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.

1This study waa financially supported in part by the Ministero Italiano della Ricerca Scientfica e Tecnologica


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