Purification and characterization of polyamine-stimulated protein kinase (casein kinase II) from bovine spermatozoa.
Casein kinase II from bovine epididymal spermatozoa was purified to apparent homogeneity by repeated chromatography with phosphocellulose and gel filtration with sephacryl S-200. The purified enzyme exhibited a molecular mass of 130 kDa by gel filtration and displayed three polypeptide bands with molecular masses of 26, 33, and 36 kDa by SDS-polyacrylamide gel electrophoresis. Antibodies raised against calf thymus casein kinase II cross reacted with the three sperm polypeptides. Incubation of the holoenzyme with either [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of the 26-kDa subunit. The enzymatic activity with casein as substrate was strongly inhibited by nanomolar heparin and greatly stimulated by micromolar spermine. With casein as substrate, the specific activity of the pure enzyme (0.5 mumol/min/mg protein) was comparable to that of casein kinase II from other sources. Endogenous substrates of the kinase were demonstrated by incubating sperm cytosolic extracts with [gamma-32P]GTP, under conditions that limit the expression of other protein kinases, and analyzing the products by SDS-PAGE and autoradiography. Similar results were obtained when sperm extracts, suitably diluted to minimize endogenous casein kinase II, were incubated with [gamma-32P]GTP and aliquots of pure sperm casein kinase II. Low concentrations (50 microM) spermine strongly enhanced the phosphorylation of 92- and 106-kDa cytosolic proteins. Our results clearly show that casein kinase II is present in spermatozoa and that it shares many of the properties of the enzyme from other sources. Further, they indicate that the enzyme plays a role in mediating the phosphorylation state of sperm proteins.
Chaudhry
P S
PS
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Nanez
R
R
Casillas
E R
ER
D000818:Animals; D047388:Casein Kinases; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010766:Phosphorylation; D011073:Polyamines / Q000494:pharmacology*; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity
D011073:Polyamines; D011494:Protein Kinases; D047388:Casein Kinases