Characterization of a protein serine kinase from yeast plasma membrane.
A casein kinase activity, which copurifies with the H+-ATPase activity during isolation of plasma membranes Saccharomyces cerevisiae and during centrifugation of the solubilized membrane extract through a sucrose gradient, is separated from the Mr = 100,000 ATPase catalytic polypeptide by subsequent DEAE-cellulose chromatography. The purified casein kinase activity exhibits a low Km of 12 microM MgATP, is maximally stimulated by 6 mM free Mg2+, and is 50% inhibited by 300 microM Zn2+, by 7.5 micrograms of heparin/ml, and by 300 microM orthovanadate. It phosphorylates only seryl residues. The purified casein kinase contains two polypeptides of Mr = 45,000 and 39,000 which yield antibodies which do not cross-react to each other. The two polypeptides seem to originate from a precursor of Mr = 85,000 which is detected by both antibodies in partly purified fractions. In the absence of casein, a zinc and heparin-sensitive phosphorylation of the ATPase polypeptide is observed in partly purified ATPase fractions, and a peptide of similar mobility is phosphorylated, among others, in isolated plasma membranes. The purified ATPase activity is markedly inhibited by incubation in the presence of acid phosphatase. In agreement with a recent report that the purified active ATPase molecule is largely phosphorylated (Yanagita, Y., Abdel-Ghany, M., Raden, D., Nelson, N., and Racker, E. (1987) Proc. Natl. Acad. Sci. U. S. A. 894, 925-929) this data suggests that dephosphorylation leads to deactivation of ATPase activity.
D047388:Casein Kinases; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D007700:Kinetics; D008970:Molecular Weight; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D006180:Proton-Translocating ATPases / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D011494:Protein Kinases; D047388:Casein Kinases; D017346:Protein Serine-Threonine Kinases; D006180:Proton-Translocating ATPases