title: Identification and purification of a novel serine/threonine messenger-independent growth-related protein kinase from lactating goat mammary gland.
abstract: A second messenger-independent serine/threonine protein kinase from lactating goat mammary gland is purified and characterized. The purification steps include: homogenization, ultracentrifugation, ammonium sulphate precipitation, DEAE-Sepharose, phosphocellulose, hydrophobic and Mono Q columns. On the final step of purification the enzyme is revealed as a single band of mol wt 45,000 on silver-stained SDS-PAGE. Mg2+ and K+ are necessary for its optimum activity. Phosvitin and casein are substrates for the enzyme but kemptide, RRREEETEEE, protamine and histone mixture are all poorly phosphorylated. The kinase is inhibited by quercetin, heparin, random tyrosine- and glutamic acid-containing polymers, Ca2+, NaF, 2,3-bis-phosphoglycerate. 1 mM Mn2+ affects positively the basal level of the kinase activity but 5 mM Mn2+ completely suppress the effect of 10 mM Mg2+. Km of this enzyme for ATP is 1.57 microM and pH optimum is from 6 to 7. Isolation of this kinase is facilitated by its unusually high affinity for phosphocellulose.
authors.affiliation: Department of Biochemistry, Institute of Biomedical Sciences, Medical Academy, Sofia, Bulgaria.
authors.forename: V I
authors.initials: VI
authors.lastname: Mitev
authors.forename: I V
authors.initials: IV
authors.lastname: Angelov
authors.forename: L N
authors.initials: LN
authors.lastname: Marekov
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D047388:Casein Kinases; D005260:Female; D006041:Goats; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007774:Lactation; D008321:Mammary Glands, Animal / Q000201:enzymology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010774:Phosvitin / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D047428:Protein Kinase Inhibitors; D010774:Phosvitin; D011494:Protein Kinases; D047388:Casein Kinases