Isolation and solubilization of casein kinase from Golgi apparatus of bovine mammary gland and phosphorylation of peptides.
Phosphate incorporation from [gamma-32P]ATP into native and dephosphorylated alpha s1-casein is catalyzed by a casein kinase localized in the Golgi apparatus of lactating bovine mammary gland. Casein kinase from the Golgi is activated with either Mg2+ or Ca2+, and increased specific activity is observed with dephosphorylated casein as the substrate. The casein kinase can be solubilized from Golgi apparatus by the non-ionic detergent, Triton X-100. Gel permeation chromatography on Sepharose CL-4B yields a Stokes radius of 10 nm for the detergent-solubilized casein kinase. Dephosphorylated beta-peptide, the amino-terminal peptide from beta-casein, is a good substrate for the solubilized casein kinase. With dephosphorylated beta-peptide, the maximal velocity is 9.1 and 12.0 nmol/min per mg protein with Mg2+ and Ca2+ activation, respectively. The Michaelis constant for beta-peptide is greater with Ca2+ than with Mg2+ (4.8 mg/ml compared to 0.97 mg/ml). However, the Michaelis constant for ATP is not greatly influenced by these metal ions. The Triton X-100-solubilized Golgi enzyme can also catalyze the phosphorylation of peptides, such as fibrinopeptide A and alpha-melanocyte stimulating hormone.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D047388:Casein Kinases; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006056:Golgi Apparatus / Q000201:enzymology*; D007700:Kinetics; D007774:Lactation; D008274:Magnesium / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000201:enzymology*; D017830:Octoxynol; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011092:Polyethylene Glycols; D011247:Pregnancy; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D002364:Caseins; D010455:Peptides; D011092:Polyethylene Glycols; D017830:Octoxynol; D011494:Protein Kinases; D047388:Casein Kinases; D008274:Magnesium; D002118:Calcium