Phosphorylation of beta-casein and alpha-lactalbumin by casein kinase from lactating bovine mammary gland.
Two milk proteins, beta-casein and alpha-lactalbumin, were compared as substrates for casein kinase from bovine mammary gland. beta-Casein could be rephosphorylated after removal of its phosphate groups, whereas alpha-lactalbumin was an effective substrate after the protein had been reduced and carboxymethylated. The native proteins could not be phosphorylated. Magnesium2+, Ca2+, and Mn2+ stimulated phosphorylation of the modified proteins. Calcium2+ was the most effective cation for alpha-lactalbumin and Mn2+ for beta-casein. Michaelis constants were 144 microM for alpha-lactalbumin in the presence of Ca2+ and 142 microM for beta-casein in the presence of Mn2+; however, the maximum velocity for alpha-lactalbumin was three times that of beta-casein. After phosphorylation with [gamma-32P] ATP, partial hydrolysis showed that only serine residues were phosphorylated in both proteins. Chymotryptic peptides of phosphorylated alpha-lactalbumin and tryptic peptides of phosphorylated beta-casein were examined by HPLC and selected peptides were analyzed for amino acid content. Comparison of the analyses with sequence data showed that serine at position 47 in alpha-lactalbumin is the major site of phosphate incorporation. Dephosphorylated beta-casein was only partially rephosphorylated. However, the sites identified correspond to the phosphorylated residues in native beta-casein, namely, serine at position 35 and the cluster of four serines between residues 15 and 20.
Bingham
E W
EW
Eastern Regional Research Center, US Department of Agriculture, Wyndmoor, PA 19118.
Parris
N
N
Farrell
H M
HM
D000818:Animals; D047388:Casein Kinases; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D005260:Female; D066298:In Vitro Techniques; D007768:Lactalbumin / Q000378:metabolism*; D007774:Lactation / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000201:enzymology*; D010766:Phosphorylation; D011247:Pregnancy; D011494:Protein Kinases / Q000378:metabolism*
D002364:Caseins; D007768:Lactalbumin; D011494:Protein Kinases; D047388:Casein Kinases