Properties of casein kinase from lactating bovine mammary gland.

Casein kinase, which catalyzes the transfer of the terminal phosphate of ATP to dephosphorylated caseins, was prepared from homogenates of lactating bovine mammary tissue by differential centrifugation, followed by extraction of the 66,000 x g particulate fraction with Triton X-100. The enzyme required divalent cations and showed comparable activities with 15 mu Ca’+, 15 mu Mg2+, and 1 mu Mn2’. Proteins with known primary structures were examined as substrates for casein kinase. Caseins ((Y.~, p, and K), pepsinogen, and pepsin (denatured) showed significant increases in rates of phosphorylation after phosphate groups were removed. The catalytic efficiency (ratio of V,, to K,) indicates that dephosphorylated pepsin was the best substrate. Dephosphorylated esland P-caseins were significantly better substrates than native a.l-casein and dephosphorylated pepsinogen. The susceptibility of pepsinogen (native and dephosphorylated) and cY-lactalbumin to enzymatic phosphorylation could be enhanced by converting the proteins to the reduced, carboxymethylated derivatives. Further studies of b-casein indicate that the dephosphorylated phosphopeptide (residues 1 to 25) was phosphorylated at a much higher rate than dephosphorylated yl-casein (residues 29 to 209). The results suggest that casein kinase catalyzes the phosphorylation of 1 to 4 serine residues in the phosphopeptide region of /3casein. Human /3-caseins, which occur in six forms differing only by zero to five phosphate groups, showed differences in specificity. The rate of phosphate incorporation in unphosphorylated human B-casein was 8 times that of human /I-casein with two and four phosphate groups. The role of acidic residues (glutamic and aspartic acids and phosphoserine) on the COOH-terminal side of serines phosphorylated by casein kinase is discussed.