CHARACTERIZATION AND PARTIAL-PURIFICATION OF MICROSOMAL CASEIN KINASE-II FROM OSTEOBLAST-LIKE CELLS - AN ENZYME THAT PHOSPHORYLATES OSTEOPONTIN AND PHOSPHOPHORYN

Microsomal casein kinase II (mCKII) is a membrane-bound enzyme present in the microsomal fractions of ROS 17/2.8 osteoblast-like cells. It p hosphorylates acidic matrix phosphoproteins such as phosphophoryn and osteopontin. Addition of 1.0% Nonidet P-40 facilitates extraction of t he optimum amount of detergent -solubilized and -activated enzyme from microsomal fractions. mCKII was partially purified over 3000-fold by sequential chromatography over DEAE-cellulose and heparin-agarose. SDS -polyacrylamide gels, showed that mCKII contained 43 kDa and 31 kDa po lypeptides, corresponding to the alpha- and beta- subunits of the enzy me, respectively. The or subunit was identified by anti-CKII antiserum and the beta subunit, by its ability to undergo autophosphorylation. The enzyme was inhibited by 50% with 0.4 mu g/ml heparin and stimulate d by 100% with 1.0 mM spermine when casein was used as a substrate. Th e phosphorylation of phosphophoryn was reduced to 50% by 0.8 mu g/ml h eparin, but was increased to 2-2.5 fold by 5 to 15 mM spermine, which may be due to substrate-directed effects. Kinetic analysis showed that the apparent K-m values for phosphophoryn (0.39 mu M) and for osteopo ntin (2.1 mu M) were lower than that for casein (21.3 mu M). V-max val ues of phosphophoryn and osteopontin were 2.2-fold and 4.6-fold higher than that of casein. Using the ratio V-max/K-m as a measure of kineti c specificity, osteopontin and phosphophoryn appear to be the more spe cific substrates than casein for mCKII. Thus, both proteins can be con sidered as physiological substrates for mCKII.