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 phosphorylates acidic matrix phosphoproteins such as phosphophoryn and osteopontin. Addition of 1.0% Nonidet P-40 facilitates extraction of the 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 polypeptides, corresponding to the alpha- and beta-subunits of the enzyme, respectively. The alpha 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 micrograms/ml heparin and stimulated by 100% with 1.0 mM spermine when casein was used as a substrate. The phosphorylation of phosphophoryn was reduced to 50% by 0.8 micrograms/ml heparin, 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 Km values for phosphophoryn (0.39 microM) and for osteopontin (2.1 microM) were lower than that for casein (21.3 microM). Vmax values of phosphophoryn and osteopontin were 2.2-fold and 4.6-fold higher than that of casein. Using the ratio Vmax/Km as a measure of kinetic specificity, osteopontin and phosphophoryn appear to be the more specific substrates than casein for mCKII. Thus, both proteins can be considered as physiological substrates for mCKII.
Wu
C B
CB
Department of Clinical Dental Sciences, Faculty of Dentistry University of British Columbia, Vancouver, Canada.
Shimizu
Y
Y
Ng
A
A
Pan
Y M
YM
D000818:Animals; D047390:Casein Kinase II; D002460:Cell Line; D008861:Microsomes / Q000201:enzymology; D010006:Osteoblasts / Q000166:cytology / Q000201:enzymology*; D053495:Osteopontin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012795:Sialoglycoproteins / Q000378:metabolism*; D013379:Substrate Specificity
D010750:Phosphoproteins; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; C017630:phosphophoryn; D053495:Osteopontin; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases