J. Biochem, 1990, Vol. 107, No. 6 802-809
© 1990 Japanese Biochemical Society
research-article |
Purification and Characterization of Calmodulin-Dependent Protein Kinase II from Rat Spleen: A New Type of Calmodulin-Dependent Protein Kinase II1
Department of Biochemistry, Asahikawa Medical College Asahikawa, Hokkaido 078
4 To whom correspondence should be addressed.
A calmodulin-dependent protein kinase has been purified from rat spleen. The enzyme showed a remarkably similar substrate specificity and kinetic parameters to those of rat brain calmodulin-dependent protein kinase II, and exhibited cross-reactivity to a monoclonal antibody against rat brain calmodulin-dependent protein kinase II, indicating that the enzyme might be a calmodulin-dependent protein kinase II isozyme. The sedimentation coefficient was 13.9S, the Stokes radius was 67 Å, and the molecular weight was calculated to be 380,000. The purified enzyme gave five polypeptides bands, corresponding to molecular weights of 51,000, 50,000, 21,000, 20,000, and 18,000, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the purified enzyme with Ca2+, cal-modulin, and ATP under phosphorylating conditions induced the phosphorylation of all five polypeptides. When the logarithm of the velocity of the phosphorylation was plotted against the logorithm of the enzyme concentration (van't Hoff plot), slopes of 0.89, 0.94, and 1.1 were obtained for the phosphorylation of the 50/51-kDa doublet, 20/21-kDa doublet, and 18-kDa polypeptide, respectively. These results indicate that the phosphorylation of the five polypeptides is an intramolecular process, and further indicate that all five polypeptides are subunits of this enzyme. Of the five polypeptides, only the 50- and 51-kDa polypeptides bound to [125I]calmodulin, the other polypeptides not binding to it. A number of isozymic forms of calmodulin-dependent protein kinase II so far demonstrated in various tissues are known to be composed of subunits with molecular weights of 50, 000 to 60, 000 which can bind to calmodulin. Thus, a new type of calmodulin-dependent protein kinase II was demonstrated in the present study.
1 This work has been supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, grants from the Ministry of Health and Welfare of Japan, the Mitsui Life Social Welfare Foundation, an Asahi Research Grant, the Daiwa Health Foundation, and the Byotai Taisha Research Foundation. This work has been submitted as partial fulfillment of the requirements for a Ph.D. byH.S.
Present addresses: 2 Sapporo Hospital of Hokkaido Railway Company, Chuo-ku, Sapporo, Hokkaido 060;
3 Department of Neurochemistry, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183