A sensitive method for detection of calmodulin-dependent protein kinase II activity in sodium dodecyl sulfate-polyacrylamide gel.
A procedure for detecting protein kinase activities of the alpha and beta subunits of calmodulin-dependent protein kinase II separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is described. After electrophoresis, the gel was immersed in 6 M guanidine HCl for 1 h and then in a buffer containing 0.04% Tween 40 for 16 h at 4 degrees C for renaturation of the resolved polypeptides. The renatured polypeptides in the gel were incubated with [gamma-32P]ATP for phosphorylation of either the substrate included in the polyacrylamide gel or the kinase itself. After removal of the unreacted [gamma-32P]ATP, the protein kinase activities were visualized by autoradiography. Two radioactive protein bands of Mr 50,000 and 60,000, which corresponded to the alpha and beta subunits, were detected only when the phosphorylation was carried out in the presence of Ca2+ and calmodulin. Approximately 0.05 micrograms of the enzyme could be detected on a gel containing no protein substrate. When microtubule-associated protein 2 was included in the gel, the sensitivity of the detection of calmodulin-dependent protein kinase II in the gel was more than one order of magnitude higher than that in the gel containing no protein substrate.
Kameshita
I
I
Department of Biochemistry, Asahikawa Medical College, Japan.
Fujisawa
H
H
D000818:Animals; D001921:Brain / Q000201:enzymology; D003598:Cytoskeletal Proteins / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D012967:Sodium Dodecyl Sulfate
D003598:Cytoskeletal Proteins; D008869:Microtubule-Associated Proteins; D011506:Proteins; D012967:Sodium Dodecyl Sulfate; D010770:Phosphotransferases; D011494:Protein Kinases; D009218:Myosins