Renaturation of calcium/calmodulin-dependent protein kinase activity after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to membranes.
A method is described for analyzing calcium/calmodulin-dependent protein kinase activity in crude or purified samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membrane. The blotted protein is denatured in situ with guanidine HCl and renatured in buffer containing NP-40. The membrane with bound protein is incubated with [gamma-32P]ATP and buffer containing the activators Ca2+ and calmodulin resulting in autophosphorylation of a subset of bound kinases. Two of the three major kinase activities detected in as little as 5 micrograms of crude brain or spinal cord homogenates are the alpha (M(r) = 50-52,000) and beta (M(r) = 58-62,000) isoforms of Ca2+/calmodulin-dependent protein kinase II. A third unidentified kinase of M(r) = 90-95,000 is not dependent on Ca2+ and calmodulin for activity. The membrane can be used for immunoblotting, phosphoamino acid analysis, or peptide mapping after the in situ renaturation and phosphorylation procedure. Detection of kinase activity in this assay is dependent on autophosphorylation of the enzyme. Therefore another procedure is described in which the blotted proteins are denatured and renatured in situ and assayed by measuring incorporation of phosphate into an exogenous peptide substrate specific for calcium/calmodulin-dependent protein kinase II.
Shackelford
D A
DA
Department of Neurosciences, University of California, San Diego, La Jolla 92093-0624.
Zivin
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D007527:Isoenzymes / Q000032:analysis*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011489:Protein Denaturation; D011494:Protein Kinases / Q000737:chemistry*; D011817:Rabbits; D051381:Rats; D013116:Spinal Cord / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D005782:Gels; D007527:Isoenzymes; D008567:Membranes, Artificial; D010446:Peptide Fragments; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases