Active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II. Purification, characterization, and structural analysis.
We report the purification and characterization of an active catalytic fragment of Ca2+/calmodulin-dependent protein kinase II, derived from autophosphorylation and subsequent limited chymotryptic digestion of the purified rat forebrain soluble kinase. The purified fragment was completely Ca2+/calmodulin-independent, existed as a monomer, and phosphorylated synapsin I at the same sites as does the native form of Ca2+/calmodulin-dependent protein kinase II. Kinetic studies with the purified fragment revealed a more than 10-fold increase in Vmax and a 50% decrease in Km for synthetic peptide substrates, compared with native Ca2+/calmodulin-dependent protein kinase II. No 32P-labeled autophosphorylated residues were detected in the purified active fragment, indicating that the autophosphorylation sites were not contained within this fragment. Comparative studies of this active fragment (30 kDa) and its inactive counterpart (32-kDa fragment) revealed certain structural details of both fragments. Calmodulin-overlay study, immunoblot analysis, and direct amino acid sequencing suggest that both fragments contain the entire NH2-terminal catalytic domain and were generated by distinct cleavage within the regulatory domain. The putative cleavage sites for both fragments are discussed.
Yamagata
Y
Y
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Czernik
A J
AJ
Greengard
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P
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D002147:Calmodulin; D010446:Peptide Fragments; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases