Functional analysis of Ca2+/calmodulin-dependent protein kinase II expressed in bacteria.
The cDNA encoding the 50-kDa subunit of Ca2+/calmodulin (CaM)-dependent protein kinase II from adult rat brain was cloned into the bacterial expression vector pK223-2 and produced in bacteria. Extensive modification of the cDNA was required to express detectable levels of enzyme. The activity of the bacterially expressed kinase was stringently dependent on Ca2+/CaM but did not exhibit cooperative activation kinetics characteristic of the forebrain enzyme and required 10-fold greater amounts of CaM for half-maximal activation. The bacterially expressed enzyme displayed an apparent Km for a synthetic peptide substrate similar to that of the forebrain enzyme (12 and 10 microM, respectively). Limited proteolysis maps of autophosphorylated peptides, and Western blot analysis demonstrated that the bacterially expressed enzyme was structurally and immunologically indistinguishable from the 50-kDa subunit of the rat forebrain holoenzyme. The bacterially expressed enzyme became Ca2+/CaM-independent after Ca2+/CaM-dependent autophosphorylation in a fashion identical to the forebrain enzyme.
Waxham
M N
MN
Department of Neurology, University of Texas Health Science Center, Houston 77225.
Aronowski
J
J
Kelly
P T
PT
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli; D066298:In Vitro Techniques; D007700:Kinetics; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000276:immunology / Q000302:isolation & purification / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D016704:Synapsins
D002147:Calmodulin; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D016704:Synapsins; D004247:DNA; D011494:Protein Kinases; D002118:Calcium