An enzymatically active cross-linked complex of calmodulin and rabbit skeletal muscle myosin light chain kinase.
The interaction between bovine testes calmodulin and rabbit fast skeletal muscle myosin light chain kinase was investigated with the zero-length cross-linking reagent N,N'-dicyclohexylcarbodiimide. A cross-linked product of 110 kDa was produced only in the presence of Ca2+. The reaction mixture was separated on diethylaminoethyl cellulose, and a fraction containing the cross-linked complex of calmodulin and myosin light chain kinase was found to have an elevated kinase activity in the absence of Ca2+, which constituted approximately 50% of the maximally stimulated kinase activity of control, and additional kinase activity in the presence of Ca2+, which constituted the remaining 50% of control activity. Calmodulin added exogenously to the cross-linked complex had no effect on the measured Ca2+ dependence or the maximal extent of kinase activity, which is consistent with the cross-linking of calmodulin in close proximity to a regulatory region of myosin light chain kinase. Moreover, the results are consistent with a mechanism whereby the association of calmodulin is sufficient to stimulate kinase activity and the binding of Ca2+ to bound calmodulin increases catalytic efficiency.
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H G
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Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Puett
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D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism*; D002234:Carbodiimides / Q000378:metabolism*; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D004024:Dicyclohexylcarbodiimide / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D009132:Muscles / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D011817:Rabbits; D013737:Testis / Q000378:metabolism
D002147:Calmodulin; D002234:Carbodiimides; D004024:Dicyclohexylcarbodiimide; D009219:Myosin-Light-Chain Kinase; D002118:Calcium