Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain.
A substrate-specific calmodulin-dependent myosin light-chain kinase (MLCK) was purified 45,000-fold to near homogeneity from bovine brain in 12% yield. Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme. The regulatory MLC present in intact gizzard myosin is also phosphorylated by the enzyme. The Mr-19,000 rabbit skeletal-muscle MLC is a substrate; however, the rate of its phosphorylation is at best 30% of that obtained with turkey gizzard MLC. Phosphorylation of all other protein substrates tested is less than 1% of that observed with gizzard MLC as substrate. SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of MLCK by the catalytic subunit of cyclic-AMP-dependent protein kinase results in the incorporation of phosphate into the Mr-152,000 protein band and a marked decrease in the affinity of MLCK for calmodulin. The presence of Ca2+ and calmodulin inhibits the phosphorylation of the enzyme. Bovine brain MLCK appears similar to MLCKs isolated from platelets and various forms of muscle.
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854.
D C
DC
Bartelt
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S
Moroney
D J
DJ
Wolff
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009219:Myosin-Light-Chain Kinase / Q000302:isolation & purification / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013379:Substrate Specificity
D002147:Calmodulin; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D009218:Myosins