Functional interactions between smooth muscle myosin light chain kinase and calmodulin.
Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].
D000255:Adenosine Triphosphate / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002794:Choline / Q000031:analogs & derivatives / Q000378:metabolism; D003619:Dansyl Compounds / Q000378:metabolism; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); C035321:9-anthroylcholine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D009219:Myosin-Light-Chain Kinase; D002794:Choline