Functional interactions between smooth muscle myosin light chain kinase and calmodulin

Abstract

Calmodulin (CaM) binding by turkey gizzard myosin L chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthrolycholine (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 Kd for the MLCK-CaM complex is increased .apprx. 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5-1.1 .mu.M in 0.2 N KCl, pH 7.3, 25.degree. C. Competition between MLCK and other CaM binding proteins and peptides was shown 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 3 proteins by the cAMP-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.