Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase

Abstract

Interactions between Ca 2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems ( Current Topics in Cellular Regulation 27, 1966–1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca 2+-binding. The determination of the Ca 2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical χ 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca 2+-binding constants. Whereas the first two stoichiometric Ca 2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca 2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca 2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca 2+ in the isotherm. Direct binding of Ca 2+-free calmodulin to myosin light chain kinase at 10 −7 M free Ca 2+ yielded a 1 1 stoichiometry and an affinity constant of 2.2 × 10 5 M −1. It is thus anticipated that in resting smooth muscle ([Ca 2+] ≤ 10 −7 M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca 2+ revealed that this Ca 2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca 3 complex.