Ca-Regulation of Mammalian Smooth Muscle Actomyosin via a Kinase-Phosphatase-Dependent Phosphorylation and Dephosphorylation of the 20000-Mr Light Chain of Myosin

The purification of actomyosin extracted from pig stomach by precipitation with a high concentration of CaCl2 (20 mM) yields an actomyosin for which the 20000-Mr L20 light chain of myosin occurs in a dephosphorylated state. This actomyosin exhibits negligible actin-activated ATPase activity. In contrast, for actomyosin obtained by precipitation with micromolar concentrations of CaCl2 the L20 light chain is phosphorylated and this actomyosin shows a very high activity but little sensitivity to Ca2+. When combined with actomyosin-free extracts obtained from smooth muscle mince or myofibrils both actomyosins may be converted to one showing both high activity and Ca-sensitivity. This is shown for the dephosphorylated actomyosin to be associated with a Ca-dependent phosphorylation of the L20 myosin light chain and in the case of the phosphorylated actomyosin with a dephosphorylation of this light chain in the absence of Ca. The phosphorylation and dephosphorylation of the light chain are further shown to be effected by a specific myosin light-chain kinase and a light-chain phosphatase present in the smooth muscle extracts. As for gizzard, the thin filaments obtained from pig stomach were found, from polyacrylamide gel electrophoresis, to lack any troponin-like components and produced a Ca-insensitive actomyosin when combined with skeletal muscle myosin. The results provide further evidence for a control mechanism in vertebrate smooth muscle in which contraction is triggered by the phosphorylation of myosin by a Ca-dependent kinase and relaxation is effected via the dephosphorylation of myosin by a Ca-independent phosphatase.