Gizzard Ca²⁺-independent myosin light chain kinase: Evidence in favor of the phosphorylation theory

Limited digestion of calmodulin (CaM)-dependent myosin light chain kinase from turkey gizzard with α-chymotrypsin in the presence of bound CaM generated an 80,000-dalton kinase fragment that was fully active in the absence of Ca²⁺. This kinase catalyzed specific Ca²⁺-independent phosphorylation of the 20,000-dalton light chain of myosin using isolated light chains, intact myosin, and actomyosin. Phosphorylation of myosin in the absence of Ca²⁺ allowed us to dissociate myosin phosphorylation from other potential Ca²⁺-dependent regulatory mechanisms, thus permitting an evaluation of the postulated central role of myosin phosphorylation in the regulation of smooth muscle contraction. Ca²⁺-independent myosin phosphorylation was found to cause loss of Ca²⁺ sensitivity of actin-activated myosin ATPase activity in a crude actomyosin preparation, and tension development in skinned smooth muscle fibers in the absence of Ca²⁺. Myosin phosphorylation is, therefore, the key event in actin activation of ATPase activity and initiation of contraction in skinned chicken gizzard fibers.