Calcium Sensitivity of Contractile Proteins from Chicken Gizzard Muscle1

Abstract

Actin, myosin, and “native” tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same way as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca²⁺. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca²⁺. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg²⁺) in the presence of Ca²⁺, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg²⁺) in the presence of Ca²⁺. In either case, the light-chain phosphorylation required Ca²⁺. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca²⁺ and NTM or tropymyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca²⁺ or in its absence, but did superprecipitate in the presence of NTM and Ca²⁺. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca²⁺ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca²⁺. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.