In Vitro Movement of Actin Filaments on Gizzard Smooth Muscle Myosin: Requirement of Phosphorylation of Myosin Light Chain and Effects of Tropomyosin and Caldesmon

• Published in: Journal of biochemistry (Tokyo) Vol. 109; no. 6; pp. 858 - 866
• Main Authors: Okagaki, Tsuyoshi, Higashi-Fujime, Sugie, Ishikawa, Ryoki, Takano-Ohmuro, Hiromi, Kohama, Kazuhiro
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 1991
• Subjects: Actins - chemistry; Animals; Biological and medical sciences; Biotransformation - drug effects; caldesmon; Calmodulin-Binding Proteins - pharmacology; Cell physiology; Chickens; Collodion; effects on; Fundamental and applied biological sciences. Psychology; gizzard; Gizzard, Avian - chemistry; Gizzard, Avian - drug effects; Gizzard, Avian - ultrastructure; light chains; Microfilament Proteins; Microscopy, Electron; Microscopy, Fluorescence; Molecular and cellular biology; Muscle contraction; Muscle, Smooth - drug effects; Muscle, Smooth - metabolism; Muscle, Smooth - ultrastructure; myosin; Myosins - chemistry; Phosphorylation; Protein Kinase C - metabolism; Rabbits; Tropomyosin - pharmacology; troponin; Vertebrata; Phosphorylation; Actomyosin; Gizzard; Myosin; Actins; light-Peptide chain; Microfilament; Smooth muscle; Chicken; Aves; Electron microscopy
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a123471

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCE) at a rate of 0.35 μm/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 μm/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.