In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon.
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 (MLCK) at a rate of 0.35 micron/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 micron/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+.
Okagaki
T
T
Department of Molecular Biology, Faculty of Science, Nagoya University, Aichi.
Higashi-Fujime
S
S
Ishikawa
R
R
Takano-Ohmuro
H
H
Kohama
K
K
D000199:Actins / Q000737:chemistry*; D000818:Animals; D001711:Biotransformation / Q000187:drug effects; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology*; D002645:Chickens; D003101:Collodion; D005895:Gizzard, Avian / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure; D008840:Microfilament Proteins; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D014335:Tropomyosin / Q000494:pharmacology*
D000199:Actins; D002148:Calmodulin-Binding Proteins; D008840:Microfilament Proteins; D014335:Tropomyosin; D003101:Collodion; D011493:Protein Kinase C; D009218:Myosins