Affinity for MgADP and force of unbinding from actin of myosin purified from tonic and phasic smooth muscle.
Smooth muscle is unique in its ability to maintain force at low MgATP consumption. This property, called the latch state, is more prominent in tonic than phasic smooth muscle. Studies performed at the muscle strip level have suggested that myosin from tonic muscle has a greater affinity for MgADP and therefore remains attached to actin longer than myosin from phasic muscle, allowing for cross-bridge dephosphorylation and latch-bridge formation. An alternative hypothesis is that after dephosphorylation, myosin reattaches to actin and maintains force. We investigated these fundamental properties of smooth muscle at the molecular level. We used an in vitro motility assay to measure actin filament velocity (nu(max)) when propelled by myosin purified from phasic or tonic muscle at increasing [MgADP]. Myosin was 25% thiophosphorylated and 75% unphosphorylated to approximate in vivo conditions. The slope of nu(max) versus [MgADP] was significantly greater for tonic (-0.51+/-0.04) than phasic muscle myosin (-0.15+/-0.04), demonstrating the greater MgADP affinity of myosin from tonic muscle. We then used a laser trap assay to measure the unbinding force from actin of populations of unphosphorylated tonic and phasic muscle myosin. Both myosin types attached to actin, and their unbinding force (0.092+/-0.022 pN for phasic muscle and 0.084+/-0.017 pN for tonic muscle) was not statistically different. We conclude that the greater affinity for MgADP of tonic muscle myosin and the reattachment of dephosphorylated myosin to actin may both contribute to the latch state.
Léguillette
Renaud
R
Meakins-Christie Laboratories, Department of Medicine, McGill University, 3626 St-Urbain St., Montreal, QC, Canada H2X 2P2.
Zitouni
Nedjma B
NB
Govindaraju
Karuthapillai
K
Fong
Laura M
LM
Lauzon
Anne-Marie
AM
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002645:Chickens; D005895:Gizzard, Avian / Q000378:metabolism*; D007834:Lasers; D018715:Microscopy, Video; D008954:Models, Biological; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms
D020033:Protein Isoforms; D000244:Adenosine Diphosphate; D018995:Myosin Heavy Chains