Cross-bridge cycling at rest and during activation. Turnover of myosin-bound ADP in permeabilized smooth muscle.
Single turnover experiments were performed on myosin-bound ADP by measuring the time course of incorporation of [3H]ADP following rapid formation of [3H]ATP by photolysis of caged [3H]ATP. Permeabilized rabbit portal veins were incubated in a solution at 20 degrees C with 1 mM MgATP, 20 mM phosphocreatine, 1 mg/ml creatine phosphokinase, and containing [14C]ATP and high specific activity caged [3H]ATP. At variable times following a UV flash, the muscle was frozen, nucleotides were extracted, and the ratio 3H:14C in ADP was compared to that in ATP. At rest, the exchange of bound ADP occurred with a rate constant of 0.004 s-1. When the myosin light chain was about 80% thiophosphorylated, and the muscle was generating maximum isometric force, there appeared a fast phase of ADP exchange (44% of the total) which had a rate constant of 0.2 s-1. The change in rate of ADP exchange on myosin is sufficient to explain the measured increase in ATPase activity upon thiophosphorylation of the myosin light chain. A simple analysis of the data suggests that there is a 50-fold increase in the cycling rate of cross-bridges in the muscle upon phosphorylation under isometric conditions. The fraction of ADP exchanged at 10 s following photolytic release of [3H]ATP was found to be approximately linearly related to the degree of thiophosphorylation of the myosin light chain. This supports the idea that phosphorylation of the light chain causes the transition of myosin from the resting (slow ATPase) cycle into the activated (fast ATPase) cycle, and that the fraction of myosin in the fast cycle is directly determined by the degree of light chain phosphorylation. The data are also consistent with the cooperativity model described previously by Vyas et al.
Vyas
T B
TB
Department of Physiology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Mooers
S U
SU
Narayan
S R
SR
Siegman
M J
MJ
Butler
T M
TM
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010766:Phosphorylation; D011169:Portal Vein; D011817:Rabbits
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins