The rates of formation and dissociation of actin-myosin complexes. Effects of solvent, temperature, nucleotide binding and head-head interactions.
The rates of formation and dissociation of actin-subfragment 1 and actin-heavy mero-myosin complexes were measured by using light-scatter and the change in fluorescence of N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine (IAEDANS)-labelled acting as probes. Association rate measurements were made at low protein concentration, where the transients approximated to single exponentials with rate constants proportional to the concentration of reactant in excess. Dissociation rate measurements were made by displacing IAEDANS-actin from myosin with excess native actin and by a salt jump. The second-order rate constant of association for actin-subfragment 1 was 3 x 10(6) M-1 . s-1 in 60 mM-KCl at 13 degree C. It was decreased 10-fold in 500 mM-KCl and in 50% (v/v) glycol. It was decreased 6-fold when MgADP or Mg[beta gamma-imido]ATP bound to myosin. The dissociation rate constant was 0.012 s-1 in 60 mM-KCl at 13 degree C. It was increased 4-fold by 500 mM-KCl, 25-fold by 50% glycol, 8-fold by MgADP binding and 170-fold by Mg[beta gamma-imido]ATP binding. Ea for association was 70 kJ . mol-1 and for dissociation 35 kJ . mol-1. Heavy meromyosin associated at twice the rate observed for subfragment 1 and dissociated at less than one-twentieth of the rate for subfragment 1 (60 mM-KCl, 25 degree C), but when Mg[beta gamma-imido]ATP bound actin-heavy meromyosin dissociated at one-half the rate for subfragment 1. There were significant correlations between increase in the dissociation rate constant, decrease in binding constant and increase in magnitude of conformational change. The association rate constant did not correlate with any property of the actin-myosin complex.
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D006018:Glycols / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009218:Myosins / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011189:Potassium Chloride / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature
D000199:Actins; D006018:Glycols; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; C006421:1,5-I-AEDANS; D009218:Myosins