Abstract
The regulation by calcium and rigor-bound myosin-S1 of the rate of acceleration of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) release from myosin-mdADP-P(i) by skeletal muscle thin filaments (reconstituted from actin-tropomyosin-troponin) was measured using double mixing stopped-flow fluorescence with the nucleotide substrate 2'-deoxy-3'-O-(N-methylanthraniloyl). The predominant mechanism of regulation is the acceleration of product dissociation by a factor of approximately 200 by thin filaments in the fully activated conformation (bound calcium and rigor S1) relative to the inhibited conformation (no bound calcium or rigor S1). In contrast, only 2-3-fold regulation is due to a change in actin affinity such as would be expected by "steric blocking" of the myosin binding site of the thin filament by tropomyosin. The binding of one ligand (either calcium or rigor-S1) produces partial activation of the rate of product dissociation, but the binding of both is required to maximally accelerate product dissociation to a rate similar to that obtained with F-actin in the absence of regulatory proteins. The data support an allosteric regulation model in which the binding of either calcium or rigor S1 alone to the thin filament shifts the equilibrium in favor of the active conformation, but full activation requires binding of both ligands.
Highlights
Summary of product dissociation kinetics (Pi and mdADP) by reconstituted thin filaments and F-actin kobs(mdADP) for the maximal rate of mdADP dissociation for the slow and fast components at saturating thin filament or actin concentrations in Figs. 3 and 4 are in boldface. kobs(Pi) is for the maximal rate of Pi dissociation for the slow and fast components at saturating thin filament or actin concentrations measured with ATP as the substrate and using phosphate-binding protein to measure Pi release [12]
Kcapp(M,Ca) ϭ kmax/(kF-actin Ϫ kmax), where kmax is the rate of the fast component of product release at saturating thin filament concentrations under the indicated conditions, and kF-actin is the maximum rate observed with F-actin in the absence of regulatory proteins
Our results indicate that the change in the tropomyosin position between the blocked and closed conformations explains only a very small part of the regulatory mechanism (2–3-fold) and that the principal mechanism of regulation is of the rate of product dissociation (ϳ200-fold)
Summary
Materials—ATP, 2Ј-deoxy-ATP, buffers, and salts were purchased from Sigma. mdATP was prepared from 2Ј-deoxy-ATP and purified according to the procedure of Hiratsuka [16] for mant-ATP. Reconstituted thin filaments were prepared from purified actin, troponin, and tropomyosin as described previously [12]. Reconstituted thin filaments displayed at least a 15-fold calcium sensitivity of the rates of steady state ATP hydrolysis (mole ratio of S1/actin ϭ 1/70 to 1/120) under identical temperature and buffer conditions used for pre-steady state assays except that the ATP concentration was 1 mM. Kinetic measurements of mdADP dissociation were observed using an excitation at 360 nm and emission Ͼ420 nm using a sharp cutoff filter. The Kintek stopped-flow was fitted with 2-ml syringes in positions A and B and a 5-ml syringe in position C so that both mixes were approximately equal volumes and to reduce the dilution of thin filaments in the final reaction mixture. The dilution of the syringe contents was 2/9 for S1 and mdATP and 5/9 for thin filaments
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