SUMMARY1. Orthophosphate (Pi, 041-2-0 mM) was photogenerated within the filament lattice of isometrically contracting glycerinated fibres of rabbit psoas muscle at 10 and 200C. The Pi was produced by laser flash photolysis of the photolabile compound 1-(2-nitrophenyl)ethylphosphate (caged Pi). Caged Pi caused a depression of tension that was much smaller than that caused by Pi.2. Photolysis of caged P1 produced a decline in isometric force composed of four phases: phase I, a lag phase (e.g. 1-4 ms at 1000) during which force did not change; phase II, an exponential decline by as much as 20 % of the pre-pulse force; phase III, a partial force recovery (0-3 % of the pre-pulse force); and phase IV, a further slow (0 5-3 s) decline to the steady value. Phases I, III and IV were largely independent of [Pi] and are likely to be indirect effects caused by the caged Pi photolysis.3. Both the rate and amplitude of phase II depended markedly on [Pi]. The amplitude of phase II was similar to the reduction of steady-state force by Pi.

Rapid laser pulse-induced photolysis of an adenosine triphosphate precursor in muscle fibers abruptly initiated cycling of the cross-bridges. The accompanying changes in tension and stiffness were related to elementary mechanochemical events of the energy-transducing mechanism. When inorganic phosphate was present at millimolar concentrations during liberation of adenosine triphosphate in the absence of calcium, relaxation was accelerated. Steady active tension in the presence of calcium was decreased but the approach to final tension was more rapid. These results suggest that, during energy transduction, formation of the dominant force-generating cross-bridge state is coupled to release of inorganic phosphate in a reaction that is readily reversible.

We screened a small-molecule library for inhibitors of rabbit muscle myosin II subfragment 1 (S1) actin-stimulated ATPase activity. The best inhibitor, N-benzyl-p-toluene sulphonamide (BTS), an aryl sulphonamide, inhibited the Ca2+-stimulated S1 ATPase, and reversibly blocked gliding motility. Although BTS does not compete for the nucleotide-binding site of myosin, it weakens myosin's interaction with F-actin. BTS reversibly suppressed force production in skinned skeletal muscle fibres from rabbit and frog skin at micromolar concentrations. BTS suppressed twitch production of intact frog fibres with minimum alteration of Ca2+ metabolism. BTS is remarkably specific, as it was much less effective in suppressing contraction in rat myocardial or rabbit slow-twitch muscle, and did not inhibit platelet myosin II. The isolation of BTS and the recently discovered Eg5 kinesin inhibitor, monastrol, suggests that motor proteins may be potential targets for therapeutic applications.

SUMMARY1. The interaction between MgADP and rigor cross-bridges in glycerol-extracted single fibres from rabbit psoas muscle has been investigated using laser pulse photolysis of caged ATP (P3-1(2-nitrophenyl)ethyladenosine 5'-triphosphate) in the presence of MgADP and following small length changes applied to the rigor fibre.2. Addition of 465 gM-MgADP to a rigor fibre caused rigor tension to decrease by 15-3 + 0 7 % (s.E.M., n = 24 trials in thirteen fibres). The half-saturation value for this tension reduction was 18+4 /SM (n = 23, thirteen fibres).3. Relaxation from rigor by photolysis of caged ATP in the absence of Ca2`was markedly slowed by inclusion of 20 /,M-2 mM-MgADP in the photolysis medium.4 10. Computer simulations of the cross-bridge reactions involving ADP release, MgATP binding, detachment, and reattachment into force-generating intermediates were fitted to the transients recorded following photolysis of caged ATP. In the absence of ADP the time course of the transients could be simulated using a simple model without strain-dependent rate constants and assuming that attached crossbridge states in rapid equilibrium with detached states ({AM.ATP} and { M ADP Pi exerted zero force. However, in the presence of MgADP the transients simulated with these assumptions showed a deeper tension dip following photolysis of caged ATP than the experimental records.11. Two explanations of this discrepancy are considered. In the first hypothesis, rigor cross-bridges are assumed to be distributed over a wide range of forces, including negative forces, and ADP dissociates more rapidly from negatively strained cross-bridges than from positively strained ones. In the presence of MgADP, rapid detachment of the negatively strained cross-bridges limits the magnitude of tension dip following ATP release.12.

The relation between the chemical and mechanical steps of the myosin-actin ATPase reaction that leads to generation of isometric force in fast skeletal muscle was investigated in demembranated fibers of rabbit psoas muscle by determining the effect of the concentration of inorganic phosphate (Pi) on the stiffness of the half-sarcomere (hs) during transient and steady-state conditions of the isometric contraction (temperature 12 degrees C, sarcomere length 2.5 mum). Changes in the hs strain were measured by imposing length steps or small 4 kHz oscillations on the fibers in control solution (without added Pi) and in solution with 3-20 mM added Pi. At the plateau of the isometric contraction in control solution, the hs stiffness is 22.8 +/- 1.1 kPa nm(-1). Taking the filament compliance into account, the total stiffness of the array of myosin cross-bridges in the hs (e) is 40.7 +/- 3.7 kPa nm(-1). An increase in [Pi] decreases the stiffness of the cross-bridge array in proportion to the isometric force, indicating that the force of the cross-bridge remains constant independently of [Pi]. The rate constant of isometric force development after a period of unloaded shortening (r(F)) is 23.5 +/- 1.0 s(-1) in control solution and increases monotonically with [Pi], attaining a maximum value of 48.6 +/- 0.9 s(-1) at 20 mM [Pi], in agreement with the idea that Pi release is a relatively fast step after force generation by the myosin cross-bridge. During isometric force development at any [Pi], e and thus the number of attached cross-bridges increase in proportion to the force, indicating that, independently of the speed of the process that leads to myosin attachment to actin, there is no significant (>1 ms) delay between generation of stiffness and generation of force by the cross-bridges.