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.

The kinetics of the force generating and Pi release steps of the actomyosin-adenosinetriphosphatase (ATPase) cycle have been compared in Ca(2+)-activated skinned fibers of rabbit soleus (slow twitch) and psoas (fast twitch) muscle. Pi was rapidly photogenerated within the fiber lattice by laser flash photolysis of caged Pi [1-(2-nitro)phenylethyl phosphate]. Pi reduces isometric tension in the steady state but is less effective in slow-twitch muscle than in fast-twitch muscle (e.g., 14 mM Pi reduces tension by 29 +/- 4.6% in slow and by 47 +/- 5.3% in fast). The tension response to a sudden increase in Pi concentration in slow-twitch muscle has four phases, but as in fast-twitch muscle, only phase II (an exponential decline in force) appears to be caused by Pi binding to cross bridges, whereas the other three phases are probably indirect effects caused by caged Pi photolysis. The amplitude of phase II is consistent with the steady-state reduction in force by Pi. The rate of phase II (kappa Pi) is 3.9 +/- 0.33 s-1 at 20 degrees C and 0.28 +/- 0.02 s-1 at 10 degrees C (1 mM Pi). kappa Pi is thus 33 times slower in slow-twitch muscle than in fast at 20 degrees C and 84 times slower at 10 degrees C. In contrast to fast-twitch muscle, in slow muscle kappa Pi is sufficiently slow to partially limit the ATPase turnover rate.

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The ATP-induced dissociation of actoSl has been studied at temperatures between -IO'C and + 30°C in a stopped-flow apparatus using ethylene glycol as antifreeze. At temperatures at and below 0°C the observed rate of the dissociation of actin shows a hyperbolic dependence on ATP concentration. This is interpreted in terms of a rapid binding of ATP followed by an isomerisation of the ternary complex which results in actin dissociation. Ethylene glycol weakens ATP binding but the rate of the isomerisation is unaffected. The second order rate constant for the dissociation shows a break in the Arrhenius plot. Actomyosin d~s~iatio~ Arrhenius plot, disco~t~~~ous Ethylene glycolSubzero temperature stooged-how

The pre-steady-state kinetics of MgATP hydrolysis by nitrogenase from Klebsiella pneumoniae were studied by stopped-flow calorimetry at 6 degrees C and at pH 7.0. An endothermic reaction (delta Hobs. = +36 kJ.mol of ATP-1; kobs. = 9.4 s-1) in which 0.5 proton.mol of ATP-1 was released, has been assigned to the on-enzyme cleavage of MgATP to yield bound MgADP + Pi. The assignment is based on the similarity of these parameters to those of the corresponding reaction that occurs with rabbit muscle myosin subfragment-1 (delta Hobs. = +32 kJ.mol of ATP-1; kobs. = 7.1 s-1; 0.2 proton released.mol of ATP-1) [Millar, Howarth & Gutfreund (1987) Biochem. J. 248, 683-690]. MgATP-dependent electron transfer from the nitrogenase Fe-protein to the MoFe-protein was monitored by stopped-flow spectrophotometry at 430 nm and occurred with kobs. value of 3.0 s-1 at 6 degrees C. Thus, under these conditions, hydrolysis of MgATP precedes electron transfer within the protein complex. Evidence is presented that suggests that MgATP cleavage and subsequent electron transfer are reversible at 6 degrees C with an overall equilibrium constant close to unity, but that, at 23 degrees C, the reactions are essentially irreversible, with an overall equilibrium constant greater than or equal to 10.

1. The fluorescence changes accompanying the binding of ATP and adenosine 5'-[gamma-thio]triphosphate (ATP gamma S) to myosin subfragment 1 (S1) and actomyosin subfragment 1 (actoS1) have been reinvestigated at 20 degrees C and 1 degree C, pH 7.0, 0.1 M-KCl. 2. Two successive fluorescence enhancements are observed following ATP binding to both S1 and actoS1. 3. The slow fluorescence change has the same rate with S1 and actoS1, and is due to the ATP cleavage step. 4. With actoS1 the fast fluorescence change occurs after dissociation, so a new intermediate, S1 ATP, is required on the actoS1 pathway. 5. The dissociation of actoS1 by ATP gamma S results in a fluorescence enhancement with the same apparent rate as dissociation, but indirect evidence suggests that this too occurs on a dissociated state.

The adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) induced dissociation of actomyosin subfragment 1 (S1) has been investigated by monitoring the light scattering changes that occur on dissociation. We have shown that ATP gamma S dissociates acto-S1 by a mechanism similar to that of ATP but at a rate 10 times slower. The maximum rate of dissociation is limited by an isomerization of the ternary actin-S1-nucleotide complex, which has a rate of 500 s-1 for ATP gamma S and an estimated rate of 5000 s-1 for ATP (20 degrees C, 0.1 M KCl, pH 7.0). The activation energy for the isomerization is the same for ATP and ATP gamma S, and both show a break in the Arrhenius plot at 5 degrees C. The reaction between acto-S1 and ATP was also followed by the fluorescence of a pyrene group covalently attached to Cys-374. We show that the fluorescence of the pyrene group reports the isomerization step and not actin dissociation. The characterization of this isomerization is discussed in relation to force-generating models of the actomyosin cross-bridge cycle.

1. The enthalpy changes during individual reaction steps of the myosin subfragment 1 ATPase were studied with the use of a new stopped-flow calorimeter [Howarth, Millar & Gutfreund (1987) Biochem. J. 248, 677-682]. 2. At 5 degrees C and pH 7.0, the endothermic on-enzyme ATP-cleavage step was observed directly (delta H = +64 kJ.mol-1). 3. ADP binding is accompanied by a biphasic enthalpy change. 4. The release and uptake of protons was investigated by the use of two buffers with widely different heats of ionization. 5. Protons are involved in all four principal steps of the myosin subfragment 1 ATPase.