2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)

Stopped-flow kinetic and fluorescence spectroscopic analyses, including solvent and temperature perturbations, of five isofunctional structural mutants of calmodulin indicate that calcium binding to calmodulin follows the order site III, site IV, site I, site II, with dissociation occurring in the reverse order. Each of the isofunctional structural mutants contains a single tryptophan residue, introduced by site-specific mutagenesis, as an internal spectroscopic reporter group that was used as a probe of local conformational change. Calcium binding was studied by using flow dialysis or by using fluorescence spectroscopy and monitoring the change in the single tryptophan residue in each calcium-binding site. Calcium removal was examined by using EDTA and monitoring tryptophan fluorescence or by using Quin 2 and monitoring the change in the chromophoric chelator. Computational analysis of the data suggests a rate-limiting step for dissociation between calcium removal from sites I/II and sites III/IV. Unexpected results with the site IV isofunctional mutant (Q135W-CaM) indicated cross-talk between the amino and carboxyl terminal halves of CaM during the calcium-binding mechanism. Studies with ethylene glycol provided empirical data that suggest the functional importance of the electrostatic potential of CaM, or the molarity of water, in the calcium-binding process. Altogether, the data allowed a kinetic extension of the sequential, cooperative model for calcium binding to calmodulin and provided values for additional parameters in the model of calcium binding to CaM, a prototypical member of the family of proteins required for calcium signal transduction in eukaryotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

There is a considerable degree of ambiguity in the literature regarding the role of the 2',3'‐cyclic phosphodiesters formed during the reaction of RNA cleavage catalysed by ribonuclease. Usually the reaction is considered to take place in two steps: in the first step there is a transphosphorylation of the RNA 3',5'‐phosphodiester bond broken yielding a 2',3'‐cyclic phosphodiester which in the second step is hydrolysed to a 3'‐nucleotide. Although in many occasions, either explicitly or implicitly, the reaction is treated as taking place sequentially, this is not the case as it has been shown that the 2',3'‐phosphodiesters are actually released to the medium as true products of the reaction and that no hydrolysis of these cyclic compounds takes place until all the susceptible 3',5'‐phosphodiester bonds have been cyclised. Comparison of the hydrolysis and alcoholysis of the 2',3'‐phosphodiesters catalysed by RNase A indicates that the hydrolysis reaction has to be considered formally as a special case of the transphosphorylation back reaction in which the R group of the R‐OH substrate is just H. It is thus concluded that the 2',3'‐cyclic phosphodiesters formed in the ribonuclease A reaction are true products of the transphosphorylation reaction and not intermediates as usually considered.