Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor

Abstract

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)

Hair-bundle movements elicited by transepithelial electrical stimulation of hair cells in the sacculus of the bullfrog

“…23 and P. Martin, D.B., Y. Choe, and A.J.H., unpublished data). To examine the role of these motors in electrically driven hair-bundle movements, we applied butanedione monoxime, a drug that interferes with the ATPase cycle of myosin by promoting weak binding to actin filaments (27). As reported (P. Martin, D.B., Y. Choe, and A.J.H., unpublished data), butanedione monoxime completely suppressed spontaneous oscillations (Fig.…”

Small-Molecule Inhibitors of Myosin Proteins

“…The fact that the inhibitory effects of this molecule are reversible further establishes blebbistatin as a powerful tool for characterizing precise cellular events. (Figure 1) [40,41]. However, the molecule has a low affinity for myosin and concentrations in the 10-mM range are required to achieve myosin inhibition [42].…”

Azidoblebbistatin, a photoreactive myosin inhibitor

“…The required precursors could be synthesized by direct aromatic nitration (16), but this reaction usually has low yield and results in degradation of the parent molecule or This article is a PNAS Direct Submission. 1 To whom correspondence may be addressed. E-mail: kovacsm@elte.hu or malna@elte.…”

“…Cell-permeable small molecules that can perturb myosin functions have greatly aided the dissection and understanding of molecular processes underlying the above phenomena. Myosin II inhibitors described to date include 2,3-butanedione monoxime (BDM) (1,2), N-benzyl-p-toluenesulphonamide (BTS) (3), and blebbistatin (4)(5)(6). As BDM has turned out to have a broad effect on many other proteins (7) and the inhibitory effect of BTS is limited to fast skeletal muscle myosin, until now blebbistatin has been the only potent tool for specific blocking of myosin II-dependent processes in various species and cell types.…”