Inhibition of myosin ATPase by vanadate ion.

Abstract

Inhibition of the myosin ATPase by vanadate ion (V1) has been studied in 90 mM NaCl/5 mM MgCl2/20 mM Tris HCI, pH 8.5, at 250C. Although the onset of inhibition during the assay is slow and dependent upon Vi concentration (kap -t 0.3 M-1 s-'), the final level of inhibition approaches iOO4o, provided the V; concentration is in slight excess over the concentration of ATPase sites. Inhibition is not reversible by dialysis or the addition of reducing agents. The source of this irreversible inhibition consists of the formation of a stable, inactive complex with the composition MADP-V1 (where M represents a single myosin active site). The complex has been isolated, and its mechanism of formation from M, ADP, and Vi has been studied. Omission of ATP increases the rate of formation by about 35-fold (kapp 11 M-1 s-'), yet this rate is still low in comparison with the rates of simple protein-ligand association reactions. This slowness is interpreted in terms of a rate-limiting isomerization step that follows the association of M, ADP, and V1: M*ADP*V1 _ Mt.ADP.V1 (t indicates the inactive product of the isomerization). (9) and Weeds and Taylor (10), respectively. The HMM fraction that precipitated between 45 and 60% saturated ammonium sulfate was dialyzed free of ammonium sulfate, centrifuged 30 min at 40,000 X g, and used within 10 days. The concentration of HMM was expressed in terms of the ATPase-site concentration, which was determined spectrophotometrically by using a value of A2801% = 6.47 (11), assuming a molecular weight of 340,000 and two ATPase sites per molecule.ATPase assays were carried out in buffer A (0.09 M NaCl/5 mM MgCl2/20 mM Tris-HCI, pH 8.5) at 250C by the addition of MgATP (final concentration, 1 mM) to HMM at 1-7 AM sites.Assay times ranged from 0.1 to 5 hr. The reaction was stopped in aliquots (1 ml) of the assay solution with 1 ml of 10% trichloroacetic acid. The aliquots were then clarified by centrifugation and half of each was analyzed for Pi by the procedure of Taussky and Shorr (12). V1 concentrations below 10 mM caused less than 1% interference.Vanadium Analysis. Stock solutions of V1 were prepared from either Na3VO4 (adjusted to pH 10 with 6 M HCl) or V205 (adjusted to pH 10 with 10 M NaOH) and then boiled to destroy yellow polymeric species such as V100286-(13). Standard solutions were prepared by volumetric dilution. In order to minimize the pH-dependent polymerization of Vi, all studies were carried out under the alkaline conditions used for the ATPase assays (buffer A). UV-visible spectra of Vi standard solutions were obtained by using a Cary 14 spectrophotometer, and the extinction coefficient was determined: Xrnax = 265 nm, C265 = 2925 M-1 cm-1. The Vi concentration was determined spectrophotometrically wherever possible.Where this was unfeasible (e.g., in the presence of protein), vanadium was determined by a modification of the colorimetric procedure of Pribil (14), using the metallochromic dye PAR.To a 1-ml sample in buffer A was added 100 Al of 1 M imidazole (pH 6.0) and ...

Decavanadate Binding to a High Affinity Site near the Myosin Catalytic Centre Inhibits F-Actin-Stimulated Myosin ATPase Activity

“…ATPase ActiVity by V 10 . Addition of decavanadate produced inhibition of the actin-stimulated myosin S1 ATPase activity ( Table 1).…”

“…More recently, fluorescent quenching studies have provided evidence for decavanadate high-affinity binding sites in myosin (18). We now show that the actin-stimulated myosin ATPase activity is indeed strongly inhibited by V 10 and that this inhibition is due to the binding of this covalently closed polyanionic species of ellipsoidal-like shape (4,19) to a specific high-affinity site in the myosin headgroup.…”

“…The rate-limiting step of the ATP hydrolysis is the release of P i from myosin, which is accelerated by the rebinding of actin (9). Vanadate inhibits myosin ATPase activity by forming, in the absence of actin, a very stable complex with MgADP that mimics either the transition state of hydrolysis or the ADP‚P i intermediate state (10). In the presence of actin, the rate of vanadate release increases by 10 5 compared to that of M‚ADP‚V 1 alone (5 × 10 -6 s -1 ) (11).…”

Inhibition of Skeletal Muscle S1-Myosin ATPase by Peroxynitrite

“…S1 contains the binding sites for ATP and actin and is the protein domain responsible for the myosin-ATPase activity during muscle contraction (29). During myosin-ATPase activity, S1 cycles through an intermediate state M**‚ADP‚P i (30,31). Vanadate inhibits myosin-ATPase activity by forming, in the absence of actin, a very stable complex with MgADP that mimics this transition M**‚ADP‚P i intermediate state (30).…”

“…During myosin-ATPase activity, S1 cycles through an intermediate state M**‚ADP‚P i (30,31). Vanadate inhibits myosin-ATPase activity by forming, in the absence of actin, a very stable complex with MgADP that mimics this transition M**‚ADP‚P i intermediate state (30). The addition of ADP‚V 1 and ADP‚AlF 4 (200 µM) along with a short incubation time (usually less than 30 min) has been used to mimic the intermediate activated M**‚ADP‚P i state (32)(33)(34).…”