Inhibition of actomyosin ATPase by vanadate.

Abstract

Actin-myosin subfragment-l (SF-1) or actin-heavy meromyosin is dissociated by the binding of ADP and vanadate (Vi) A recent study demonstrated the inhibition of myosin ATPase activity by vanadate (Vi) (1). The formation of an extremely stable myosin-ADP-vanadate complex (t112 of 1-2 days) provides an explanation of the mechanism of inhibition. Further studies to be published elsewhere have shown that the normal myosin-ADP complex binds vanadate weakly and undergoes a slow isomerization to form a stable complex that is a competitive inhibitorin which M indicates myosin. Considering the similarity in structure of vanadate and phosphate and that vanadate is bound stoichiometrically at or near the active site, the M-ADP-Vit complex may be a stable analogue of the myosin-ADP-phosphate intermediate, which is believed to be a key intermediate in the myosin and actomyosin ATPase mechanism (2).In the present study we have examined the reactions of vanadate with actomyosin to test the hypothesis that M-ADP-Vit is indeed an analogue of the myosin-products intermediate.This state, which is generally referred to as the M.ADP.Pi**, state is characterized by the following properties. The complex is much more wealdy bound to actin than is myosin alone or M-ADP (3, 4). The rates ofdissociation ofADP and Pi are greatly increased by forming a complex with actin, and this increase in rate accounts for the activation of myosin ATPase activity by actin. The control ofthe ATPase activity ofregulated actomyosin (AM) by calcium ion involves a change either in the association constant of M.ADP-Pi** to the actin-tropomyosin-troponin complex or in the rate of release of products from the AM.ADP.Pi** complex (5-7). The evidence to be presented here shows that the AM.ADP.Vit complex has the same properties. MATERIALS AND METHODSMyosin was prepared from rabbit back and leg muscles by the method described by Perry (8); actin was made by the procedure of Spudich and Watt (9); heavy meromyosin (HMM) and subfragment-1 (SF-1) were obtained by chymotryptic digestion ofmyosin and purified by the method ofWeeds and Taylor (10); native tropomyosin (the one-to-one complex of troponin and tropomyosin) was made by the procedure of Hitchcock (11).The myosin-ADP-vanadate complex was formed by incubation ofthe protein with 0.5 mM ADP and 1 or 2 mM vanadate for 15 min (1). Longer incubation times had no further effect on the state of the protein or the ability to quantitatively regenerate the ATPase activity. Free ADP and vanadate were removed when necessary by passage of the protein solution in pH 8.5 or pH 7.0 buffer over a 1-cm Dowex-1 X 8 column. In all experiments vanadate was added by dilution from a concentrated stock solution at pH 10 to avoid the possible formation of complex ions.Free vanadate was determined colorimetrically by reaction with 4-(2-pyridylazo)resorcinol (PAR) as described (1). The rate of color development was first order in vanadate and PAR concentrations over the ranges used in the experiments. Because protein-bound van...

Substructure of the outer dynein arm

“…Enzymatic studies of dynein (45,47,58) and other ATPases (20,41) have concluded that vanadate does not prevent either the ATP binding or ATP hydrolysis steps of the ATPase cycle. However, an ADP.V complex forms that cannot be released from the enzyme, preventing further rounds of hydrolysis.…”

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

“…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). Although the actin-stimulated myosin ATPase activity has been described to be about 90% inhibited by ADP‚V 1 (12), it was suggested that this inhibition could be due in part to the presence of polymeric vanadate ions, because high vanadate concentrations (> 900 µM) are needed (13). In fact, it has been suggested that tetrameric vanadate (V 4 ) interacts with myosin, being responsible for the vanadate-induced photolytic cleavage of myosin subfragment 1 (14,15).…”

Suppression of muscle contraction by vanadate. Mechanical and ligand binding studies on glycerol-extracted rabbit fibers.

“…V; blocks the myosin and actomyosin ATPase reactions by forming a stable M -ADP -V; complex (Goodno, 1979). This intermediate dissociates very slowly (rate constant~z2.5 x 10-6 s-', Goodno, 1982), but binding to actin promotes V; and ADP release (Goodno and Taylor, 1982). V; also suppresses the contraction of skinned muscle fibers (Goody et al, 1980 ;Magid and Goodno, 1982).…”