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 ...
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...
The structural stabilities of all the familiar proteolytic fragments of myosin have been investigated in melting studies over the pH ranges 5.5-7.0 in 0.5 M KCl. All fragments except subfragment 2 undergo a melting transition manifested by the cooperative uptake of protons in the temperature range 34-47 degrees C, and these fragments experience an increase in transition temperature, Tm as the pH is increased. Subfragment 2 undergoes a melting transition in the 43-55 degrees C range, manifested by the dissociation of protons, and it experiences a decrease in Tm as the pH is increased. These results suggest that pH changes can modulate the relative stabilities of the light meromysin, subfragment-1, and subfragment-2 regions of the myosin molecule.
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