Yuji Tonomura

Influence of solvent composition on the molecular shape and the enzymic activity of myosin A

Abstract In 0.5 M KCl, the molecular weight, radius of gyration, sedimentation constant, and intrinsic viscosity of myosin A were respectively 5.94 × 10 5 , 1058 A., 6.11 S , and 2.30. In the ethanol-water-KCl mixture, the ATPase activity and the radius of gyration were almost unchanged, though the molecular weight increased with time. In the chloroethanol-water-KCl mixture, at the first stage the molecular weight increased, and at the second stage the ATPase activity decreased accompanied by an increase in the viscosity and a decrease in the radius of gyration. In the dioxane-water-KCl solution, at the first stage the ATPase activity increased accompanied by a decrease in the viscosity and an increase in the radius of gyration, but at the second stage the decrease in ATPase activity with time was accompanied by an increase in viscosity and a decrease in the radius of gyration.

Inhibition of myosin B-adenosinetriphosphatase by excess substrate.

Abstract 1. 1. The inhibition by excess substrate of myosin B-ATPase commences at a lower concentration of ATP as the ionic strength increases. 2. 2. A marked inhibition by substrate is observed in the presence of Mg ++ and a weak one in the presence of Mn ++ . In the presence of other bivalent cations, no inhibition can be observed. 3. 3. The inhibition by substrate disappears on the addition of a small amount of PCMB or Salyrgan. 4. 4. The inhibition by substrate is remarkably promoted by chelate compounds. Their promoting effect parallels closely with their relaxing effect of muscle model. 5. 5. It is demonstrated that the ATPase activity of superprecipitated myosin B is very much higher than that of a nonsuperprecipitated one, and it is concluded that the inhibition by substrate is due to the inhibition of superprecipitation by a high concentration of ATP. 6. 6. The ATP level, where the inhibition commences, is increased by an increase of the actin content of synthetic actomyosin, and at a sufficiently high ATP ATPase activity of synthetic actomyosin approaches that of myosin A.

Physicochemical studies on denaturation of myosin-adenosinetriphosphatase

Abstract Denaturation of myosin A- and myosin B-ATPase was investigated in 0.5–0.6 M KC1 and at 30 °C. and pH 7.0. The denaturation of myosin A proceeded in accordance with the first-order law until ATPase disappeared, while that of myosin B proceeded according to the first-order law to a constant value of ATPase. From the results on denaturation of the three parts of myosin B separated by ultracentrifugation and of synthetic actomyosin, it was shown that the rapid denaturation of the first-order myosin B was due to myosin A contaminant in the preparation and/or released from the “heavy” components of myosin B. Under present conditions, the denaturation of myosin A was not affected by the addition of cysteine and by passing nitrogen or oxygen through the solution. By salting-out analysis, it was found that the main peak of myosin A moved from 38.5 to 32% saturation of ammonium sulfate with denaturation, and that a subunit, occupying about 10% of the total, was revealed in the precipitation range of 50–60%. This subunit was water soluble and considerably heat stable and traveled in the ultra-centrifuge as one peak, its s20w being 2.1–2.8. The reduced viscosity of myosin A and the weight-average molecular weight of myosin A increased with decrease of ATPase. When PP was added and ATPase was assayed using Ca++ as an activator of ATPase, a lag phase appeared in the denaturation of ATPase. After the lag phase, the denaturation proceeded with a smaller velocity than in the absence of PP, while, when Mg++ was used as a modifier of ATPase, the effect of PP could not be observed. From these results a reaction scheme was proposed which satisfactorily explained the course of the denaturation under various conditions.

On the active site of myosin A-adenosine triphosphatase IV. Properties of binding of trinitrobenzenesulfonate and p-chloromercuribenzoate to myosin a

Abstract The addition of ATP or PP i retarded conspicuously the rate of specific binding of trinitrobenzenesulfonate to one mole of lysine residue in 2.1·10 5 g of myosin A. The rate of specific binding of trinitrobenzenesulfonate to myosin A was increased in 4M LiBr which melts almost completely the helical structure of myosin A. The rate was also remarkably influenced by the treatment of myosin A with 1.5 M LiBr which inactivated ATPase (ATP phosphohydrolase, EC 3.6.1.3) without changing significantly the helical content of the myosin A molecule as a whole. Furthermore, it was demonstrated that actomyosin reconstituted from myosin A treated with p -chloromercuribenzoate and β-mercaptoethanol does not show a clearing response on addition of high concentrations of ATP and its ATPase activity is not inhibited by the substrate or by EDTA. The p -chloromercuribenzotae added was completely removed from myosin A by the further addition of excess β-mercaptoethanol and the optical rotatory dispersion of myosin A was insignificantly altered by the treatment with p -chloromercuribenzoate and β-mercaptoethanol.

Hydrolysis by myosin A of several synthetic adenosine triphosphate analogues

Abstract The behaviours of three synthetic ATP analogues, 6-dimethylamino-9-β- d -ribofuranosylpurine 5′-triphosphate (dimethyl-ATP), 9-(4′-hydroxybutyl)-6-aminopurine 4′-triphosphate (BTP) and ribose 5-triphosphate (RTP), on hydrolysis by myosin A were investigated mainly in 0.5 M KCl and 5 m M CaCl 2 (pH7.0, 20°), together with those of ATP, ITP and inorganic tripolyphosphate (TP i ). The following results were obtained. 1. 1. The effect of divalent cations, such as Mg 2+ , Mn 2+ , Ca 2+ , Sr 2+ and Ba 2+ , on the rate of hydrolysis of BTP, TP i subtrates, the relations between the ionic radii ( r ) of various added divalent cations and the rates of hydrolysis were expressed by a bell-shaped curve, which showed a maximum at r = 0.95 A . 2. 2. The Michaelis constants of hydrolysis of ITP, TP i , BTP, RTP, dimethyl-ATP and ATP were 3, 2.5, 2.5, 1.7, 1.4 and 0.2 and m M , respectively, while the ratios of the maximum velocity of hydrolysis of ITP, BTP, dimethyl-ATP, TP i and RTP to that of ATP were, 7, 3, 1.3, 1 13.5 and 1 20 , respectively. 3. 3. The Michaelis constant and the maximum velocity of hydrolysis of TP i showed a remarkable increase with decreasing pH, while the rate of hydrolysis of RTP depended slightly on the pH. The dependence of the rate of hydrolysis of BTP on the pH showed a slight depression at the neutral pH and its Michaelis constant was independent of the pH. 4. 4. p -Chloromercuribenzoate and EDTA inhibited the hydrolysis of TP i , RTP, ITP, dimethyl-ATP and BTP, although they activated ATPase. These results have been interpreted on the basis of the reaction between myosin A and nucleoside triphosphates which has been proposed by one of the present authors 7,24 and is further discussed below.

Physicochemical studies on denaturation of myosin—Adenosinetriphosphatase. II. Changes in chromatographic profile and optical rotation

Abstract The inactivation of myosin A-ATPase proceeds according to the first-order law. The rate constants of the inactivation were proportional to the 1.3, 0, and −3.3 powers of [H+], respectively, in the ranges of pH 5.2–6.0, 7.5–8.5, and 10.0–10.5. In these three ranges of pH, ΔH‡ values were 31.6, 52.6, and 42.1 kcal./mole, respectively. During the incubation at 36 °C. the α component in chromatographic profiles on diethylaminoethylcellulose decreased, the β component increased, and finally the β component decomposed. However, specific ATPase activities of both the α and the β components were not constant and decreased with time. Under three conditions (pH 7.0 and 30 °C., pH 5.7 and 20 °C., and pH 10.3 and 20 °C.), the change in optical rotatory dispersion with time was measured and compared with that of ATPase activity. The helical content decreased very slowly and only by a few per cent, even after ATPase activity disappeared.

III. Interaction between synthetic adenosine triphosphate analogues and actomyosin systems

Abstract The following compounds were synthesized as analogues of ATP:6-morpho 5′-triphosphate (III) and 2,6-dimethlymercapto-9-β- d -ribofuranosylpurine 5′-triphosphate (V). The interactions of these analogues with actomyosin system were investigated, together with those of 3′-deoxythymidine 5′-triphosphate (I), thymidine 5′-triphosphate (II)and 2′,3′-O-isopropylidene adenosine 5′-triphosphate (IV). The degrees of decrease in light-scattering of myosin B on addition of these analogues were similar to that induced by ATP, except in the case of Compound I. The rates of hydrolysis of analogues by myosin B in 0.6 M KCl and 7 mM Ca72+ were in the decreasing order of ATP > IV ≈ II > V > I ≈ III, while the order of hydrolysis in 0.075 M KC1 and 2mM Mg2+ was II > ATP > IV > I > III > V. Compounds II and IV, as well as ATP, induced contraction of myofibrils, while Compounds I, II and V did not. It was concluded that hydrogen bondings at the 6-N or O of the base and the 3′-O of ribose with myosin are necessary for the rapid hydrolysis fo an ATP analogue and for contraction of myofibrils by the analogue.

The optical-rotatory dispersion of myosin A: II. Effect of dioxane and p-chloromercuribenzoate

Effects of dioxane and p-chloromercuribenzoate (PCMB) on the optical-rotatory dispersion of myosin A were measured in 0.6 M KCl at pH 7.0 and compared with those on the ATPase activity. The α-helical content of myosin A estimated from the b0 term of the Moffitt-Yang plot was 57–61%. On addind 8–10 volume percent of dioxane, increase in the helical content by several percent and pronounced activation of ATPase were firstly observed and were followed by gradual decreases in the helical content and the ATPase activity. 2 h after the addition of dioxane, the helical content decreased only by a few percent, while the ATPase activity disappeared completely. Immediately after the addition of dioxane, specific rotatory power at 5000 A showed its maximum at about 10% of dioxane in accordance with the activation of ATPase. The helical content of the alkaline-inactivated myosin A, however, remained constant on the addition of dioxane. On adding PP1 before and at various times after the addition of dioxane, shift in the helical content caused by dioxane was depressed completely and the content remained constant during the measurements. On adding 3–4 moles PCMB per 105 g of myosin A, the maximum velocity and the Michaelis constant of ATPase at 20° were increased, respectively, from 0.22 to 0.44 mmoles P1/min/g and from 1.3 to 1.5·104M, and temperature dependence of the maximum velocity was increased significantly, while on adding 8 moles PCMB the ATPase activity was completely inhibited. The helical content of myosin A increased by several percent on addition of 4 moles PCMB and decreased by several percent on 8 moles PCMB per 105 g. On adding PCMB in the presence of ATP or PP1, the helical content fell in between those in the presence of either of the two. On the basis of these and other observations, it was suggested that conformation of the active site is very susceptible to influences of dioxane and PCMB and that, accordingly, a minute change in the helical content induces a pronounced change in the ATPase activity.

Binding of p-nitrothiophenol to myosin a

Abstract The p -nitrothiophenol-myosin A compound was produced by reaction of p -nitrothiophenol with myosin A in the presence of Mg 2+ and ATP, and was isolated by gel filtration through a Sephadex column. The change in absorption spectrum by the binding of p -nitrothiophenol to myosin A was similar to that of the formation of a thiolester bond between p -nitrothiophenol and succinic anhydride. The p -nitrothiophenol-myosin A compound was stable at acidic and neutral pH, but was unstable at alkaline pH. The stability was not affected by replacement of air with N 2 gas. The p -nitrothiophenol bound to myosin A was easily displaced by cysteine and H 2 S. p -Nitrothiophenyl-peptides were isolated with 24% recovery after proteolysis and chromatography on talc, since the proteolysis did not accelerate the liberation of p -nitrothiophenol. The hydromate was formed quantitatively by the addition of NH 2 OH to p -nitrothiophenyl-peptides. The amino acid composition of the p -nitrothiophenyl-peptides was determined, and Arg, Asp, Ser, Glu, Ileu and Leu were found as amino acid residues whose mole ratios to p -nitrothiophenol were nearly equal to or higher than 1. These results establish the covalent binding of p -nitrothiophenol to myosin A in the presence of Mg 2+ and ATP, and strongly suggest the binding of p -nitrothiophenol on a carboxyl side-chain of Glu or Asp in myosin A as an acylthiol bond. Effects of divalent cations, pH and modifiers of the ATPase activity of the p -nitrothiophenyl-myosin A were also investigated. The pH-activity curve of the p -nitrothiophenyl-myosin A was similar to those of the p -chloromercuribenzoate-myosin A compound and the trinitrophenyl-myosin A, and showed no depression at neutral pH. The ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the p -nitrothiophenyl-myosin A was activated by p -chloromercuribenzoate or EDTA, whereas the ATPase of the trinitrophenyl-myosin A was not activated. Actomyosin reconstituted from the p -nitrothiophenyl-myosin A showed ATPase activity of the typical actomyosin type.

THE OPTICAL ROTATORY DISPERSION OF MYOSIN A. IV. CONFORMATIONAL CHANGES IN MEROMYOSINS.

The effect of 8% dioxane on the optical rotatory dispersion of myosin A purified by treatment with DEAE-cellulose was measured in 0.6 M KCl and at pH 7.0 and 20°. The increase in the —b0 term by 5% was followed by a gradual decrease to 0.88 of the original. The —b0 term of L1-meromyosin in 0.6 M KCl and at pH 7.0 and 20% was 575 and was unchanged by the addition of dioxane, ATP of PP1. On the other hand, the —b0 term of H2-meromyosin in 0.3 M KCl and at pH 7.0 and 20% was 263. It increased by several percent immediately after the addition of 8% dioxane and decreased gradually with time. It increased by several percent on the addition of 4 moles p-chloromercuribenzoate, but decreased by several percent on the addition of 8 moles p-chloromercuribenzoate per 105 g protein. It was also decreased by the binding of H2-meromyosin with F-actin. ATP increased the —b0 term of H2-meromyosin by several percent, though PP1 decreased the term by several percent. Thus these reagents change the helical content of H2-meromyosin in ways similar to the case of myosin A. The extents of the changes in the helical content of H2-meromyosin by F-actin and ATP were, however, higher than those observed in myosin A.