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At least 19 recordsLinked to original sources
PubMed1977

Calorimetric studies of the ADP binding to myosin subfragment 1, heavy meromyosin, and to myosin filaments.

A calorimetric titration method was used to study the ADP binding to the chymotryptic subfragments of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1), and to myosin aggregated into filaments at low ionic strength. The binding constant (K) and heat of reaction (deltaH, kiloJoules (moles of ADP bound)-1) were determined. For HMM in 0.5 M KCl, 0.01 M MgCl2, 0.02 M Tris (pH 7.8) at 12 degrees, log K = 5.92 +/- 0.13 and deltaH = -70.9 +/- 3.6 kJ mol-1. These results agree with our previous findings for myosin in 0.5 M KCl at 12 degrees. When the KCl concentration was reduced to 0.1 M, the binding constant did not change significantly (log K = 6.09 +/- 0.06) but the binding was more exothermic (deltaH = -90.1 +/- 3.3 kJ mol-1). Similar results were obtained for myosin filaments in 0.1 M KCl and also for both the isoenzymes of S-1(S-1(A1) and S-1(A2) in 0.1 M KCl. In 0.5 M KCl, the binding curves suggest that about one ADP is bound per active site, but as 0.1 M KCl, the apparent stoichiometry drops from 0.7 to 0.75. The most probable explanation is that there is some site heterogeneity which is more evident at lower ionic strength.

Adenosine Diphosphate↗
PubMed1979

[Study of the Mg2+-ATPase reaction of myosin using the NMR-31P method. Detection of adenylate kinase activity in a purified myosin subfragment I].

Isolated myosin heads (HMM-S1) were concentrated by ultrafiltration in an "Amicon" cell to a concentration of 120 mg/ml. Incubation of HMM-S1 with ATP in the presence of Mg2+ produced an AMP peak in the spectrum of NMR-31P whose size was increasing linearly with the reaction time. It was demonstrated that after heating of the sample the appearance of AMP in the incubation mixture was due to the presence of a small amount of adenylate kinase (or myokinase) in the purified preparation of HMM-S1.

Adenosine Triphosphatases↗
PubMed1977

Flexibility of myosin rod, light meromyosin, and myosin subfragment-2 in solution.

Myosin rod was prepared by papain proteolysis of myosin. The components of rod, light meromyosin (LMM) and subfragment-2 (S-2), were prepared by proteolysis of myosin and rod, respectively, using trypsin treated with tosylphenylalanine chloromethyl ketone. S-2, thus prepared, was of greater molecular weight than obtained previously, so that the combined molecular weights of LMM and S-2 were equal to that of rod, and S-2 contained virtually all of the region of the rod susceptible to trypsin. Electro-optical measurements were made on the three fragments in 2 mM sodium pyrophosphate, pH 9.3 at 3 degrees, over a large range of protein concentrations. Analysis of the relaxation of birefringence, at low protein concentration where there was no aggregation, showed that LMM (relaxation time 13.1 micros) behaves as a rigid cylinder. Rod (relaxation time 41.2 micros) and S-2 (relaxation time 6.0 micros) had relaxation rates that were too fast for rigid molecules of their dimensions, and therefore are not straight rods. This implies that myosin rod is flexible in the S-2 portion, presumably in the region susceptible to proteolysis. The implications of rod flexibility for the mechanism of muscle contraction are discussed.

Birefringence↗
PubMed1975

Electric birefringence of myosin subfragments.

Electric birefringence measurements have been made on aqueous solutions of myosin subfragments, heavy meromyosin, subfragments 1 and 2 (S-1 and S-2). All of these showed positive electric birefringence. Heavy meromyosin and S-2 showed a large intrinsic Kerr constant. From the analysis of the build up and decay process of the birefringence, the contribution of the slow induced dipole moment was concluded in heavy meromyosin and S-2, although the existence of the permanent dipole moment was not completely excluded. The decay process of the birefringence of heavy meromyosin was found to consist of two components; the fast one of which had a relaxation time of the same order as that of S-1. This is probably due to the presence of a flexible hinge in heavy meromyosin.

Adenosine Triphosphate↗
PubMed1978

The effects of divalent cations on the rotational mobility of myosin, heavy meromyosin and myosin subfragment-1 and on the binding of heavy meromyosin to actin.

The effects of the divalent cations Mg2+, Mn2+ and Ca2+ on the Brownian rotational motion of fluorescently labeled myosin, heavy meromyosin and myosin subfragment-1 were measured by the method of time-resolved fluorescence depolarization. When Mg2+ was added to solutions of myosin or heavy meromyosin and EDTA, their rotational mobility increased. Ca2+ had no effect. Mn2+ increased the mobility of heavy meromyosin but decreased that of myosin. None of these divalent cations effected the mobility of subfragment-1. The binding of heavy meromyosin to actin was affected very little by Mg2+ or EDTA over a wide range of conditions. Divalent cations appear to change the swivel about which the heads of myosin rotate, presumably by binding to light chain 2 (also called DTNB light chain). However, the heads are still able to bind actin in nearly the same way whether Mg2+ is present or not. The concentration of free Mg2+ for the mid-point of the change in heavy meromyosin mobility is in good agreement with that for EDTA activation of ATPase activity. This suggests that EDTA activation is due to removal of Mg2+ bound to myosin itself.

Actins↗
PubMed1979

Ca2+ bindings of pig cardiac myosin, subfragment-1, and g2 light chain.

Ca2+ binding to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4 x 10(5) M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1 x 10(-5) M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6 x 10(5) M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111--120). The Ca2+-binding constant was obtained as 4.1 x 10(3) M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.

Animals↗
PubMed1976

Interactions of the actin and nucleotide binding sites on myosin subfragment 1.

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K'A, KN, and K'N in the scheme (see article), were determined for the magnesium salts of ADP, adenyl-5'-yl imidodiphosphate AMP-P(NH)P, and PPi. The nucleotide binding site on S-1 was "mapped" with respect to its interaction on the actin binding site. The subsites were the beta- and gamma-phosphoryl groups of ATP bind had the largest effects. A quantitative measure of the interaction, the interaction free energy, was defined as -RT ln (KA/K'A). For ADP, K'A was 2.7 X 10(5) M-1 and the interaction free energy was -4.67 kJ M-1. For AMP-P(NH)P and PPi it was much larger. A ternary complex was shown to exist for ADP, S-1, and actin in the presence of Mg2+ and evidence from AMP-P(NH)P and PPi measurements indicated that ATP also likely forms a ternary complex. The mechanism of (S-1)-actin dissociation is discussed in light of these results.

Actins↗
PubMed1978

Preparation of a myosin-extracted "ghost" myofibril Sephadex conjugate column and its application to the separation of myosin subfragment-1 giving and not giving the initial burst of phosphate.

A "ghost" myofibril (myosin-extracted myofibril) Sephadex conjugate which specifically binds myosin, HMM and S-1 in the absence of Mg-ATP or Mg-PP can be prepared in a few days by conjugating "ghost" myofibrils to Sephadex beads. Binding ability is retained for over a month. It is used, therefore, for actin-affinity chromatography of myosin and its active fragments. It is under debate whether the two heads of the myosin molecule are functionally identical. Recently several reports have indicated that S-1 could be separated into two kinds of S-1, one giving the initial burst of phosphate and the other not, by assuming a difference in the affinity of the two kinds of S-1 to F-actin. Attempts are reported here to obtain these two components of S-1 separately by using the "ghost" myofibril Sephadex conjugate column. The method of S-1 separation reported by Shibata-Sekiya and Tonomura ((1976) J. Biochem, 80, 1371-1380), which used S-1 treated with CMB, was applied to the "ghost" myofibril Sephadex conjugate column. This resulted in the successful separation of S-1 modified with CMB giving no initial burst of phosphate and unmodified S-1 giving the initial burst of phosphate. A separation method based essentially on the principle employed by Taniguichi and Tawada ((1976) J. Biochem. 80, 853-860) gave an unsuccessful result.

Actins↗
PubMed1976

Separation of myosin subfragment 1 into two fractions, one having the burst site and the other having the non-burst site.

During Mn(II)-ATP hydrolysis by myosin, the predominant intermediate formed at the burst site of the enzyme below 10 degrees is the myosin-ADP complex formed by adding ADP to myosin, while above 10 degrees it is the myosin -ADP-P1 complex generated by ATP hydroolysis (Yazawa, Morita, & Yagi (1973) J. Biochem. 74, 1107; Hozumi & Tawada (1975) Biochim. Biophys. Acta 376, 1; Tawada & Yoshida (1975) J. Biochem. 78, 293). It is suggested that the second (non-burst) site of myosin predominantly forms the myosin-ATP complex (Hozumi & Tawada, ibid.). From these findings, it is expected that (i) myosin subfragment 1 (S1) having the burst site is bound to actin in Mn(II)-ATP solution containing ADP below 10 degrees, because it forms the S1-ADP complex even in the presence of ATP; (ii) the other S1, i.e., that having the non-burst site, is dissociated from actin, because it forms the S1-ATP complex. These two expectations were confirmed by viscosity measurements of acto-S1 solutions, giving a basis for the separation of S1 into two fractions: one having the burst site and the other having the non-burst site. S1 having the non-burst site could be extracted from partially papain [EC 3.4.22.2]-digested myofibrils of rabbit skeletal muscle with a solution containing MnCl2, ATP, and ADP at 0 degrees. S1 having the burst site was extracted from myofibrils already used for the extraction of S1 having the non-burst site, with a solution containing MgCl2 and ATP at 20 degrees. The former S1 fraction had Mg-ATPase [EC 3.6.1.3] activity, but scarcely showed any initial burst of Pi liberation. The latter S1 showed a Pi burst of more than 0.5 (M/M). The steady state ATPase activity of the former S1 was slightly higher than that of the latter. The burst size of normal S1, i.e., that extracted from papain-digested myofibrils with Mg-PPi or Mg-ATP, was 0.5 (M/M). The ultraviolet absorption spectrum of the non-burst type S1 was not changed by ADP but was changed by ATP, though the difference spectrum was distinct from that of normal S1 and the difference molar extinction coefficient at 289 nm was only 20% of that of normal S1. No significant difference was seen in the compositions of these two S1's and normal S1, as determined by SDS gel electrophoresis.

Adenosine Diphosphate↗
PubMed1976

Pre-steady-state kinetic evidence for a cyclic interaction of myosin subfragment one with actin during the hydrolysis of adenosine 5'-triphosphate.

A single cycle of adenosine 5'-triphosphate (ATP) hydrolysis by a complex of actin and myosin subfragment one (acto-S-1) was studied in a stopped-flow apparatus at low temperature and low ionic strength, using light scattering to monitor the interaction of S-1 with actin and fluorescence to detect the formation of fluorescent intermediates. Our results show that the addition of a stoichiometric concentration of ATP to the acto-S-1 causes a cycle consisting of first, a rapid dissociation of the S-1 from actin by ATP; second, a slower fluorescence change in the S-1 that may be related to the initial phosphate burst; and third, a much slower rate limiting recombination of the S-1 with actin. This latter step equals the acto-S-1 steady-state adenosine 5'-triphosphatase (ATPase) rate at both low and high actin concentrations, and like the steady-state ATPase levels off at a V max of 0.9s-1 at high actin concentration. Therefore, the release of adenosine 5'-diphosphate and inorganic phosphate is not the rate-limiting step in the acto-S-1 ATPase. Rather, a slow first-order step corresponding to the previously postulated transition from the refractory to the nonrefractory state precedes the rebinding of the S-1 to the actin during each cycle of ATP hydrolysis.

Actins↗
PubMed1978

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.

Actins↗
PubMed1979

Active site trapping of nucleotides by crosslinking two sulfhydryls in myosin subfragment 1.

Studies with reagents that crosslink two thiol groups have shown that it is possible to trap nucleotides at the active site of myosin chymotryptic subfragment 1. Subfragment 1 incorporates nearly stoichiometric quantities of [14C]ATP or [14C]ADP in a manner that depends linearly on the extent of inactivation by either N,N'-p-phenylenedimaleimide or Co(II)phenanthroline/[Co(III)(phenanthroline)2CO3]+ complexes. The incorporated radioactive nucleotide is retained after gel filtration, even when the enzyme derivatives are stored in the presence of EDTA or nonradioactive nucleotides (t 1/2 approximately 5 days). The nucleotide incorporated is not covalently bound because HClO4 denaturation allows immediate release of bound nucleotide. The nucleotide retained is ADP because the gamma-phosphate of [gamma-32P]ATP is lost after trapping. Subfragment 1 inactivated as above does not bind the competitive inhibitor adenosine 5'-[beta, gamma-imido]triphosphate, indicating that the active site is blocked. It is proposed that a jawlike nucleotide cleft closes on MgADP or MgATP, which can be locked shut by crosslinking two thiol groups by reaction with N,N'-p-phenylenedimaleimide or cobalt phenanthroline complexes.

Adenosine Diphosphate↗
PubMed1975

A study of the binding of adenosine diphosphate to myosin subfragment-1.

The binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 10(5) g of protein, and the dissociation constant, 1.6 x 10(-6) M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 25 degrees. Similar maximum numbers, about 0.5 mole per 10(5) g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature. Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 10(5) g of subfragment-1, and the dissociation constant, less than 10(-7) M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8 degrees. The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP. The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46 +/- 0.06 to 0.30 +/- 0.09 mole of Pi per 10(5) g of subfragment-1 on adding ADP to a level of 0.6 mM. Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP. On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.

Actins↗
PubMed1977

Mechanism of oxygen exchange in actin-activated hydrolysis of adenosine triphosphate by myosin subfragment 1.

The gamma-phosphoryl groups of two intermediates (M-ATP and M-ADP-P1) in the pathway of MgATP hydrolysis by myosin undergo extensive oxygen exchange with water. Actin activates the overall rate of hydrolysis at a rate-limiting step which follows these exchange reactions. Thus, actin, by decreasing the turnover time of hydrolysis, would be expected to proportionately decrease the time available for oxygen exchange. Using subfragment 1 of myosin, the turnover time of hydrolysis can be varied over a wide range by changing the concentration of actin. An estimate for the rate constant of exchange can then be obtained by relating these turnover times to measured values for oxygen exchange (incorporation of 18O from H218O into the inorganic phosphate (Pi) released by hydrolysis). The results of such an experiment, with turnover times between 0.2 and 25 s, indicate that, for each gamma-phosphoryl group, one oxygen from the medium is added rapidly (to cleave the phosphoryl group or form a pentacoordinate phosphroyl complex); two more oxygens exchange with a rate constant, kc, of about 1 s-1; and a fourth oxygen exchanges slowly with ke about 0.2 s-1. The higher value is about 18 times smaller than the rate constant, 5-3, for the reverse cleavage step of the myosin pathway, which is postulated to be responsible for oxygen exchange. The data, then, indicate that the rate-limiting step for oxygen exchange is not k-3, but may be the rate of rotation of oxygens around the phosphorus atom, with one oxygen severely restricted by its binding to the active site. The finding that kc differs for the four oxygens in each phosphate group is related to past observations on myosin-catalyzed oxygen exchange.

Actins↗