UMLS:C0027960 - FACTA Search

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Query: UMLS:C0027960 (mole)
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The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.
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PMID:An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements. 1 88

1. While below 10 degrees C, the initial burst of Pi liberation in the hydrolysis of Mn(II)-ATP by heavy meromyosin or myosin subfragment 1 was inhibited by the pre-addition of ADP without any change in the steady-state activity, it was not inhibited above 10 degrees C. The burst size was about one mole per two moles of myosin active sites. 2. Above 10 degrees C, the ultraviolet absorption spectrum of heavy meromyosin induced by ATP in MnCl2 was similar to that induced in MgCl2 and the spectral decay to the ADP-induced level occurred only after all the ATP in the solution was depleted. In contrast, below 10 degrees C the spectrum induced by ATP in MnCl2 decayed to the ADP-induced level within a few seconds after the addition of ATP, although ATP was present in the solution. 3. These two results indicate that in Mn-ATP above 10 degrees C at the burst site there is a myosin*-ADP-Pi complex generated by ATP hydrolysis while below 10 degrees C there is a myosin-product complex identical with the one generated by adding ADP (and Pi) to myosin. 4. At tempertures both above and below 10 degrees C, the Mn-ATP hydrolysis of heavy meromyosin was activated by actin and superprecipitation of actomyosin occurred. Characteristics of these phenomena showed a transition at around 10 degrees C.
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PMID:Temperature-dependent transitions of the myosin-product intermediate at 10 degrees C in the Mn(II)-ATP hydrolysis. 12 63

Studies were carried out to elucidate the nature of biphasic ATP hydrolysis by myosin at low temperature. 1. The rate of ATP splitting decreased sharply at 3--5 min after initiation of the reaction below a critical temperature (25 degrees and 30 degrees in the presence of Ca-2+ and EDTA, respectively). On the other hand, Mg-2+-ATPase [ED 3.6.1.3] did not exhibit such biphasic kinetics. 2. The Arrhenius plot of the second phase of the reaction after the rate transition gave a straight line whether the temperature of assay was above or below the critical one, giving 5.7 kcal/mole as the activation energy of Da-2+-ATPase showed features similar to those of Ca-2+-ATPase. 3. Michaelis constants for the two phases at 8 degrees were also different. In addition, the first phase of EDTA-ATPase was shown to have two different constants, depending on ATP concentration. 4. The profiles of the dependence of ATPase activity on KCl concentration were essentially the same for both phases, while bending of the time curve was scarecly observed obove pH 8 for Ca-2+-ATPase or at pH 6 for EDTA-ATPase. 5. 2, 4-Dinitrophenol abolished the phase transition for Ca-2+-ATPase and EDTA-ATPase, and heat treatment also minimized the transition for the former.
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PMID:Biphasic ATP splitting of myosin at low temperature. 12 18

H-Meromyosin (CMB leads to betaME-H-meromyosin) was prepared by tryptic digestion of myosin, which had been treated with CMB bound to H-meromyosin and the extent of desensitization of the substrate inhibition of acto-H-meromyosin ATPase [EC 3.6.1.3.] was investigated. Both the dissociation of acto-H-meromyosin induced by ATP and substrate inhibition decreased with increase in the amount of bound CMB to a minimum value at about 1 mole of CMB bound per mole of H-meromyosin. The substrate inhibition of acto-H-meromyosin ATPase was restored to the original level by complete removal of the bound CMB by further treatment of CMB leads to beta ME-H-meromyosin with a large excess of beta-mercaptoethanol. The dissociation constant of acto-H-meromyosin in the presence of ATP decreased markedly on modification with CMB, while the maximum ATPase activity ar a sufficiently high concentration of F-actin remained essentially unchanged. Acto-H-meromyosin was reconstituted from F-actin and CMB LEADS TO beta ME-H-meromyosin, containing less than the stoichiometric amount of bound CMB. Its ATPase activity and the extent of dissociation of acto-H-meromyosin induced by ATP were explained as those of a mixture of unmodified H-meromyosin and CMB leads to beta ME-H-meromyosin containing 1 mole of CMB per mole of H-meromyosin. Half of the light chains (g2), with a molecular weight of 18,000, were removed from myosin by treatment with CMB and beta-mercaptoethanol. After this treatment, on further incubation of the myosin with a large excess of beta-mercaptoethanol, the myosin contained only half of the g2, but the substrate inhibition of acto-H-meromyosin ATPase was restored completely. The initial burst of P1 liberation and the EDTA-ATPase activity decreased to almost zero on specific modification of the SH1-groups with NEM, while the initial burst decreased to some extent and the EDTA-ATPase activity to 50% of the original value on binding of 1 mole CMB per mole of H-meromyosin. The actomyosin-type of ATPase activity was strongly inhibited by modification with CMB. The extent of the dissociation of acto-H-meromyosin induced by ATP was unaffected by modification with NEM, while it decreased on further treatment of NEM-myosin with CMB FOLLOWED BY BETA-MERCAPTOETHANOL.
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PMID:Desensitization of substrate inhibition of acto-H-meromyosin ATPase by treatment of H-meromyosin with rho-chloromercuribenzoate. Relation between the extent of desensitization and the amount of bound rho-chloromercuribenzoate1. 12 73

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.
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PMID:A study of the binding of adenosine diphosphate to myosin subfragment-1. 12 50

In 2 mM MgATP, 0.08 ionic strength and 1 mM free Mg++ cardiac myofibrils bound 3.5 nmoles Ca/mg protein at maximal ATPase activation. Significant amounts of Ca were also bound to cardiac myosin with these same conditions. By subtraction of this myosin-bound Ca we obtained an estimate of 4 moles Ca bound per mole of myofibrillar troponin at maximal ATPase. We found, however, that Ca activation of myofibrillar ATPase could be estimated assuming that only two of troponin's Ca-binding sites are engaged in regulation of crossbridge activity. Increases in MgMTP from 0.3 to 5.0 mM raised the free Ca, giving half-maximal isomteric tension or ATPase. Although part of this shift is most probably due to changes in the number of rigor (nucleotide-free) actin-myosin linkages, the rightward shift of the free Ca++-activation relation with increase in MgATP from 2 to 5 mM appears to be due to effects of active (nucleotide-containing) actin-myosin linkages.
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PMID:Calcium regulation of cardiac myofibrillar activation: effects of MgATP. 12 91

Two types of canine cardiac myosins, from the free wall of the left ventricle and from the free wall of the right ventricle, were compared with canine skeletal muscle myosin from gastrocnemius. For K+ -activated myosin the Vmax values in mumoles of Pi/mg.min were: right ventricle, 0.57 +/- 0.02; left ventricle, 0.72 +/- 0.09; gastrocnemius, 0.92 +/- 0.04. For Ca++ -activated myosin the Vmax values were: right ventricle, 0.32 +/- 0.04; left ventricle, 0.42 +/- 0.03; gastrocnemius, 0.52 +/- 0.02; (p greater than 0.01 for all defferences). For all three types of tissues the Vmax values for NH4+ -activated myosin were the same (2.30 +/- 0.11). Corresponding to kinetic changes there were significant changes in the proportion and type of myosin subunits. In the two cardiac ventricles where heavy chains were immunologically identical, 81% of the total nitrogen of right ventricular myosin was present in the heavy chains whereas in left ventricular myosin 90% of the total nitrogen of myosin was present in the heavy chains. Quantifications were made on polyacrylamide gels were dye binding was directly related to nitrogen concentration for each of the myosin chains. In canine skeletal muscle gastrocnemius where the myosin heavy chains were immunologically nonidentical with those of cardiac myosin, 87% of the total nitrogen was present in the heavy chains. The data suggest that there are 2 moles of myosin light chains per mole of myosin heavy chains in right ventricular myosin where the adenosine triphosphatase (ATPase) activity is low and 1 mole of myosin light chains per mole of myosin heavy chains in left ventricula myosin where ATPase activity is elevated; for skeletal muscle myosin there were 1.5 moles of myosin light chains per mole of myosin heavy chains. Proportion of myosin light chain C1 to light chain C2 was the same in both left and right ventricular myosin. In skeletal muscle myosin the proportion of light chain C1 to light chain C2 was significantly different from that of cardiac tissue. It appears that the proportion of myosin light chain C1 to light chain C2 is directly related to the type of myosin heavy chain present since the immunologically identical heavy chains of cardiac tissue were immunologically nonidentical with those of skeletal muscle myosin.
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PMID:Comparative analyses of skeletal and cardiac myosins. 12 33

Mild pulmonic stenosis was performed in dogs to evaluate the effect of systolic pressures overloading on the activity and subunits of myosin in the early hypertrophied right ventricle. Three weeks following pulmonary constriction, six hypertrophied dogs were sacrificed and compared to six sham-operated dogs which served as controls. In the right ventricular free wall of hypertrophied right ventricles (HRV), the heart/body weight was 46% greater than that of normal right ventricles (NRV) (p less than 0.01). Myosin ATPase activity (Vmax values) in mumoles phosphate/mg/min, was elevated significantly in the stressed ventricle for both K+ and Ca++ activity in hypertrophied right ventricles. Associated with the increase in myosin activity, there was an increase in proportion of heavy to light chains in myosin from HRV. There were approximately 2 moles of myosin light chains per mole of myosin heavy chains in NRV and approximately 1 mole of myosin light chains per mole of myosin heavy chains in HRV. The proportion of light chain C1 to C2, did not change in myosin from NRV and HRV. Of the C1 light chains, according to two-dimensional gel electrophoresis, there was less C1d as compared to C1c in HRV as compared to NRV. Thus K+- and Ca++- activated myosin is elevated in early canine HRV by pressure overload. It is suggested taht the augmented myosin activity is due to a reduction of light chain inhibition of myosin ATPase activity, which appears to result from the slower turnover rate of myosin light chains relative to heavy chains. Furthermore, when myosin light chains are added to hypertrophied right ventricular myosin, the ATPase activity is lowered.
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PMID:Modulation of myosin in right ventricular hypertrophy. 12 38

Asakura, Taniguchi and Oosawa [1]proposed that muscle actin polymer under sonic vibration is in a different state from that of the ordinary double stranded helical structure (F-actin), characterised by partially interrupted structures of F-actin, a state of "f-actin". In order to confirm different states for actin polymers [1, 2], physicochemical studies were made by measurements of viscosity, flow birefringence, electric birefringence, fluorescence, electron microscopy, quasielastic light scattering and ATP splitting. The following results were obtained. (1) F-actin polymers can undergo two processes of depolymerization upon treatment with urea and various salts as judged by measurements of flow birefringence and viscosity: one is a rapid process in a solution containing K+ or Ca2+ and urea; the other is a slow process following a brief rapid one in a solution containing Mg2+ and urea. (2) In the presence of Mg2+ and a suitable concentration of urea, F-actin (FMU-actin) appeared to exhibit different properties than ordinary F-actin; it had lower viscosity and lower flow birefringence and it had on the whole a more flexible polymer structure, also judging from experiments of quasielastic light scattering, electric birefringence. The different structure was confirmed directly be electron microscopic observation. The aromatic side chains of FMU-actin were also more mobile than those of F-actin judging from fluorescence measurements. The transformation between F-actin and FMU-actin was reversible. (3) The state of FMU-actin polymers was also characterized by ATP splitting; FMU-actin split about one mole of ATP into ADP and inorganic phosphate per mole of actin monomer at room temperature, where F-actin did not. A molar excess of Mg2+ with respect to actin monomer at room temperature, where F-actin did not. A molar excess of Mg2+ with respect to actin monomer is required for ATP splitting. F-actin in solutions containing K+ or Ca2+ and urea did not split ATP. FMU-actin activated on Mg-ATP-ase of myosin at nearly the same rate as that of F-actin. (4) We have postulated a flexible filament model (f-actin). The relationships between the structure of f-actin and its functional role for force generation during contraction are discussed.
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PMID:Diphasic transformations of F-actin. Effects of urea and MgCl2 on F-actin. 13 Sep 28

H-Meromyosin (HMM) was digested with insoluble papain [EC 3.4.22.2]. Neither the size of the initial burst of Pi liberation (0.5 mole/mole of myosin head) nor the Mg2+-ATPase [EC 3.6.1.3] activity of HMM in the steady state was affected by this treatment. Acto-S-1 was obtained by mixing F-actin with HMM digested with insoluble papain (HMM-S-1). The size of the initial burst of Pi liberation of acto-S-1 was 0.35 mole/mole of S-l at an ATP concentration of 0.5 mole/mole of S-1, and 0.5 mole/moleof S-1 at ATP concentrations above 1 mole/mole of S-1...
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PMID:Separation of subfragment-1 of H-meromyosin into two equimolar fractions with and without formation of the reactive enzyme-phosphate-ADP complex. 13 97

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