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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The assembly mechanism of synthetic thick filaments of purified myosin formed at pH 8.0 has been extensively studied. These filaments were chosen for experimentation since they share a number of structural features, as well as aspects of the kinetics of their assembly, with native filaments. C-protein copolymerization consistently favours the formation of longer synthetic filaments with the diameter of the crossbridge region remaining comparable to that of the native filament. At moderate concentrations the close-to-symmetrical length distribution typical of pH 8.0 filaments is altered to a distribution with a steep rising, and extended tailing edge towards longer filament lengths. The asymmetric length distributions probably originate from an at least partial exclusion of C-protein from the equivalent of the accessory-protein binding stripes adjacent to the bare zone from which C-protein is apparently excluded in vivo. An outer limit to C-protein binding exists in native filaments. This does not appear to be the case in vitro since filaments significantly longer than the native appear stabilized by C-protein. A minimum of three types of C-protein binding can be resolved. Physiological stoichiometries of C-protein (0 to approximately 0.3 mole ratios) lower the critical concentration of myosin (not length equilibrated) and increase filament length. The lack of a significant change in filament turbidity as these high-affinity sites are occupied is indicative of a C-protein-induced change in the structure of the synthetic filaments. A second set of binding sites occupied at higher mole ratios of C-protein: myosin (approximately 0.3-1.0) are typified by a marked increase in the specific turbidity of the filaments; a result consistent with the addition of weight to such a structure. The precedent of C-protein binding to the subfragment-2 portion of the myosin molecule provides a plausible basis for these observations. A third phase characterized by a less marked increase in turbidity occurs between 1-2:1 (and possibly higher) C-protein: myosin mole ratios. The molecular basis of this process is not immediately apparent.
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PMID:Interaction of C-protein with pH 8.0 synthetic thick filaments prepared from the myosin of vertebrate skeletal muscle. 341 55

The effect of MgATP on myosin filament assembly has been studied. Filaments were assembled by a standard dilution procedure involving two steps, dilution from 0.6 to 0.3 M KCl and from 0.3 to 0.15 M KCl with a different rate of dilution in each step. This standard dilution procedure gives filaments which are structurally similar to native filaments in that they have a sharp length distribution around 1.5 micron, a diameter of 16 nm and they vary in length with KCl concentration in a similar manner to native filaments. The addition of 1 mM MgATP leads to a sharpening of the length distribution around 1.5 micron without change in the 16 nm diameter. Filaments assembled by dialysis or by rapid dilution are not similarly affected by the presence of MgATP indicating that the standard dilution procedure produces filaments which are more closely similar to native filaments than those produced by these other methods. MgAMPPNP and magnesium pyrophosphate have the same effect as MgATP thus eliminating the possibility that phosphorylation of the myosin is involved in the effect. The effect of MgATP is not directly related to its binding to the active site of the myosin molecule since a 500:1 mole ratio of MgATP to myosin is required for the effect. It is therefore likely that the effect of MgATP is related to other binding sites on the myosin molecule. The presence of MgATP leads to molecular rearrangements which finely tune the molecular organization of the filaments formed by the standard dilution procedure in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The myosin filament. XII. Effect of MgATP on assembly. 349 33

Contraction and myosin light-chain phosphorylation were measured in electrically stimulated tracheal smooth muscle. Latencies for the onset of force, stiffness, and light-chain phosphorylation were 500 milliseconds. Myosin light chain was phosphorylated from 0.04 to 0.80 mole of phosphate per mole of light chain with a pseudo-first-order rate of 1.1 per second with no evidence of an ordered or negatively cooperative process. Following the period of latency, stiffness increased with phosphorylation and both increased more rapidly than isometric force. The linear relation between stiffness and phosphorylation during activation suggests independent attachment of each myosin head upon phosphorylation.
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PMID:Activation of smooth muscle contraction: relation between myosin phosphorylation and stiffness. 375 63

The role of orthophosphate ions (Pi) in crossbridge kinetics was investigated by parallel measurements of the ATP hydrolysis rate and tension transients in maximally activated, chemically skinned rabbit psoas fibers. The hydrolysis rate of the standard activation at 20 degrees C was measured at 1.25 nmole X s-1 X m-1 X fiber-1, which corresponds to the hydrolysis of 3 moles ATP per mole of myosin head per second. The isometric tension, stiffness extrapolated to the infinite frequency, and the ATPase rate progressively decreased when increasing concentrations of Pi (0-16 mM) were added to the activating saline. The decrease was greatest with tension, followed by stiffness and the ATPase rate. Both the apparent rate constant and the magnitude parameters of exponential process (B) increased with Pi concentration resulting in a significant increase in the oscillatory power output. The effects of Pi on processes (A) and (C) were only marginal. When fibers were oscillated at 1 Hz [close to the characteristic frequency of process (A)], no significant increase in the ATP hydrolysis rate was observed. However, a small increase was noticed at 10 Hz [1%, process (B)], and at 100 Hz [6%, process (C)]. We interpret these results in terms of a crossbridge scheme which adds a branch pathway to the conventional hydrolysis cycle. In the proposed scheme, the number of crossbridges entering the branch pathway increases at higher Pi concentrations and in the presence of imposed oscillations at the proper frequency.
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PMID:The effect of inorganic phosphate on the ATP hydrolysis rate and the tension transients in chemically skinned rabbit psoas fibers. 382 68

A double-flash microphotographic technique has been used to follow the variation with temperature of the following kinetic parameters related to the contraction and re-extension of the ciliate Stentor coeruleus, namely the rate of contraction, the initiation time before contraction, the rate of re-extension and the initiation time before re-extension, all described by first order kinetics. Activation enthalpies, entropies and free energies related to the above mentioned parameters were calculated from the variation of the rate constants with temperature. The enthalpies and entropies appear to be of minor interest compared to the free energies. For the contraction and the initiation of contraction the delta G transition state values obtained were 14 and 15 kcal/mole, respectively, while the re-extension and the initiation of re-extension both were represented by a value of delta G transition state about 19 kcal/mole. These results are compared to activation parameters for different motile systems and for the formation and breakdown of ATP-myosin complexes. A model for the contraction and re-extension processes is proposed in accordance with the results measured.
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PMID:Temperature effects upon the time course of contraction and re-extension in Stentor coeruleus. 393 May 18

The fluorophore, N(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS), incubated with glycerinated psoas fibers primarily labels the S-1 moieties of such fibers, but it does not impair fiber contractility even when the degree of labeling is as high as 0.8 moles fluorophore per mole myosin. The polarization of the on-axis fluorescence from either the IAEDANS fluorophore, or the intrinsic tryptophane fluorophore, depends on whether the fiber is relaxed, in rigor, or developing isometric tension; furthermore, the changes in polarization on going from one state to another are much the same with either tryptophane or IAEDANS fluorophores. The foregoing is true whether the plane of the exciting light is parallel or perpendicular to the fiber axis. Also, if a fiber is first freed of its myosin by extraction, and is then incubated with IAEDANS-labeled S-1 the resulting polarization approaches that observed with a labeled, unextracted fiber in rigor. By contrast, incubation with the fluorophore, 7-nitro-4-chlorobenz-2-oxa-1,3-diazole (NBD-Cl) confers fluorescence only on actin, without impairing contractility, but the polarization of such fluorescence changes in a different direction and magnitude from myosin-originating fluorescence. It is concluded from these various observations that whether the fluorophore is IAEDANS or tryptophane the polarization change with change in physiological state originates in the S-1 moieties of fibers, and relates to the space attitude of these moieties.
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PMID:Use of fluorescence polarization to observe changes in attitude of S-1 moieties in muscle fibers. 413 95

It has been previously demonstrated that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II is inhibited by phosphorylation of its two heavy chains (Collins, J. H., and Korn, E. D. (1980) J. Biol. Chem. 255, 8011-8014). In this paper, it is shown that a partially purified kinase preparation from Acanthamoeba catalyzes the incorporation of 3 mol of phosphate into each mole of myosin II heavy chain. Tryptic digestion of the 32P-myosin, followed by two-dimensional peptide mapping, indicates that two of the three sites phosphorylated by the kinase in vitro correspond to the two major phosphorylation sites on the myosin heavy chain in vivo. Phosphorylation of myosin II in vitro by the kinase fraction completely inhibits the actin-activated Mg2+-ATPase activity of myosin II. Myosin II can be isolated in a highly phosphorylated, enzymatically inactive form, then dephosphorylated to an active form, and finally rephosphorylated to an inactive form. The Acanthamoeba kinase fraction catalyzes the phosphorylation of all three sites on the heavy chain of myosin II at virtually the same rate. From a comparison of the decrease in actin-activated Mg2+-ATPase activity with the amount of phosphate incorporated into myosin II, and from the results obtained previously by dephosphorylating myosin II (Collins, J. H., and Korn, E. D., (1980) J. Biol. Chem. 255, 8011-8014), it can be inferred that two of the sites phosphorylated in vitro act in a synergistic manner to inhibit the actin-activated myosin II Mg2+-ATPase.
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PMID:Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II. 611 66

Using mixed anhydride of AMP and mesitylene carboxylic acid carrying a fluorescent or radioactive label, it was found that the previously established irreversible inhibition of myosin ATPase is a result of protein covalent binding to the nucleotide residue of the inhibitor. The stoichiometry of the affinity labelling of heavy meromyosin is 1 mole of nucleotide residue of mixed anhydride per 1 mole of protein, that of subfragment 1-0.5 mole per 1 mole of protein. The lack of irreversible inhibition of the ATPase activity of subfragment 1 is suggestive of an existence of a regulatory substrate-binding site in the myosin molecule.
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PMID:[Affinity modification of heavy meromyosin and subfragment 1 by mixed anhydrides of [14C] AMP, epsilon AMP and mesitylene carboxylic acid]. 621 86

To investigate the regulation of actin-myosin interaction in rabbit phagocytic cells, purified myosin and a partially purified cofactor protein were obtained from polymorphonuclear leukocytes (PMN) and alveolar macrophages (ALM) by molecular sieve filtration over an agarose column. ALM cofactor enhanced the Mg++-ATPase activity of ALM myosin by actin to 0.15 +/- 0.04 mumoles Pi per mg myosin per min and PMN cofactor enhanced PMN myosin to 0.43 +/- 0.03 mumoles Pi per mg myosin per min. The crude cofactor preparations isolated from the two types of leukocyte extracts were interchangeable with the leukocyte myosins. When ALM cofactor was added to a PMN actomyosin complex, the Mg++-ATPase activity of the PMN myosin was 3-fold higher than with ALM cofactor and its own actomyosin complex. In contrast, PMN cofactor did not enhance ALM actomyosin Mg++-ATPase activity beyond that observed with ALM cofactor and ALM actomyosin. Cofactor protein from the PMN was further purified on a DEAE-Sephagel column. After electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, the isolated fraction weighed 70,000 daltons. This fraction stimulated the actinmediated myosin Mg++-ATPase. In the presence of Mg++ and [gamma 32P]ATP, the 70,000 dalton protein phosphorylated the 20,000 dalton light chain of PMN myosin, leading to the incorporation of 0.62 +/- 0.09 moles of Pi per mole myosin. On the basis of these results, we propose that phagocytic cofactor is a kinase which regulates the enzymatic activity of phagocytic cell myosin.
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PMID:Purification of myosin light chain kinase from rabbit polymorphonuclear leukocytes. 626 Dec 11

Cibacron Blue F3GA and its immobilized derivatives have been shown before to bind and inhibit nucleotide-dependent enzymes and, among them, myosin subfragment 1. Experiments have been carried out to examine the mechanism of the subfragment 1--dye interaction. Binding of subfragment 1 to immobilized dye (Affi-Gel Blue) does not involve the ATP binding site on myosin. Subfragment 1 hydrolyzes MgATP and CaATP while bound to the Affi-Gel Blue column. Inactivated subfragment 1, which contains [3H]ADP noncovalently trapped at the active site, binds and elutes from the Affi-Gel Blue column in the same manner as unmodified, active protein. Free Cibacron Blue inhibits the ATPase activity of subfragment 1. The inhibition is pH, salt, and time dependent. Complete inhibition correlates with the noncovalent binding of four to five dye molecules per mole of subfragment 1. Three to four of these dye molecules can be preferentially removed from subfragment 1 in the presence of 1 M KCl without relieving the inhibition. This inhibition, which can be traced to one dye molecule per subfragment 1, is reversible and is facilitated in the presence of MgADP and MgATP, suggesting that the dye does not bind at the active site of subfragment 1. Our observations are explained in terms of hydrophobic and electrostatic protein--dye interactions.
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PMID:Interaction of myosin subfragment 1 with Cibacron Blue F3GA. 645 18


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