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Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).

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We reinvestigated whether the native myosin LC2-free-subfragment 1 (S1) dimer exists by using viscometry, capillary electrophoresis, and laser light scattering. We found that the intrinsic viscosity of the monomer is [eta]m = 6.7 cm3/g and its translation diffusion coefficient is (c = 0) = 4.43 x 10(-)7 cm2/s. For the dimer, [eta]d = 19.8 cm3/g and (c = 0) = 2.54 x 10(-)7 cm2/s. Using the Svedberg equation and introducing the values of the sedimentation coefficients (5.05 S for the monomer and 6.05 S for the dimer), we find the following molecular weights: Mr,m = 108 000 Da and Mr,d = 213 000 Da, which agree well with previous determinations. Capillary electrophoresis successfully separated S1(A1) and S1(A2), in a monomer buffer, and S1(A1) and S1(A2) and a heterodimer S1(A1)-S1(A2), in a dimer buffer. An interesting feature of the monomer-dimer equilibrium is the presence of temperature transitions, whose positions and widths depend upon the buffer conditions. At low temperatures, a pure dimer was observed, whereas at high temperatures only the monomer was present. The dimerization site on both myosin and S1 is extremely labile.

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The globular heads of skeletal muscle myosin have been shown to exist as isoenzymes S1 (A1) and S1 (A2), and there are also isoforms of the heavy chains. Using capillary electrophoresis, we found two dominant isoenzymes of the whole native myosin molecule, in agreement with what has previously been found by various techniques for native and nondenatured myosin from adult rabbits. Findings about possible states of aggregation of myosin and its heads are contradictory. By analytical ultracentrifugation, we confirmed the existence of a tail-tail dimer. By laser light scattering, we found a head-head dimer in the presence of MgATP. Capillary electrophoresis coupled with analytical ultracentrifugation and laser light scattering led us to refine these results. We found tail-tail dimers in a conventional buffer. We found tail-tail and head-head dimers in the presence of 0.5 mM MgATP and pure head-head dimers in the presence of 6 mM MgATP. All the dimers were homodimers. Naming the dominant isoenzymes of myosin a and b, we observed tail-tail dimers with isoenzyme a (TaTa) and with isoenzyme b (TbTb) and also head-head dimers with isoenzyme a (HaHa) and with isoenzyme b (HbHb).

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Irrespective of the exact shape and length of myosin heads, it is shown that radial compressive forces occur in contracting muscle, which tend to bring the myofilaments closer to each other. This is in contradiction with common observation: a muscle that shortens swells laterally. The presence of these forces is model-dependent and they appear only in the case of the traditional concepts of force generation. Here we present an overview and a historical account of the problem.

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Using sheep cerebellum microsomes previously loaded with 45Ca2+ or 90Sr2+, we measured the dependence of inositol 1,4,5-trisphosphate (InsP3)-induced efflux of these ions on Ca2+ or Sr2+ on the cytosolic side. At a low InsP3 concentration, Ca2+ in the submicromolar range only poorly activated 45Ca2+ or 90Sr2+ efflux, and higher Ca2+ concentrations were inhibitory. In contrast, Sr2+ in the micromolar range activated release efficiently, while only very high Sr2+ concentrations were inhibitory. Experiments were repeated in the presence of a high InsP3 concentration, which allowed increasing free Ca2+ to micromolar concentrations without inducing complete inhibition of the InsP3-dependent efflux. Under these conditions, micromolar Ca2+ was found to activate efflux to a large extent, similar to that previously found with Sr2+. Optimal activation by Ca2+ of the InsP3-dependent channel occurs at micromolar rather than submicromolar free Ca2+ concentrations, but at too low an InsP3 concentration, Ca(2+)-induced activation is counteracted by Ca(2+)-induced inactivation. Separate measurements of [3H]-InsP3 binding at a low concentration showed that Sr2+ and Ca2+ did not enhance the amount of bound [3H]-InsP3, implying that the activating effect of Sr2+ and Ca2+ in cerebellar microsomes is mediated by an increase in the channel opening probability and not by an increase in the receptor's affinity for InsP3. A similar relationship also holds in the case of the activating effect of nucleotides.

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Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor's apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.

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Using microsomal membrane vesicles derived from sheep cerebellum, we measured the rate of inositol 1,4,5-trisphosphate (InsP3)-dependent 45Ca2+ efflux from 45Ca(2+)-loaded compartments during rapid perfusion with a medium containing InsP3 and various concentrations of free 40Ca2+ on the cytosolic side (pH 7.1, 5 mM Mg2+, in the absence of ATP at 20 degrees C). At 0.15 microM InsP3 and pCa 6.5, half-45Ca2+ release was attained within less than 200 ms. At low Ca2+ concentrations, the initial rate of 45Ca2+ release depended smoothly on InsP3 concentration, and InsP3 activated release with moderate positive cooperativity. Preliminary experiments performed at various free 40Ca2+ concentrations were consistent with a bell-shaped 40Ca2+ dependence of 45Ca2+ release. In the range of micromolar or higher free 40Ca2+ concentrations, the apparent inhibition of 45Ca2+ release was dependent on InsP3 concentration, and 45Ca2+ release for intermediate InsP3 concentrations was transient; under selected conditions, a second perfusion period, identical to the first one but separated from it by a short recovery period, was found to allow renewed 45Ca2+ efflux. At high Ca2+ concentration, fast reversible Ca(2+)-dependent desensitization of the channel, and not heterogeneity, was therefore responsible for the termination of InsP3-triggered 45Ca2+ efflux at submaximal concentrations of InsP3. At lower Ca2+ concentrations, a large fraction of the apparent activating effect of submicromolar 40Ca2+ concentrations on 45Ca2+ efflux that we had observed in the preliminary experiments proved to be the artifactual consequence of an inhibitory effect exerted by metal-free Ca2+ chelators on InsP3-dependent efflux at nanomolar 40Ca2+ concentrations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid, EGTA, and fluo-3 were all effective inhibitors. When this inhibition was taken into account, a rise in free 40Ca2+ concentration from 30 to 300 nM only weakly enhanced 45Ca2+ fluxes in the presence of a low concentration of InsP3. As a result, submicromolar free 40Ca2+ appears to be only a poor activator of InsP3-induced Ca2+ release under these experimental conditions.

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For a number of years, the isometric force per myosin head has been taken as 1 pN by the authors who studied in vitro movement. This value was deduced from the value of the isometric tension exerted by a living or a skinned fibre at approximately 0 degree C and for the frog (1-3 kg cm-2). Starting from an isometric tension exerted by a single unit cell of approximately 10 kg cm-2, it has been shown that the isometric force per myosin head is (8 +/- 1)pN. The value of approximately 10 kg cm-2 was deduced from theoretical and semi-empirical reasonings independent of the mechanical roles of the crossbridges. Here, we discuss this latest value by using simple geometrical considerations.

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There is controversy concerning the shape and length of myosin heads. In the present paper we try to analyse the data and to draw clear conclusions in this field. When the myosin heads are isolated (S1) from the rest of the molecule, their length is approximately 12 nm and their shape is close to that of a prolate ellipsoid with an axial ratio approximately 2.3 (in solution) or close to that of a comma when attached to F-actin (with a length of 12-13 nm). When the myosin heads are observed on a whole molecule, their length is approximately 19 nm and they are pear-shaped. Here we suggest that all these observations are compatible. We believe that, for a whole myosin molecule, a large part of the head-rod joint (S1/S2 joint) is measured with the head, owing to a particularly heavy staining or shadowing of this joint. On the other hand, S1 is probably built up of a head part plus the S1/S2 joint, which is not revealed by the usual techniques (hydrodynamics, X-ray and neutron scattering). Finally, the comma shape would be related to a flexible part in the head region of S1, which is significantly bent when S1 is attached to F-actin, but which would be less bent for S1 in solution. A similar bending also occurs in crystalline S1.

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We attempted to establish whether lanthanide ions, when added to sarcoplasmic reticulum (SR) membranes in the absence of nucleotide, compete with Ca2+ for binding to the transport sites of the Ca(2+)-ATPase in these membranes, or whether they bind to different sites. Equilibrium measurements of the effect of lanthanide ions on the intrinsic fluorescence of SR ATPase and on 45Ca2+ binding to it were performed either at neutral pH (pH 6.8), i.e. when endogenous or contaminating Ca2+ was sufficient to nearly saturate the ATPase transport sites, or at acid pH (pH 5.5), which greatly reduced the affinity of calcium for its sites on the ATPase. These measurements did reveal apparent competition between Ca2+ and the lanthanide ions La3+, Gd3+, Pr3+, and Tb3+, which all behaved similarly, but this competition displayed unexpected features: lanthanide ions displaced Ca2+ with a moderate affinity and in a noncooperative way, and the pH dependence of this displacement was smaller than that of the Ca2+ binding to its own sites. Simultaneously, we directly measured the amount of Tb3+ bound to the ATPase relative to the amount of Ca2+ and found that Tb3+ ions only reduced significantly the amount of Ca2+ bound after a considerable number of Tb3+ ions had bound. Furthermore, when we tested the effect of Ca2+ on the amount of Tb3+ bound to the SR membranes, we found that the Tb3+ ions which bound at low Tb3+ concentrations were not displaced when Ca2+ was added at concentrations which saturated the Ca2+ transport sites. We conclude that the sites on SR ATPase to which lanthanide ions bind with the highest affinity are not the high affinity Ca2+ binding and transport sites. At higher concentrations, lanthanide ions did not appear to be able to replace Ca2+ ions and preserve the native structure of their binding pocket, as evaluated in rapid filtration measurements from the effect of moderate concentrations of lanthanide ions on the kinetics of Ca2+ dissociation. Thus, the presence of lanthanide ions slowed down the dissociation from its binding site of the first, superficially bound 45Ca2+ ion, instead of specifically preventing the dissociation of the deeply bound 45Ca2+ ion. These results highlight the need for caution when interpreting, in terms of calcium sites, experimental data collected using lanthanide ions as spectroscopic probes on SR membrane ATPase.

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To evaluate the distribution of an amphiphile or its binding to membranes whose properties are affected by such binding, it is only necessary to establish to what extent the dose-response to the amphiphile depends on the membrane concentration. The measured response only needs to reflect local events. This method of evaluation does not depend on the precise shape of the dose-response curve and is particularly useful for amphiphiles devoid of properties like fluorescence or radioactivity which would allow their direct assay. In this work, we establish the validity of this approach by comparing it with direct conventional determinations. Two parameters are especially suitable for such evaluation: the perturbation of an enzyme's activity, produced by many amphiphiles, and the fluorescence quenching of membrane-embedded proteins by chromophoric amphiphiles through long-range Förster transfer. We illustrate this approach in sarcoplasmic reticulum membranes containing Ca2(+)-ATPase as the main protein constituent. The equilibrium distribution of the antioxidant 4-nonylphenol was deduced from its inhibition of ATPase activity, whereas the equilibrium distribution of the calcium ionophore calcimycin (A23187) and of its brominated analog 4-bromo-A23187 were determined from their quenching of ATPase fluorescence. Apparent partition coefficients K* in the range of 10(5) (expressed as (moles of lipid/liter)-1) were obtained for these highly hydrophobic molecules.

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