It is shown, by means of analytical ultracentrifugation, that skeletal myosin S-1 exists in the form of a monomer-dimer mixture, in rapid reversible equilibrium, sensitive to the hydrostatic pressure, the temperature, and the composition of the buffer (at least, pH, ionic strength, presence or absence of a Mg-(phosphate compound), and presence or absence of Mg2+). The dimer is predominant at high pH, at low ionic strength, in the presence of a Mg-(phosphate compound), at high pressure, and at low temperature. The monomer is predominant in the reverse conditions. At atmospheric pressure and at room temperature, in a buffer having a composition close to that of the physiological medium, but containing no Mg-(phosphate compound), the monomer is largely predominant (more than 90% at 1 mg/mL S-1). At atmospheric pressure and at room temperature, in a buffer containing a Mg-(phosphate compound) and having a composition close to that of the physiological medium, S-1 exists in the form of a monomer-dimer mixture, with a noticeable proportion of dimer (more than 25% at 1 mg/mL S-1 in the presence of 2 mM MgADP and 3 mM Mg2+). In such buffers, the monomer:dimer ratio is extremely sensitive to both the pH and the ionic strength. The sedimentation coefficients of the monomer and the dimer are respectively 5.05 +/- 0.05 S and 6.05 +/- 0.05 S. The two protomers making up the dimer are stuck together in an end-to-end arrangement. Both the monomer and the dimer are highly hydrated (about 0.9 g of water/g of protein for the monomer and probably more for the dimer).

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Hydrodynamic calculations lead to the conclusion that chymotryptic (or ethylenediaminetetraacetic acid) myosin S1 in solution (hydrated), at 1-5 degrees C, can be modeled as a prolate ellipsoid, with an axial ratio lying between p = 1.0 and 2.5 (major axis between 100.5 A, for p = 1.0, and 162.5 A, for p = 2.5). The degree of hydration is considerable (1.24 g/g for p = 2.5 and 2.02 g/g for p = 1.0). The dehydrated myosin head is pear-shaped under the electron microscope, and its narrowest part is located near the junction with the tail [Elliott, A., & Offer, G. (1978) J. Mol. Biol. 123, 505-519]. Mendelson & Kretzschmar [Mendelson, R. A., & Kretzschmar, K.M. (1980) Biochemistry 19, 4103-4108] have shown that the pear-shaped molecule does not predict the experimental X-ray scattering curve. Nor is this model able to predict the hydrodynamic values. The three-dimensional model for S1 used by Mendelson and Kretzschmar gives a rather good fit to the experimental X-ray scattering curve, but it does not predict the hydrodynamic values. In order to try to reconcile the three models and to fit the X-ray scattering curve and the hydrodynamic data, we suggest that, in solution, the S1 monomer has the shape of a prolate ellipsoid and that an inclusion of bound water exists at one extremity of the protein. The rest of bound water surrounds the protein. As first approximation, the dry protein and the hole are assumed to have the same shape as the hydrated molecule (prolate ellipsoid; p).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cell swelling on intracellular calcium concentration ([Ca2+]i) was studied in newborn rat cardiomyocytes. Hypotonic cell swelling induced a fast and transient [Ca2+]i increase (hypotonically induced calcium increase, HICI; 388±47 nM, n=14). HICI was not inhibited by cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic Ca2+-ATPase, nor ryanodine (an inhibitor of calcium-induced calcium release), whereas it was abolished (11±19 nM, n=5) in the absence of external calcium. Thus, HICI appeared to depend exclusively on entry of external calcium. Gadolinium ion (Gd3+), a generic inhibitor of stretch-activated cation channels (SACs), was unable to affect HICI (353±79 nM, n=6). Similarly, HICI was unaffected by internal Na+ depletion and external Na+ omission. These results suggest that neither Gd3+-sensitive SACs nor Na+-Ca2+ exchange is responsible for HICI. Conversely, HICI was inhibited by diltiazem (42±4 nM, n=3) and by membrane predepolarization (40±18 nM, n=5), suggesting an involvement of L-type voltage-activated calciumchannels. Cardiomyocyte swelling was followed by a regulatory volume decrease (RVD). The putative role of HICI in volume regulation was studied by removal of external calcium. This procedure significantly slowed RVD but did not abolish it. In conclusion, newborn rat cardiomyocytes exhibit an external-calcium-dependent HICI which contributes partially to the RVD.

Several conflicting experiments have been carried out concerning the existence of a refractory state for myosin subfragment 1 (S1; i.e. unable to bind to F‐actin), in the presence of Mg‐nucleotide compounds, at low temperature. Contradictory experiments have been published on the existence of a S1 dimer, under the same conditions. By taking into account some elementary, but crucially important, precautions in the preparation of myosin and S1, it is possible to maintain intact the head‐to‐head sites of dimerisation on both myosin and S1. Moreover, it has been shown that Mg2+ alone, at low temperature, is able to induce a reversible S1‐S1 dimerisation [Morel, J. E. & Garrigos, M. (1982) Biochemistry 21, 2679–2686). Here, we have studied the intrinsic viscosity of S1, in the presence of 2 mM MgC12, versus temperature, between 0.5–20°C. At approximately 0.5–2.0°C, the intrinsic viscosity of S1 was found to be 19.8 cm3/g. Above 2.5–3.0°C, a steep decrease in the intrinsic viscosity was observed. Between 7°C and 20°C, a constant intrinsic viscosity of 6.7 cm3/g was measured. Therefore, there is a dramatic temperature transition between approximately 2.5–7.0°C (width, 4.5°C; midpoint, 5°C): below 2.5°C we observed the presence of a S1 dimer (confirmed by analytical ultracentrifugation performed at 1°C) and above 7–8°C we observed the presence of a S1 monomer (confirmed by analytical ultracentrifugation performed at 8°C). We have also studied the interactions of F‐actin with S1, under the same conditions of temperature, and in the presence of 2 mM MgCl2, by using preparative ultracentrifugation. At 1–2°C, S1, which is in the dimeric form, is unable to bind to F‐actin. At 6–20°C, F‐actin and S1 bind stoichiometrically (1S1/1actin), with an equilibrium constant of 0.5 μM−1, under our experimental conditions. We also performed binding experiments at 3–5°C. Although we were unable to clearly reach the asymptote corresponding to stoichiometry, by assuming a 1:1 ratio, we found a temperature transition in the equilibrium constants, between 3–5°C (width, 2°C; midpoint, 4°C). These phenomena are comparable to those observed for the dimer. Thus, we have shown that there is also a temperature transition in the F‐actin‐S1 binding process. We conclude that the dimeric and the refractory states of S1 are identical.