Localization of binding sites of F-protein (phosphofructokinase) on the myosin molecule

Summary To determine the location of F-protein binding sites on myosin, the interaction of F-protein with myosin and its proteolytic fragments in 0.1 m KCl,10 mm potassium phosphate, pH 6.5, has been investigated using sedimentation, electron microscopy and optical diffraction methods.Sedimentation experiments show that F-protein can bind to myosin and myosin rod rather than to light meromyosin or subfragment-1. The F-protein binding to myosin and rod is of a similar character. The calculated values of the constants of F-protein binding to myosin and rod are 2.6 × l0⁵M^–1 and 2.1 × 10⁵ m^–1, respectively. The binding sites are probably located on the subfragment-2 portion of the myosin molecule. The number of the F-protein binding sites calculated per chain weight of 80 000 is 5 ±1.Electron microscopic observations confirm the sedimentation results. F-protein does not bind to light meromyosin paracrystals, but decorates myosin and rod filaments with the interval of 14.3 nm regardless of whether F-protein is added prior to or after filamentogenesis. The comparison of optical diffraction patterns obtained from myosin and rod filaments with those from decorated ones reveals the marked enhancement of meridional reflection at (14.3 nm)^–1 in the latter case.Neither the increase in ionic strength from 0.1 to 0.15 and pH from 6.5 to 7.3 nor substitution of potassium phosphate buffer by imidazole-HCl buffer, or Tris-HCl influences F-protein binding to myosin and rod filaments as visualized by electron microscopy.The possible significance of F-protein location in the thick filament structure is discussed.     

Visualization of caldesmon binding to synthetic filaments of smooth muscle myosin

We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca²⁺ and in the presence of ATP) to visualize the mode of caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on caldesmon binding to both actin and myosin and electron microscopic observations on the mode of caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by caldesmon. This revised version was published online in July 2006 with corrections to the Cover Date.     

Autoantibodies to myosin light chains in the blood as early marker of myocardial injury after aortocoronary bypass surgery

Serum concentration of autoantibodies to myosin light chains was measured after resumption of the bloodflow recovery in patients who underwent hypothermic aortocoronary bypass surgery. The patients were divided into 3 groups according to postoperative hemodynamic parameters and degree of myocardial injury. The studies showed significant differences in the concentrations of autoantibodies to myosin light chains between the groups. High correlation was shown between the duration of aorta clamping and concentration of autoantibodies to myosin light chains. Some factors characterizing initial severity of the disease can modulate blood concentration of autoantibodies to myosin light chains. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 8, pp. 208–212, August, 2007     

The effect of Ca2+ on the structure of synthetic filaments of smooth muscle myosin

Using electron microscopy and negative staining we have studied the effect of Ca²⁺ on the structure of synthetic filaments of chicken gizzard smooth muscle myosin under conditions applied by Frado and Craig (1989) for demonstration of the influence of Ca²⁺ on the structure of synthetic filaments of scallop striated muscle myosin. The results show that Ca²⁺ induces the transition of compact, ordered structure of filaments with a 14.5 nm axial repeat of the myosin heads close to the filament backbone (characteristic of the relaxing conditions) to a disordered structure with randomly arranged myosin heads together with subfragments-2 (S-2) seen at a distance of up to 50 nm from the filament backbone. This order/disorder transition is much more pronounced in filaments formed of unphosphorylated myosin, since a substantial fraction of phosphorylated filaments in the relaxing solution is already disordered due to phosphorylation. Under rigor conditions some of the filaments of unphosphorylated and phosphorylated myosin retain a certain degree of order resembling those under relaxing conditions, while most of them have a substantially disordered appearance. The results indicate that Ca²⁺-induced movement of myosin heads away from the filament backbone is an inherent property of smooth muscle myosin, like molluscan muscle myosin regulated exclusively by Ca²⁺ binding, and can play a modulatory role in smooth muscle contraction.