Elementary Steps of the Cross-Bridge Cycle in Bovine Myocardium with and without Regulatory Proteins

Abstract

The role of regulatory proteins in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed by gelsolin and the actin filament was reconstituted without tropomyosin or troponin. Further reconstitution was achieved by adding tropomyosin and troponin. The effects of MgATP and phosphate (Pi) on the rate constants of exponential processes were studied in control, actin filament-reconstituted, and thin filament-reconstituted myocardium at pCa < or = 4.66, pH 7.00, 25 degrees C. In control myocardium, the MgATP association constant was 9.1 +/- 1.3 mM(-1), and the Pi association constant 0.14 +/- 0.04 mM(-1). The equilibrium constant of the cross-bridge detachment step was 2.6 +/- 0.4, and the equilibrium constant of the force generation step was 0.59 +/- 0.04. In actin filament-reconstituted myocardium without regulatory proteins, the MgATP association constant was approximately the same, and the Pi association constant increased to 2.8x. The equilibrium constant of cross-bridge detachment decreased to 0.2x, but the equilibrium constant of the force generation step increased to 4x. These kinetic constants regained control values after reconstitution of the thin filament. These results indicate that tension/cross-bridge in the presence of regulatory proteins is approximately 1.5-1.7x of that in the absence of regulatory proteins. These results further indicate that regulatory proteins promote detachment of cross-bridges.

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“…The slow EMB-fiber data, on the other hand, fit a configuration in which the rate-limiting step is an isomerization of myosin before MgADP release ( Fig. 3 A and C), consistent with reaction schemes proposed for other slow striated muscles (11,12,21).…”

“…For both IFI and EMB, elevating [MgATP] increased the frequency of optimum work production ( fw max ) to a saturating level (Fig. 1 A), a result qualitatively similar to that observed in other muscle types (11,12,21). However, Ϸ15 mM MgATP was required to reach maximal fw max in IFI compared with Ϸ2 mM for EMBexpressing fibers, which suggests that the affinity of MgATP for myosin is much lower in IFI than in EMB.…”

“…These trends are opposite to the Pi-dependent rise in fw max observed in EMB fibers and all other striated muscle fiber types studied by using this technique (10)(11)(12)21). The lack of IFI fw max sensitivity to [Pi] at saturating [MgATP] strongly suggests a departure from the typical myosin cross-bridge scheme, where, with MgADP rate limiting for oscillatory work production, fw max is not independent of [Pi] in working muscle (9)(10)(11)(12)(21)(22)(23).To identify the correct rate-limiting step for IFI, we examined the qualitative effects of [MgATP], [MgADP], and [Pi] on the characteristic frequencies of the work-producing (B) and workabsorbing (C) processes of IFI fibers undergoing smallamplitude sinusoidal oscillations (see Materials and Methods and Fig. 4, which is published as supporting information on the PNAS web site).…”

“…In IFI fibers at saturating levels of MgATP (15-20 mM), fw max was insensitive to changes in [Pi]; at 10 mM MgATP, fw max showed a slight tendency to decrease with increasing [Pi]; at 5 mM MgATP, fw max decreased significantly with increasing [Pi]. These trends are opposite to the Pi-dependent rise in fw max observed in EMB fibers and all other striated muscle fiber types studied by using this technique (10)(11)(12)21). The lack of IFI fw max sensitivity to [Pi] at saturating [MgATP] strongly suggests a departure from the typical myosin cross-bridge scheme, where, with MgADP rate limiting for oscillatory work production, fw max is not independent of [Pi] in working muscle (9)(10)(11)(12)(21)(22)(23).…”

“…The prolongation is essential for coupling enzyme chemical kinetics, which normally occur rapidly, to the slower movements of the sarcomere during normal muscle function. In most vertebrate striated muscle types, the comparatively slow release of MgADP (one of the products of MgATP hydrolysis) is thought to be the ratelimiting step (8)(9)(10)(11)(12)(13). However, recent studies suggest MgADP release may not be rate limiting for faster muscle types (14)(15)(16).…”