The effects of chemical cross-linking agents on calcium-induced structural changes in skinned muscle fibers: Origin within thick filaments detected by optical diffraction methods

Abstract

We have reported earlier (Sabbadini, R.A., Rieser, G.D. and Paolini, P.J. (1979) Biochim. Biophys. Acta 578, 526–533) that physiological levels of calcium (p Ca 6.95-5.49) can produce structural changes in thick filaments which are detectable as an intensity loss of the first-order optical diffraction lines from chemically skinned skeletal muscle fibers stretched beyond myofilament overlap. We now show that the calcium-induced intensity decrease results from structural changes within, rather than between, thick filaments. Glycerinated, detergent-treated fibers from frog semitendinosus muscle were incubated in 1–10 mM concentrations of dimethylsuberimidate (DMS), dithiobis(succinimidylpropionate) (DTSP) or dimethyl-3,3′-dithiobispropionimidate (DTBP) for 4 h. These substances are homobifunctional lysine-modifying cross-linking reagents known to restrict movement of S-1 heads and limit changes in the association of myosin rods within the core of the thick filament without affecting interfilament lattice spacing. Diffraction patterns from cross-linked cells in relaxing solution were identical to those in control cells, but Ca 2+ (p Ca 5.49) totally failed to produce the typical 50–70% attenuation of first-order line intensity. Cleavage of the disulfide bond in DTBP-treated cells with dithiothreitol fully restored the Ca 2+ sensitivity. Lysine group modification with methylacetimidate, a monofunctional lysine modification reagent equivalent to DMS, did not block the Ca 2+ sensitivity. We observed that intensity reductions can also be produced by numerous other agents and mechanisms, such as nonionic polymeric solutions of polyvinylpyrrolidone, which reduces the lattice spacing, and alkaline pH, which probably displaces the S-1 heads from a resting position close to the thick filament surface. However, the prevention of the Ca 2+ effect by cross-linkers indicates that intrafilament rather than interfilament changes in structure are responsible for the light diffraction intensity decrease accompanying activation.