Effects of sulphydryl modification on skinned rat skeletal muscle fibres using 5,5'-dithiobis(2-nitrobenzoic acid)

1. The sulphydryl groups of skinned skeletal muscle fibres have been reacted with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in order to determine whether the effects of modifications to the contractile proteins are reflected in changes in the physiological properties of the contractile apparatus and Ca(2+)-regulatory system. 2. Results obtained from fast-twitch and slow-twitch rat fibres which were treated with DTNB (10 mM, pH 8.6, 5 degrees C) for various periods of time under relaxing conditions showed that a major effect of the modification was to reduce the level of maximally Ca(2+)-activated force and fibre stiffness. Force and fibre stiffness were found to decline in proportion. Treatment with DTNB under these conditions did not cause a rise in force or fibre stiffness in relaxed fibres of either type. 3. The effects induced by DTNB under relaxing conditions were substantially reversed by exposure to the reducing agent dithiothreitol (DTT) (10 mM, pH 7.1, 23 degrees C). Force abolished by 30-35 s treatment with DTNB recovered after subsequent DTT treatment to 67 +/- 3% (mean +/- S.E.M., n = 4) in fast-twitch fibres and to 91 +/- 2% (n = 7) in slow-twitch fibres. These results were significantly different (t test, P less than 0.001) indicating that the level of force recovery depended upon the fibre type. 4. DTNB was found to affect not only the maximal Ca(2+)-activated force, but also the force-pCa (pCa = -log10[Ca2+]) relationships of the fibres in a complex, fibre-type specific way. DTT treatment partially reversed these DTNB effects. 5. The skinned fibre preparations reacted differently with DTNB under rigor conditions than under relaxing conditions, indicating that rigor modifies the reactivity of the functional sulphydryl groups to the thiol-targeted agents. 6. When superprecipitation assays (an in vitro analogue of fibre contraction) were carried out with recombined myofibrillar proteins which had been previously reacted with DTNB it was found that modification of myosin, but not modification of thin filament proteins, led to changes in the superprecipitation reaction. 7. Both the skinned fibre results and the superprecipitation results indicate that the effects of DTNB upon the fibre characteristics are primarily due to modifications of the sulphydryl groups of myosin. Therefore, these results show that myosin is not only involved in determining the ability of the contractile apparatus to develop force but also in determining the Ca(2+)-regulatory characteristics of the muscle fibre.