Kinetics of reaction in calcium-activated skinned muscle fibres

Abstract

A PROCEDURE reducing the diffusion-limited equilibration time of the Ca2+ concentration within skinned muscle fibres1 activated from outside has been used to investigate the kinetics and the stoichiometry of Ca2+ in the process of tension development in frog skeletal muscle. The diffusion problems are minimised by activating (or relaxing) the myofibrillar preparation in a solution with a very high Ca2+-buffering capacity compared to that of the fibre as a whole2,3 (solution in the interfilament space, sarcoplasmic reticulum, myofibrillar proteins);by using only thin fibres; and by using an efficient pH buffer (TES: N-Tris (hydroxymethyl) methyl-2-aminoethanesulphonic acid) and a high ATP concentration in conjunction with a powerful ATP regenerative system (creatine phosphate (CP) and creatine kinase (CK)). Great care has been taken in preparing bathing solutions with similar concentrations of monovalent metallic cations ( 150 mM), protons (pH 7.1 )4 and [Mg2+](1 mM) (refs 5 and 6) to those in vivo. For this purpose, the apparent binding constants of the various ligands for Ca2+ and Mg2+ have been measured by a pH metric method (Table 1).

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