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 whole^2,3 (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).