Kinetic Studies of Calcium Binding to Parvalbumins from Bullfrog Skeletal Muscle1

Abstract

In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stopped-flow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20°C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25 μM CaCl₂ metal-deprived PA-1 or PA-2, various concentrations of Mg²⁺ and KCl to adjust the ionic strength of the medium to 0.106 The results can be explained in terms of the following rate constants under the conditions mentioned above when a secondorder kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca²⁺ are 1.5×10⁷ M⁻¹·S⁻¹ and 1.5 S⁻¹, respectively, or more. The rate constants for Mg²⁺ are 7,500 M⁻¹·S⁻¹ and 5–6 S⁻¹, respectively. For PA-2, the rate constants for Ca²⁺ are 7×10⁸ M⁻¹·S⁻¹ and 1.16 S⁻¹ respectively, and those for Mg²⁺ are 3,500 M⁻¹·S⁻¹ and 3.5–4 S⁻¹, respectively. Increased affinities for Ca²⁺ and Mg²⁺ at 10°C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.