Kinetic Studies of Calcium Binding to Parvalbumins from Bullfrog Skeletal Muscle

抄録

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 stoppedflow 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, 25μM CaCl₂, metaldeprived 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, 500M^-1•s^-1 and 5-6s^-1, respectively. For PA-2, the rate constants for Ca²⁺ are 7×10⁶ 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.