Steady-State Properties of Calcium Binding to Parvalbumins from Bullfrog Skeletal Muscle: Effects of Mg²⁺, pH, Ionic Strength, and Temperature

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To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I=0.106, pH 6.80, at 20°C) containing various concentrations of Mg²⁺ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca²⁺ in the presence of Mg²⁺ changed in the manner expected if Ca²⁺ and Mg²⁺ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, Kcal×10⁷ M^-1, KMg=900 M^-1; for PA-2, KCa= 6×10⁶ M^-1, KMg= 830 M^-1 (I=0.106, pH 6.80, at 20°C). The apparent binding constants are strongly dependent on temperature: at 10°C for PA-1, KCa=2×10⁸ M^-1, KMg=10⁴ M^-1; for PA-2, KCa=5×10⁷ M^-1, KMg=5×10³ M^-1 (I=0.106, pH 6.80). The dependence of the affinities for Ca²⁺ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca²⁺ and Mg²⁺ of parvalbumins are unchanged between pH 6.5 and 7.2.