Steady-State Properties of Calcium Binding to Parvalbumins from Bullfrog Skeletal Muscle: Effects of Mg2+ pH, Ionic Strength, and Temperature1

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Abstract
To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI 4.97) 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, KCa=1×107 M−1 KMg=900M−1; for PA-2, KCa=6×108 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.