FIGURE 2.

Reversibility, divalent cation dependence and concentration dependence of the effects of Ca²⁺. A, cerebellum microsomes were incubated for 5 min in the absence (lane 1) or presence (lane 2) of 2.2 μm free Ca²⁺ for 5 min, followed by a 5-min digestion with trypsin (4 μg/ml). 1 mm EGTA was added 1 min or 30 s prior to trypsin digestion in lanes 3 and 4, respectively. In lane 5, the trypsin digestion was carried out first for 5 min in the absence of Ca²⁺ (0.5 mm HEDTA). The digestion was stopped by the addition of 10 μg/ml soybean trypsin inhibitor, and 0.4 mm CaCl₂ was added to give a final free concentration 2.2 μm. B, microsomes were incubated for 5 min with 1 mm Mg²⁺, 2.2 μm Ca²⁺, or the indicated free concentrations (μm) of Sr²⁺ or Ba²⁺ (as described under “Experimental Procedures”). The membranes were then digested for 5 min with trypsin. C, cerebellum microsomes were incubated for 5 min with the indicated concentrations of buffered free Ca²⁺ in the presence or absence of 10 μm IP₃. The samples were digested for 5 min with trypsin and immunoblotted with CT-1 Ab. The top panel shows a representative immunoblot of the 95-kDa fragment V, and the lower panel is a compilation of the data from three separate experiments (mean ± S.E.).