Effects of Divalent Cations and ATP on the Kinetic Properties of the Sulfhydryl Groups of Sarcoplasmic Reticulum Membranes and Purified Ca-ATPase

The modification of SR membranes and purified Ca-ATPase with 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-C1) reveals about 11 mol of SH-groups per mol enzyme. The sulfhydril groups of both preparations are divided into two classes (kinetic sets). These classes of SH-groups of SR membranes display pseudo-first-order rate constants (k) of 0.16 and 0.015 min"' (25 °C, pH 7.0, 0.75 mmol/1 NBD-C1). The corresponding values of k for modification of these sulfhydryl groups of purified Ca-ATPase are 0.11 and 0.027 min"', respectively. In the presence of Mg (E-state of enzyme) all fast sulfhydryl groups of SR membranes are modified with k 0,09 min"'. Under these conditions, however, there are but little changes in the reactivity of fast SH-groups of the purified enzyme. Binding of Ca to SR membranes (E-state) results in an enhancement of the fast SH-groups number by 1, and all fast SH-groups are modified with k 0.34 min"'. In the presence of this ion the number of fast SH-groups of purified Ca-ATPase is decreased by 2, and their modification constant is increased to 0.17 min ' . The addition of ATP to both preparations results in similar effects. At low ATP concentration (30 umol/1, E-state) the number of fast SH-groups is decreased by 1 in both preparations, modification constants of these groups being of 0.12 and 0,09 min" for SR membranes and purified Ca-ATPase, respectively. In the presence of high ATP concentration (3 mmol/1, EATP-state) the number of the fast SH-groups is decreased more: by 2 for SR membranes and by 1 for purified Ca-ATPase. Ca-ATPase activity of SR membranes and the purified enzyme is lost as a result of NBD-C1 modification. The pseudo-first-order constants of modification exceed by the factor of 4 the values of k for modification of fast SH-groups. In the presence of high ATP concentrations, the value of k for Ca-ATPase inactivation in SR membranes is decreased to 0.07 min", that is equal to the modification constant of the fast SH-groups. The rate of the Ca-ATPase inactivation in the purified enzyme does not change virtually in the presence of ATP.