BINDING OF CA2-MG2+)-ATPASE OF SARCOPLASMIC-RETICULUM - KINETIC-STUDIES( TO THE (CA2+)

Stop-flow fluorescence and rapid-filtration methods have been used to establish the kinetics of Ca2+ binding to, and dissociation from, the (Ca2+-Mg2+)-ATPase of skeletal-muscle sarcoplasmic reticulum and to de fine the effects of H+ and Mg2+ on Ca2+ binding and dissociation rates . The kinetics have been interpreted in terms of the scheme: E2 revers ible arrow E1 reversible arrow E1Ca reversible arrow E1'Ca reversible arrow E1'Ca,. The kinetics of the E2 reversible arrow E1 transition ha ve been determined by measuring the rate of change of the fluorescence of the ATPase labelled with 3-nitrobenzo-2-oxa-1,3-diazole after a pH jump or the addition of Ca2+ to the labelled ATPase in the presence o f thapsigargin or thapsivillosin A. It has been shown that Mg2+ has a marked effect on Ca2+ dissociation at pH 7.2 and that changes in the t ryptophan fluorescence of the ATPase follow the same time course as th e dissociation of Ca-45(2+). It is proposed that the effect of Mg2+ fo llows from binding to a 'gating' site, as detected by changes in the f luorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxyc oumarin. The rate of dissociation of Ca2+ from the ATPase increases wi th increasing pH. The rate of dissociation of Ca2+ decreases with incr easing Ca2+ concentration in the medium, with an apparent affinity for Ca2+ greater than that seen for the change in fluorescence amplitude. It is shown that this follows if the first, inner, Ca2+-binding site on the ATPase has a lower affinity for Ca2+ than the second, outer, si te. Effects of H+ and Mg2+ on Ca2+ dissociation can be treated by the quasiequilibrium approach. Mg2+ and H+ also affect the rate of Ca2+ bi nding to the ATPase, and effects of H+ and Mg2+ on the E2 reversible a rrow E1 equilibrium explain the results of experiments in which the co ncentrations of H+ and Mg2+ are jumped.