PROTONS INDUCE CALSEQUESTRIN CONFORMATIONAL-CHANGES

Calsequestrin, a high-capacity, intermediate-affinity, calcium-binding protein present in the lumen of sarcoplasmic reticulum, undergoes ext ensive calcium-induced conformational changes at neutral pH that cause distinct intrinsic fluorescence changes. The results reported in this work indicate that pH has a marked effect on these calcium-induced in trinsic fluorescence changes, as well as on calorimetric changes produ ced by the addition of Ca2+ to calsequestrin. The addition of Ca2+ at neutral pH produced a marked and cooperative increase in calsequestrin intrinsic fluorescence. In contrast, at pH 6.0 calsequestrin's intrin sic fluorescence was not affected by the addition of Ca2+, and the sam e intrinsic fluorescence as that measured in millimolar calcium at neu tral pH was obtained. The magnitude and the cooperativity of the calci um-induced intrinsic fluorescence changes decreased as either [H+] or [K+] increased. The evolution of heat production, determined by microc alorimetry, observed upon increasing the molar ratio of Ca2+ to calseq uestrin in 0.15 M KCI, decreased markedly as the pH decreased from pH 8.0 to pH 6.0, indicating that pH modifies the total heat content chan ges produced by Ca2+. We propose that protons bind to calsequestrin an d induce protein conformational changes that are responsible for the o bserved proton-induced intrinsic fluorescence and calorimetric changes .