Protons induce calsequestrin conformational changes.
Calsequestrin, a high-capacity, intermediate-affinity, calcium-binding protein present in the lumen of sarcoplasmic reticulum, undergoes extensive 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 intrinsic fluorescence changes, as well as on calorimetric changes produced by the addition of Ca(2+) to calsequestrin. The addition of Ca(2+) at neutral pH produced a marked and cooperative increase in calsequestrin intrinsic fluorescence. In contrast, at pH 6.0 calsequestrin's intrinsic fluorescence was not affected by the addition of Ca(2+), and the same intrinsic fluorescence as that measured in millimolar calcium at neutral pH was obtained. The magnitude and the cooperativity of the calcium-induced intrinsic fluorescence changes decreased as either [H+] or [K+] increased. The evolution of heat production, determined by microcalorimetry, observed upon increasing the molar ratio of Ca(2+) to calsequestrin in 0.15 M KCl, decreased markedly as the pH decreased from pH 8.0 to pH 6.0, indicating that pH modifies the total heat content changes produced by Ca(2+). We propose that protons bind to calsequestrin and induce protein conformational changes that are responsible for the observed proton-induced intrinsic fluorescence and calorimetric changes.
Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Santiago. ch@cecs.cl
C
C
Hidalgo
P
P
Donoso
P H
PH
Rodriguez
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002151:Calorimetry; D002155:Calsequestrin / Q000737:chemistry* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018482:Muscle, Skeletal; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D002155:Calsequestrin; D011189:Potassium Chloride; D002118:Calcium