CALCIUM SEQUESTRATION BY ISOLATED SARCOPLASMIC-RETICULUM - REAL-TIME MONITORING USING RATIOMETRIC DUAL-EMISSION SPECTROFLUOROMETRY AND THE FLUORESCENT CALCIUM-BINDING DYE INDO-1

This study demonstrates a simple, rapid, and reproducible microassay for real-time monitoring of Ca2+-sequestration by isolated sarcoplasmic reticulum (SR) using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1. The SR membranes were isolated by differential centrifugation and suspended in a medium including Ca2+, indo-1, ATP and oxalate. As Ca2+ was sequestered by SR, Ca2+-bound indo-1 fluorescence decreased equivalently but reciprocally to the increase in Ca2+ -free indo-1 fluorescence. The kinetic and thermodynamic properties of Ca2+-transport measured fluorometrically were similar to those measured radiometrically by 45Ca2+, with the exception that the former monitors changes in free Ca2+ whereas the latter monitors total Ca2+. An estimate of the maximal rate of change in total Ca2+ could be made by multiplying the maximal rate of change in free Ca2+ by the ratio of initial total Ca2- to free Ca2- concentration. © 1990 Kluwer Academic Publishers.