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Calcium sequestration by isolated sarcoplasmic reticulum: real-time monitoring using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1

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Abstract

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.

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  1. Department of Pathology, Ontario Veterinary College, University of Guelph, N1 G 2W1, Guelph, Ontario, Canada

    P. J. O'Brien

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  1. P. J. O'Brien
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O'Brien, P.J. Calcium sequestration by isolated sarcoplasmic reticulum: real-time monitoring using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1. Mol Cell Biochem 94, 113–119 (1990). https://doi.org/10.1007/BF00214118

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  • DOI: https://doi.org/10.1007/BF00214118

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