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Estimation of the free [Ca2+] gradient across endoplasmic reticulum membranes by a null-point method
A P Dawson1, G T Rich, J W Loomis-Husselbee
1School of Biological Sciences, University of East Anglia, Norwich, U.K.
The Biochemical Journal
|September 1, 1995
Summary
Researchers measured the calcium (Ca2+) electrochemical gradient across rat liver microsomal vesicles. They found that internal Ca2+ concentrations increase linearly with loading, revealing insights into cellular calcium transport.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Calcium ions (Ca2+) are critical intracellular messengers.
- Disruptions in cellular calcium homeostasis are linked to various pathologies.
- Understanding calcium transport mechanisms is vital for cell function.
Purpose of the Study:
- To quantify the electrochemical gradient of Ca2+ across rat liver microsomal vesicles.
- To determine the relationship between Ca2+ loading and internal free Ca2+ concentration.
- To investigate Ca2+ levels in the endoplasmic reticulum of L1210 cells.
Main Methods:
- Utilized 45Ca-labeled rat liver microsomal vesicles.
- Employed thapsigargin to induce Ca2+ efflux.
- Measured external free Ca2+ concentration at zero net efflux to determine internal Ca2+.
- Applied a similar technique to permeabilized L1210 cells.
Main Results:
- Unidirectional Ca2+ efflux was not inhibited by external Ca2+, allowing for gradient measurement.
- At 7.9 nmol/mg protein loading, internal free Ca2+ was 21 microM.
- Internal free Ca2+ increased linearly with loading, reaching 47 microM at 21.6 nmol/mg.
- Endoplasmic reticulum free Ca2+ in L1210 cells was 12.5 microM.
Conclusions:
- Established a method to measure the Ca2+ electrochemical gradient in vesicles.
- Demonstrated a linear relationship between Ca2+ loading and internal concentration in microsomes.
- Provided baseline Ca2+ levels for rat liver microsomes and L1210 cell endoplasmic reticulum.