Abstract
Calcium binding to isolated adipocyte microsomes enriched in endoplasmic reticulum has been characterized. Binding was concentration-dependent, saturable, and totally dissociable. Steady state was reached within 20 min at all calcium concentrations tested. Three apparent classes of binding sites were identified in kinetic and steady state studies using calcium concentrations from 1 muM to 10 mM. The affinity constants (and maximum binding capacities) as determined by computer analysis for the three classes were 2.1 X 10(5) M-1 (0.28 nmol of calcium/mg of protein), 1.3 X 10(4) M-1 (1.1 nmol/mg), and 1.3 X 10(2) M-1 (35 nmol/mg). The dissociation rate constants for the high and intermediate affinity classes of sites were 1.6 X 10(-3) S-1, respectively, and the association rate constant for the high affinity sites was 8 X 10(2) M-1 S-1. The affinity constant calculated from the rate constants was 5.0 X 10(5) M-1 for the high affinity sites in agreement with the value obtained in studies at steady state. The three classes of binding sites were specific for calcium. Magnesium was a noncompetitive inhibitor of calcium binding to all three classes of sites with a Ki of 9 to 12 mM. Calcium binding at 1 muM calcium was 50% inhibited by 18 muM La3+, 600 muM Sr2+, or 2.7 mM Ba2+. These data represent the first analysis of passive calcium binding to endoplasmic reticulum from nonmuscular cells and the first report of corresponding rate constants for either endoplasmic or sarcoplasmic reticulum. The characteristics of the binding are consistent with the properties of calcium transport by endoplasmic reticulum of adipocytes. The characteristics and specificity of the calcium binding constitute further evidence that endoplasmic reticulum plays an important role in cellular calcium homeostasis.
Highlights
Binding was concentration-dependent, saturable, and totally dissociable
Determination of Binding Constants-Calcium binding to adipocyte microsomes was a concentration-dependent satura
The K, for calcium binding to the high affinity sites was calculated to be 2 to 5 x lo-” M which corresponds to the estimated level of calcium in the cytosol of a variety of cells [4]
Summary
Calcium binding to isolated adipocyte microsomes enriched in endoplasmic reticulum has been characterized. The characteristics and specificity of the calcium binding constitute further evidence that endoplasmic reticulum plays an important role in cellular calcium homeostasis. Recently have reports identified active calcium transport systems in endoplasmic reticulum vesicles of liver (lo), kidney (ll), and adipocytes [8]. The properties of these transport systems have strongly suggested that the endoplasmic reticulum in these cells, like the sarcoplasmic reticulum in muscle, plays an important role in controlling cytosol calcium concentrations. The data reported contribute to an understanding of the mechanism of active calcium transport by endoplasmic reticulum [13] and re-emphasize the importance of this organelle in cellular calcium homeostasis
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