Calcium binding to the rat liver plasma membrane

Abstract

Calcium binding to isolated rat liver plasma membranes shows saturation kinetics, has a pH optimum of 7.8–8.0, and is independent of metabolic energy. scatchard analysis shows two classes of calcium binding sites. The higher affinity sites, with an association constant of 4.0·10 2 M −1, accomodate 22 ± 2 ( S.D.) , nmoles of calcium/mg membrane protein. The lower affinity sites with an association constant of 3.2·10 2 M −1 accomodate 120 ± 16 ( S.D.) nmoles of calcium/mg membrane protein. Mg 2+ competes with Ca 2+ only for the low affinity binding sites. K + and Na + do not inhibit calcium binding. EDTA decreases the amount of calcium bound to the membranes. The ATP effect on calcium binding depends on the calcium concentration. At 1 mM calcium, ATP at 0.3 mM inhibits binding but at 3 mM calcium, ATP stimulates biding. ADP and AMP at 0.3 mM have no effect on calcium binding at 1 mM calcium but ADP stimulates binding at 3 mM calcium. Cyclic AMP at 10 −3 M increases calcium bnding at both 1 and 3 mM levesl of calcium. Phospholipases, neuraminidase and proteases were used to determine the role of phospholipids, neuraminic acid and proteins in the binding of calcium. Of the total extrapolated maximum calcium binding sites ( 142 + 18 ( S.D.) nmoles/mg membrane proten), acidic phospholipids accounted for approximately 100 nmoles/mg membrane protein while neuraminic acid residues accounted for approximately 50 nmoles/mg membrane protein. Treatment of the membrane with a variety of functional group reagents showed that agents which react with amino groups or hydroxyl groups of proteins have a small or no effect on calcium binding. However, SH agents increase calcium biding but only when the calcium concentration in the medium was 1 mM or greater. Sodium dodecyl sulfate (0.1%) increases calcium binding very markedly, Triton X-100 (0.1%) gave a 3-fold increase in calcium binding and Lubrol (0.1%) had no influence on calcium binding. Triton may unmask certain acidic phospholipids. Sodium dodecyl sulfate is believed to become incorporated into the membrane and convert it to a highly charged form which now binds calcium. This detergent may also unmask acidic phospholipids.