Distribution of plasma membrane Ca 2+-ATPase and inositol 1,4,5-trisphosphate receptor in human platelet membranes

Abstract

Human platelet plasma membranes were prepared by the glycerol lysis method of Harmon et al. [Harmon JT. Greco NJ. Jamieson GA. (1992) Isolation of human platelet plasma membranes by glycerol lysis. Meth. Enzymol., 215, 32–36]. The membranes were observed to contain a Ca 2+-ATPase with different properties than those of internal membranes. The specific activity of Ca 2+-ATPase was lower in plasma membranes (10–40 nmol ATP hydrolyzed/min/mg), but the ATPase was less sensitive to thapsigargin (41% inhibition at 500 nM) and more sensitive to vanadate (50% inhibition at 4 μM) than the Ca 2+-ATPase in internal platelet membranes. The plasma membranes contained a Ca 2+-ATPase detectable by monoclonal and polyclonal antibodies against erythrocyte Ca 2+-ATPase that had a molecular mass of 144 kD. However, an anti-peptide antibody against an N-terminal sequence of the inositol 1,4,5-trisphosphate receptor recognized this protein in internal membranes, but not plasma membranes.