Characterization of high-capacity low-affinity calcium binding protein of liver endoplasmic reticulum: Calsequestrin-like and divergent properties

Abstract

It had been previously demonstrated that endoplasmic reticulum membranes from rat hepatocytes contain a major calsequestrin-like protein, on account of electrophoretic and Stains All-staining properties (Damiani et al. , J. Biol. Chem. 263, 340–343). Here we show that a Ca 2+-binding protein sharing characteristics in size and biochemical properties with this protein is likewise present in the isolated endoplasmic reticulum from human liver. Human calsequestrin-like protein was characterized as 62 kDa, highly acidic protein (pl 4.5), using an exctraction procedure from whole tissue, followed by DEAE-Cellulose chromatography, that was originally developed for purification of skeletal muscle and cardiac calsequestrin. Liver calsequestrin-like protein bound Ca 2+ at low affinity (Kd=4 mM) and in high amounts (Bmax=1600 nmol Ca 2+/mg of protein), as determined by equilibrium dialysis, but differed strikingly from skeletal muscle calsequestrin for the lack of binding to phenyl-Sepharose resin in the absence of Ca 2+, and of changes in intrinsic fluorescence upon binding of Ca 2+. Thus, these results suggest that liver 62 kDa protein, in spite of its calsequestrin-like Ca 2+-binding properties, does not contain a Ca 2+-regulated hydrophobic site, which is a specific structural feature of the calsequestrin-class of Ca 2+-binding proteins.