The Role of Calcium on the Activity of ERcalcistorin/Protein-disulfide Isomerase and the Significance of the C-terminal and Its Calcium Binding

ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI) shows a 55% identity with mammalian protein-disulfide isomerase (PDI) (Lucero, H. A., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112-23119) is a high capacity low affinity Ca2+-binding protein and behaves as a Ca2+ storage protein in the ER of a living cell (Lucero, H. A., Lebeche, D., and Kaminer, B. (1998) J. Biol. Chem. 273, 9857-9863). Here we show that recombinant ECaSt/PDI bound 26 mol of Ca2+/mol and a C-terminal truncated mutant bound 14 mol of Ca2+/mol, both with a Kd of 2.8 mM in 50 mM KCl and 5.2 mM in 150 mM KCl. The percentage reduction in Ca2+ binding in the mutant corresponded with the percentage reduction of deleted pairs of acidic residues, postulated low affinity Ca2+-binding sites. 5 mM Ca2+ moderately increased the PDI activity of both ECaSt/PDI and the C-terminal truncated mutant on reduced RNase and insulin. Surprisingly, ECaSt/PDI in the absence of Ca2+ prevented the spontaneous reactivation of reduced bovine pancreatic trypsin inhibitor. In the presence of 1-5 mM Ca2+ (or 10 microM polylysine) ECaSt/PDI augmented the bovine pancreatic trypsin inhibitor reactivation rate. In contrast, the C-terminal truncated ECaSt/PDI augmented rBPTI reactivation in the absence of Ca2+ and 1-5 mM Ca2+ further accelerated the reactivation rate, responses similar to those obtained with mammalian PDI.