CALCIUM-DEPENDENT AND PH-DEPENDENT AGGREGATION OF CARBOXYPEPTIDASE-E

Carboxypeptides E (CPE) is involved with the biosynthesis of numerous peptide hormones and neurotransmitters. Several forms of CPE have been previously detected in neuroendocrine cells, including a form which i s soluble at pH 5.5 (S-CPE), a form which can be extracted from membra nes with 1 M NaCl at pH 5.5 (M1-CPE), and a form which requires both 1 % Triton X-100 and 1 M NaCl for extraction from membranes at pH 5.5 (M 2-CPE). Like other peptide processing enzymes, CPE is known to be sort ed into peptide-containing secretory vesicles of the regulated pathway . One mechanism that has been proposed to be important for sorting of regulated pathway proteins is Ca2+ and pH-induced aggregation. CPE pur ified from bovine pituitary membranes aggregates at pH 5.5 when the co ncentration of CPE is 0.3 mu g/mu l or higher, but not when the concen tration is 0.01 mu g/mu l. Aggregation of CPE is pH-dependent, with ve ry little aggregation occurring at pH 6 or above. At pH 5.0-5.5, the M 2 form of CPE shows a greater tendency to aggregate than the other two forms. At pH 6, Ca2+ concentrations from 1-30 mM increase the aggrega tion of M1- and M2-CPE, but not S-CPE. The aggregation of M2-CPE does not explain the apparent membrane binding of this protein since the ag gregate is solubilized by 1% Triton X-100 at pH 5.5 or by pH 6.0, wher eas M2-CPE is not extracted from membranes under these conditions. Tak en together, these results are consistent with a model in which the de creasing pH and increasing Ca2+ levels in the trans Golgi network indu ce the aggregation of CPE, which contributes to the sorting of this pr otein into regulated pathway secretory vesicles.