Calcium- and pH-dependent aggregation of carboxypeptidase E

Carboxypeptidase 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 is soluble at pH 5.5 (S-CPE), a form which can be extracted from membranes 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 (M2-CPE). Like other peptide processing enzymes, CPE is known to be sorted 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 purified from bovine pituitary membranes aggregates at pH 5.5 when the concentration of CPE is 0.3 μg/μl or higher, but not when the concentration is 0.01 μg/μl. Aggregation of CPE is pH-dependent, with very little aggregation occurring at pH 6 or above. At pH 5.0-5.5, the M2 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 aggregation 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 aggregate is solubilized by 1% Triton X-100 at pH 5.5 or by pH 6.0, whereas M2-CPE is not extracted from membranes under these conditions. Taken together, these results are consistent with a model in which the decreasing pH and increasing Ca²⁺ levels in the trans Golgi network induce the aggregation of CPE, which contributes to the sorting of this protein into regulated pathway secretory vesicles.