Purification and Characterization of Carboxypeptidase D, a Novel Carboxypeptidase E-like Enzyme, from Bovine Pituitary

Abstract

Carboxypeptidase E (CPE) is involved in the biosynthesis of most neuropeptides and peptide hormones. Until recently, CPE was the only intracellular carboxypeptidase thought to be involved in neuroendocrine peptide processing. However, the finding that fat/fat mice, which have a mutation within the CPE gene that inactivates the enzyme, are capable of a reduced amount of insulin processing suggests that another carboxypeptidase is present within the secretory pathway. We have detected a CPE-like enzyme, designated CPD, which has many properties in common with those of CPE. Like CPE, CPD is a metallocarboxypeptidase that has a pH optimum of 5.5-6. The Km and Kcat values for a series of short peptide substrates show only minor differences between CPD and CPE. Several active site-directed inhibitors also show generally similar potency toward the two enzymes, although guanidinoethylmercaptosuccinic acid is approximately 10-fold more potent, and hippuryl-Arg is approximately 100-fold more potent as an inhibitor of CPD than of CPE. A major difference between the two enzymes is the molecular masses; CPE is 50,000-56,000, whereas CPD is approximately 180,000. Also, CPD does not elute from a substrate affinity column when the pH is raised to 8, which elutes CPE, although CPD can subsequently be eluted by arginine. Both CPE and CPD are present in purified bovine anterior pituitary secretory vesicles, but the tissue distribution of CPD is more uniform than that of CPE. Antisera to the N- and C-terminal regions of CPE do not recognize CPD. The partial N-terminal amino acid sequence of bovine CPD shows 30-40% homology with an N-terminal region of bovine and rat CPE and 70% homology with a duck protein known as gp180, a hepatitis B virus particle binding protein that shows 47% homology to CPE. Taken together, these results suggest that CPD is a novel secretory pathway enzyme that may be the bovine homologue of gp180.

Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

“…In addition, another group identified a protein designated AEBP1 as a novel member of the carboxypeptidase gene family (35). Of these proteins, only CPD and carboxypeptidase Z demonstrate activity toward standard CPE substrates (27,(32)(33)(34). The cellular distribution of carboxypeptidase Z is restricted to specific cell types, such as the leptomeningeal cells in brain (36).…”

“…However, low levels of mature peptides are detected, indicating that another enzyme is able to partially compensate for the absence of CPE activity. A search for novel CPE-like enzymes led to the identification of CPD, carboxypeptidase Z, and proteins designated CPX-1 and CPX-2 (27,(32)(33)(34). In addition, another group identified a protein designated AEBP1 as a novel member of the carboxypeptidase gene family (35).…”

“…CPD was recently discovered in a search for CPE-like enzymes that could contribute to peptide processing in the Cpe fat / Cpe fat mouse (27). These mice lack active CPE due to a point mutation that converts a Ser into a Pro (28).…”

Enzymic characterization of a novel member of the regulatory B-like carboxypeptidase with transcriptional repression function: stimulation of enzymic activity by its target DNA

“…The CP activators NiCl # , CoCl # and ZnCl # (CP-N [22], CP-E and CP-D [23], and CP-M [11]) were all found to enhance AEBP1 CP activity. At an activator concentration of 1 mM, a 1.4-fold activation with NiCl # , a 1.6-fold activation with CoCl # and a 1.9-fold activation with ZnCl # was observed after a 20-min assay period (results not shown).…”

“…AEBP1 had a greater affinity for ZnCl # (K a l 0.29 mM) than the other metals, CoCl # (K a l 0.55 mM) and NiCl # (K a l 0.71 mM). The general CP inhibitor and zinc chelator, o-phenanthroline (CP-M [24], CP-N [25], CP-E and CP-D [23], and CP-M [11]), and the CP-specific competitive inhibitor, captopril (CP-N [25]), inhibited AEBP1 CP activity. Captopril (1 µM) reduced AEBP1 CP activity to 54 % after a 20-min assay period, and a concentration of 10 µM completely abolished enzyme activity (results not shown).…”