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Carboxypeptidase E (CPE; EC 3.4.17.10) is a carboxypeptidase B-like enzyme involved with the biosynthesis of numerous peptide hormones and neurotransmitters, including the enkephalins. Reflex splanchnic stimulation of the rat adrenal medulla, which has previously been found to substantially increase enkephalin mRNA and enkephalin peptide levels, was examined for an influence on CPE mRNA and enzymatic activity. Several hours after insulin-induced reflex splanchnic stimulation, the levels of CPE activity in rat adrenal medulla are reduced to 40-60% of control. CPE activity returns to the control level 2 days after the treatment and then continues to increase, reaching approximately 200% of control 1 week after the treatment. The time course of the changes in CPE activity is different from those of the changes in epinephrine levels and the previously reported changes in enkephalin peptide levels. CPE mRNA is also influenced by the insulin shock, with levels increasing to 155% of the control level after 6 h and 170% after 2 days. The time course of the change in CPE mRNA levels is similar to that previously found for proenkephalin mRNA. However, the magnitude of the change is much different: Proenkephalin mRNA has been reported to increase by 1,600%. The changes in CPE mRNA and enzymatic activity are consistent with the proposal that CPE is not a rate-limiting enzyme in the biosynthesis of enkephalin.

Publisher Summary This chapter examines the activity, specificity and structural chemistry of metallocarboxypeptidase D. It is found that when the cDNA encoding gpl80 was cloned and sequenced, it became apparent that this protein represented a 180 kDa member of the metallocarboxypeptidase family. Carboxypeptidase D (CPD) removes C-terminal +Lys or + Arg residues from a variety of substrates, with no detectable activity towards nonbasic residues. It prefers an Ala in the penultimate position. In comparing N-terminally blocked small peptides, CPD shows lower activity with dipeptides than with tripeptides. The optimal pH for CPD activity is 5–7. CPD is stimulated by millimolar concentrations of Co2+ and inhibited by 1,10-phenanthroline. The enzyme is partially inhibited by some thiol-directed reagents, such as 7-chloromercuriphenylsulfonate or HgCl2. CPD is a single-chain protein that exists in several forms, with the major form being 180 kDa. It is likely that CPD contains 2–3 mol of zinc per mol of protein based on the homology between CPD and other metallocarboxypeptidases. CPD contains three carboxypeptidase domains of about 390 amino acids, which are followed by a transmembrane domain and a short 60 residue sequence that forms the cytosolic tail.

Publisher Summary This chapter describes the activity, specificity and structural chemistry of carboxypeptidase Z (CPZ). CPZ is a single-chain protein of approximately 71 kDa. Different splice variants have been detected, with the major form of human CPZ 641 amino acids in length. The crystal structure of CPZ has not been determined, but predictions of the structure have been made based on amino acid sequence similarities to other proteins. As with all members of the metallocarboxypeptidase family, CPZ contains a 300 residue domain that has approximately 20%–50% amino acid sequence identity with other family members. Following this, carboxypeptidase domain is an 80 residue region that has 40–50% amino acid sequence identity with the corresponding region of metallocarboxypeptidase D; this region of metallocarboxypeptidase D has been found to fold into a β sheet-rich structure that has homology with transthyretin. Based on the amino acid sequence similarity between CPZ and this region of metallocarboxypeptidase D, it is likely that the corresponding domain on CPZ also folds into a transthyretin-like domain.Upstream of the carboxypeptidase domain is a 120 residue Cys-rich region that has approximately 20%–30% amino acid sequence identity with a Cys-rich domain present in a number of proteins that bind Wnts.

Cultured astrocytes have been shown to secrete various neuropeptides and the neuropeptide processing enzyme, carboxypeptidase E (CPE). The secretion of CPE enzymatic activity from astrocytes has been shown previously to be increased approximately twofold by treatment with tetradecanoylphorbol 13-acetate (TPA), a phorbol ester. In this study, metabolic labeling with [35S]Met was utilized to examine the effect of TPA on the biosynthesis of CPE protein in cultured astrocytes and in AtT-20 cells, a pituitary-derived cell line. Treatment of astrocytes with 0.1 micrograms/ml TPA for 24 h caused an 80% increase in the level of radiolabeled CPE in both the media and the cells, indicating that the synthesis of CPE was stimulated by the TPA. AtT-20 cells also secreted more radiolabeled CPE in response to TPA, but this increase was offset by a proportional decrease in the cellular level of radiolabeled CPE, and synthesis of CPE was not stimulated in this cell line. Northern blot analysis demonstrated that 0.1 micrograms/ml TPA elevated CPE mRNA by approximately 50% in cultured astrocytes but not in AtT-20 cells. Quantitative in situ hybridization studies demonstrated that the TPA-induced increase in CPE mRNA expression was largely due to increases in the number of cells expressing CPE mRNA, although for astrocytes from some brain regions the average level of CPE mRNA per cell was also elevated by TPA. These results suggest that astrocytes can be induced to express CPE, which is consistent with a role for astrocytes in intercellular signaling.

Carboxypeptidase E (EC 3.4.17.10) is a carboxypeptidase B-like enzyme associated with the biosynthesis of many peptide hormones and neurotransmitters. Media collected from cultured astrocytes contain a carboxypeptidase E-like activity. Cultured astrocytes secrete approximately 73% of their cellular level of carboxypeptidase E per hour, and secretion is not substantially influenced by 35 mM KCl. In contrast, neurons secrete only 29% of their cellular carboxypeptidase E per hour, but secretion increases to 86% on stimulation with 35 mM KCl. Secretion of carboxypeptidase E activity from both neuronal and astrocyte cultures is relatively selective; neither acid phosphatase or acetylglucosaminidase is secreted in appreciable amounts. Cultured neurons and astrocytes express a carboxypeptidase E mRNA of a similar size. The levels of this mRNA differ in astrocytes cultured from different brain regions, with high levels in striatal, cortical, hippocampal, and hypothalamic astrocytes and low levels in cerebellar astrocytes. The level of carboxypeptidase E mRNA in hypothalamic astrocyte cultures is four- to fivefold higher than the level in hypothalamic neuronal cultures. These results indicate that cultured astrocytes express carboxypeptidase E mRNA and enzymatic activity and thus contain one of the enzymes required in the biosynthesis of many peptide hormones and neurotransmitters.

Carboxypeptidase E (CPE) is the carboxypeptidase B-like enzyme associated with the biosynthesis of numerous peptide hormones and neurotransmitters. This enzyme has been previously purified to homogeneity from bovine tissues, and cDNA clones (non-full length) isolated from a bovine pituitary cDNA library. In the present study, cDNA encoding full-length rat CPE has been isolated and sequenced. Both the nucleotide and amino acid sequences of rat CPE show substantial homology with the bovine sequences. The bovine and rat nucleotide sequences are homologous within the entire coding region, as well as within several portions of the 3'-untranslated region. The predicted amino acid sequence of rat CPE is greater than 90% homologous with the bovine enzyme. Northern blot analyses indicate a single species of CPE mRNA approximately 2100 nucleotides in length to be present in many neural and endocrine tissues. High levels of CPE mRNA are present in rat hypothalamus, hippocampus, midbrain, striatum, and cerebral cortex; and moderate levels are present in the brain stem, cerebellum, heart, adrenal, and eye. Low levels are detected in testis and duodenum, but not in liver or thymus. This tissue-specific expression of CPE mRNA is consistent with the proposed role for this enzyme in the production of numerous peptide hormones and neurotransmitters.