Peptide neurotransmitters and hormones regulate many physiological processes, and may be involved with the biological mechanisms underlying drug addiction. In order to fully understand the regulation of the biosynthesis of this important group of intercellular messengers, it is necessary to study the enzymes responsible for neuropeptide production. Most neuropeptides are initially synthesized as precursor proteins that are subsequently cleaved at specific amino acid residues to produce the bioactive peptides. These studies will focus on the neuropeptide processing enzyme carboxypeptidase E (EC 3.4.17.10), which has been alternatively designated carboxypeptidase H and enkephalin convertase. This enzyme removes C-terminal basic amino acids from a wide range of neuropeptide precursors which is an essential step for the generation of many biologically active peptides. The rime distribution of carboxypeptidase E (CPE) suggests that this enzyme is involved in the production of many peptide hormones and neurotransmitters. CPE has been previously characterized and purified to homogeneity from bovine brain, pituitary, and adrenal (Fricker and Snyder, 1983). Several forms of CPE exist, including both soluble and membrane bound forms. cDNA clones encoding bovine and rat CPE have been isolated and sequenced (Fricker, et al, 1986 and 1989). The predicted amino acid sequence indicates that CPE is initially produced as a precursor ('proCPE'), which is posttranslationally processed into the soluble and membrane forms of CPE. The overall objective of these studies is to examine the mechanisms by which CPE is regulated. In cell culture systems, various treatments will be examined for an effect on the levels of CPE mRNA and enzymatic activity (both soluble and membrane-bound). Since membrane-bound CPE is less enzymatically active than the soluble form (Fricker, 1988), the processing of CPE is a potential mechanism for regulating CPE activity. The post-translational modification responsible for the difference between the soluble and membrane-bound forms of CPE will be determined, and the enzymes that proteolytically process proCPE into the various forms of CPE will be identified and characterized. Finally, the 5'-flanking region of the rat CPE gene will be sequenced, and the enhancer and/or promoter elements that confer the tissue-specific expression of CPE will be determined. The results of this multi-level analysis will provide a better understanding of the mechanisms responsible for the regulation of CPE expression and activity.
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