REGULATION OF ENKEPHALIN CONVERTASE--CARBOXYPEPTIDASE E

Peptide neurotransmitters and hormones regulate many physiological processes. In order to understand the mechanisms governing the production of these neuropeptides, it is necessary to study the enzymes involved in neuropeptide biosynthesis. Most neuropeptides are initially synthesised as precursor proteins that are subsequently cleaved at specific amino acid residues to produce the bioactive peptides. Many of the proteolytic cleavage sites are pairs of basic amino acids, and the sequential actions of trypsin-like and carboxypeptidase B-like enzymes would produce the neuropeptide. 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 tissue distribution of carboxypeptidase E (CPE) suggests that this enzyme is involved in the production of many diverse neuropeptides. CPE has been previously characterized and purified to homogeneity from bovine brain, pituitary, and adrenal (Fricker and Synder, 1983). Recently, cDNA clones encoding CPE have been isolated and sequenced from a bovine pituitary cDNA library (Fricker, et al, 1986). The predicted amino acid sequence suggests that CPE is initially synthesized as a precursor protein, which must be enzymatically processed to produce the active form of CPE. Furthermore, there are several pairs of basic amino acids within CPE, which are similar to the cleavage sites within neuropeptide precursors. Cleavage at these pairs of basic amino acids could give rise to different forms of the protein. The objectives of this study will be to examine the control of CPE activity at several levels. Antibodies to CPE will be used to quantitate levels of CPE, the CPE precursor (proCPE), and any other forms of the protein. Enzymes that process CPE, such as the enzyme that converts proCPE to CPE, will be isolated and characterized. Treatments that have been reported to alter neuropeptide biosynthesis will be examined for an effect on the levels of CPE mRNA, enzymatic activity, and different protein forms in both cell culture and in vivo systems. Finally, the structure of the rat CPE gene will be determined, and the 5' region will be sequenced. This, together with the regulation studies, will provide a background for future studies investigating the regulatory and/or promoter elements of the CPE gene.