Our goal is to characterize, compare, and study the regulation of the trypsin-like processing enzyme(s) that cleave proenkephalin and Beta-protachykinin. These studies are important because it is only the mature enkephalin and substance P peptides, and not their precursors, which function as neurotransmitters. Thus, the processing enzymes which convert the biologically inactive forms into the active peptides are crucial for the control of neuropeptide production. Furthermore, the different proenkephalin-derived products found in brain and peripheral tissues show that these enzymes are responsible for tissue specific processing of a peptide precursor. The trypsin-like processing enzyne(s) that cleave enkephalin and substance P precursors are poorly understood. Thus, the purpose of this grant is to characterize these proteases and investigate their regulation.
The specific aims are: (1) Identification of the trypsin-like endopeptidase(s) that cleave proenkephalin and protachykinin in enkephalin- and tachykinin-containing secretory vesicles of bovine adrenal medulla and posterior pituitary. Enzyme activity will be assayed by following the conversion of authentic (35S-methionine)-proenkephalin and (35S-methionine)-protachykinin into their correct peptide products. (2) Purification of the processing enzyme(s) identified in specific aim 1 by protein chromatographic methods. This will determine if one or several enzymes are responsible for cleaving each precursor, and will indicate if the two precursors are processed by similar or different enzyme(s). (3) Biochemical and immunochemical characterization of the purified enzyme(s). This will include determination of mol. wt., pH optimum, effect of inhibitors and activators, site of cleavage, and substrate specificity. Immunochemical studies will develop polyclonal and monoclonal antibodies against the enzyme(s). These studies will demonstrate if these enzyme(s) are unique or similar to other proteases, and will provide insight as to what factors may regulate their activity-(ies) in vivo. (4) Investigation of the regulation of trypsin-like processing enzyme activity during elevated enkephalin biosynthesis in cultured chromaffin cells. These studies are fundamental for understanding the role of processing enzymes involved in neurotransmitter production. Knowledge of regulatory mechanisms that modify processing enzyme activities will provide further understanding of molecular abnormalities underlying many neurological and mental disorders.
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