The biochemical basis for the addictive properties of opiate drugs may result from deficiencies in the biosynthetic capacity for opioid peptides; it is therefore extremely important to thoroughly understand the enzymatic mechanisms responsible for endogeneous opioid production. Active opioid peptides, like other neuropeptides and hormones, are formed through the action of intracellular processing enzymes which cleave and then modify precursor proteins. One of the most critical- and least understood- steps during this process is proteolytic cleavage at paired basic residues. Since within proenkephalin these paired basic cleavage sites lie adjacent to enkephalin tyrosine residues, and since free amino terminal tyrosines are required for opiate activity, this type of cleavage plays an especially important role in the liberation of bioactive enkephalins. The experiments described within this proposal represent a concerted effort to identify and biochemically characterize the proteolytic processing enzymes responsible for the cleavage of proenkephalin. We propose to provide a primary amino acid sequence for the adrenal trypsin-like processing enzyme which we have previously described as well as to characterize and purify other potential calcium-activated processing enzymes present in adrenal chromaffin granules. Standard biochemical purification procedures, including affinity chromatography on proenkephalin- agarose columns, will be used. We will also employ our new radiozymographical technique which yields molecular weight information on proenkephalin-cleaving proteinases. The initial cleavage sites of recombinant proenkephalin will be identified through Western blot and sequencing procedures. In addition, we will take advantage of the powerful technique of PCR (polymerase chain reaction) to explore the potential presence of unique serine proteinases within tissues capable of generating active opioid peptides. This work will provide insight into the enzymatic mechanisms controlling endogenous enkephalin production; this knowledge might ultimately lead to the development of enzyme-based drugs serving as therapeutic agents in drug addiction.
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