PROENKEPHALIN IN PROCESSING IN THE ADRENAL MEDULLA

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The ultimate goal of this project is to understand the processing of proenkephalin and the regulation of that processing. Recently the enkephalin precursor in adrenal medulla has been identified and sequenced via DNA cloning. From this sequence and the sequences of enkephalin-containing polypeptides (ECP's) we have isolated, it is clear that the post-translational processing of proenkephalin requires a trypsin-cleavage like enzyme. Using chromaffin cells in culture we will use a pulse chase to determine the order of these cleavages. We have identified an enzyme(s) in adrenal extracts, which has a pH optimum consistent with the low pH within chromaffin granules and which has been shown to cleave at proenkephalin cleavage sites using peptide substrates. We propose to purify this enzyme and study its physiochemical properties, its relationship to other proteases, and whether it is present in other enkephalin-producing tissues such as gut and brain. This enzyme also appears to be regulated in some manner as all possible cleavage sites are not cleaved normally but rupture of the granules and dilution in buffer results in much greater cleavage. How this regulation occurs will be determined. In addition to the regulation of this enzyme we have evidence suggesting that epinephrine containing granules have a greater proportion of enkephalins and ECP's than do nonepinephrine granules. Whether this is due to regulation of the trypsin-like enzyme or regulation of proenkephalin synthesis will be determined using chromaffin cell primary cultures. The overall result will be an understanding of how the post-translational processing of proenkephalin occurs and how it is regulated to result in the normally produced ECP's in adrenal tissue and enkephalins in neural tissue.