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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS020185-06
Application #
3400414
Study Section
Endocrinology Study Section (END)
Project Start
1983-12-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1991-03-31
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Wyoming
Department
Type
Earth Sciences/Resources
DUNS #
069690956
City
Laramie
State
WY
Country
United States
Zip Code
82071
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Kraemer, W; Noble, B; Robertson, K et al. (1985) Response of plasma proenkephalin peptide F to exercise. Peptides 6 Suppl 2:167-9
Brandau, D T; Ray, P; Stern, A S et al. (1985) New bovine adrenal medullary peptide and its precursor. Int J Pept Protein Res 25:238-41
Kraemer, W J; Noble, B; Culver, B et al. (1985) Changes in plasma proenkephalin peptide F and catecholamine levels during graded exercise in men. Proc Natl Acad Sci U S A 82:6349-51
Micanovic, R; Ray, P; Kruggel, W et al. (1985) Purification and sequence of a novel ovine adrenal medullary peptide and its precursor. Neuroendocrinology 41:197-200
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