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How do gastrin‐releasing peptide (GRP) and endopeptidase 24.11 (EP) co‐exist in human seminal plasma?
Author(s) -
Anderson Cheryl D.,
Ryan James W.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1469-a
Subject(s) - endopeptidase , enzyme , substrate (aquarium) , biochemistry , chemistry , peptide , recombinant dna , biology , hydrolysis , microbiology and biotechnology , ecology , gene
Human seminal plasma (HSP) contains immunoreactive GRP (iGRP) and its reputed inactivating enzyme, EP; both in remarkable abundance. The iGRP is presumably secreted by neuroendocrine cells, and EP is disposed on prostasomes secreted by acinar epithelial cells. Both are present in HSP hours after semen liquification. To clarify how iGRP survives under conditions in which rapid activation might expected, we examined for the similarities of the iGRP with authentic GRP and examined the metabolic fate of 125I‐GRP on incubation in HSP. We also assayed iGRP concentrations and EP activities: the latter assay using glutaryl‐A‐A‐F‐[3H]anilide as substrate. These assays revealed an expected inverse correlation between iGRP and EP activity. Similarly, 125I‐GRP was metabolized to yield an 125I‐product of about 1,000 Da. Unexpectedly, the metabolism was slow, with an apparent half‐life of about 80 min. when EP concentration was remarkably high; near 0.5 micromolar. As a further unexpected finding, HSP iGRP was found by molecular sieving to exist in two forms, one significantly larger than GRP and a second about the size of neuromedin C (itself a likely substrate for EP). The immunoassay antibody recognizes a C‐terminal epitope in which the amide must be intact, thus the larger iGRP cannot be pre‐pro‐ or pro‐GRP unless amidated. Post‐translational modification of GRP is not ruled out and could confer resistance to hydrolysis by EP. EP has erroneously been called ‘neutral endopeptidase’. Its pH optimum is in fact near 5.7, and the slow hydrolysis of 125I‐GRP observed here might be attributable to the slightly alkaline pH of HSP. Nonetheless, both the large and small iGRPs are stable in HSP samples maintained at room temp. for several hours. Both iGRPs are present in quantities (~180 pM) sufficient for definitive analysis by mass spectrometry.