Evidence for a two-step mechanism of gonadotropin-releasing hormone metabolism by prolyl endopeptidase and metalloendopeptidase EC 3.4.24.15 in ovine hypothalamic extracts
The metalloendopeptidase EC 3.4.24.15 is believed to degrade gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) by cleavage at the Tyr5-Gly6 bond. We compared the ability of crude and partially purified endopeptidase 24.15 from ovine hypothalamus with recombinant ra...
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Published in | The Journal of biological chemistry Vol. 269; no. 17; pp. 12626 - 12632 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Bethesda, MD
American Society for Biochemistry and Molecular Biology
29.04.1994
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Subjects | |
Online Access | Get full text |
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Summary: | The metalloendopeptidase EC 3.4.24.15 is believed to degrade gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2)
by cleavage at the Tyr5-Gly6 bond. We compared the ability of crude and partially purified endopeptidase 24.15 from ovine
hypothalamus with recombinant rat testicular endopeptidase 24.15 to degrade synthetic GnRH. Both soluble and membrane hypothalamic
fractions degraded GnRH to GnRH1-5, with some production of GnRH1-9 and GnRH1-3. Generation of the smaller fragments was blocked
by a specific endopeptidase 24.15 inhibitor (CFP-AAY-pAB), but production of GnRH1-9 was reciprocally enhanced, suggesting
this peptide may be an intermediate generated by prolyl endopeptidase. Indeed, both bacitracin and Z-Pro-prolinal, inhibitors
of this enzyme, markedly reduced GnRH degradation to any product. Degradation of synthetic GnRH1-9 was more rapid than that
of GnRH and was inhibited by CFP-AAY-pAB but not bacitracin. Activity against either substrate was greater in the soluble
fraction. Repeated washing of the membrane fraction followed by extraction with Triton X-114 suggested that both endopeptidase
24.15 and prolyl endopeptidase, although predominantly soluble, can be peripherally associated with membranes. When fractionated
by hydrophobic interaction chromatography, soluble endopeptidase 24.15 degraded GnRH only in fractions that also exhibited
prolyl endopeptidase activity. In contrast, maximal degradation of GnRH1-9 was observed in adjacent fractions, which also
contained the highest levels of immunoreactive endopeptidase 24.15. The affinity of recombinant endopeptidase 24.15 for GnRH
was low (Km = 1.35 mM), was improved 10-15-fold by removal of the COOH-terminal amide or glycinamide (Km = 90 and 119 microM,
respectively), and could be inhibited by CFP-AAY-pAB but not bacitracin. Taken together, these results suggest that GnRH metabolism
in the hypothalamus may occur via a two-step process involving first removal of Gly10-NH2 by prolyl endopeptidase, followed
by cleavage by endopeptidase 24.15 at the Tyr5-Gly6 bond. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)99922-9 |