EVIDENCE FOR A 2-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 gonadotro pin releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly -NH2) by cleavage at the Tyr(5)Gly(6) bond. We compared the ability of crude and partially purified endopeptidase 24.15 from ovine hypothala mus with recombinant rat testicular endopeptidase 24.15 to degrade syn thetic GnRH. Both soluble and membrane hypothalamic fractions degraded GnRH to GnRH(1-5) with some production of GnRH(1-9) and GnRH(1-3). Ge neration of the smaller fragments was blocked by a specific endopeptid ase 24.15 inhibitor (CFP-AAY-pAB), but production of GnRH(1-9) was rec iprocally enhanced, suggesting this peptide may be an intermediate gen erated by prolyl endopeptidase. In deed, both bacitracin and Z-Pro-pro linal, inhibitors of this enzyme, markedly reduced GnRH degradation to any product. Degradation of synthetic GnRH(1-9) was more rapid than t hat of GnRH and was inhibited by CFP-AAY-pAB but not bacitracin. Activ ity against either substrate was greater in the soluble fraction. Repe ated washing of the membrane fraction followed by extraction with Trit on X-114 suggested that both endopeptidase 24.15 and prolyl endopeptid ase, although predomi nantly soluble, can be peripherally associated w ith membranes, When fractionated by hydrophobic interaction chromatogr aphy, soluble endopeptidase 24.15 degraded GnRH only in fractions that also exhibited prolyl endopeptidase activity. In contrast, maximal de gradation of GnRH(1-9) was observed in adjacent fractions, which also contained the highest levels of immunoreactive endopeptidase 24.15. Th e affinity of recombinant endopeptidase 24.15 for GnRH was low (K-m = 1.35 mM), was improved 10-15-fold by removal of the COOH-terminal amid e or glycinamide (K-m = 90 and 119 mu M, respectively), and could be i nhibited by CFP-AAY-pAB but not bacitracin. Taken together, these resu lts suggest that GnRH metabolism in the hypothalamus may occur via a t wo step process involving first removal of Gly(10)-NH2, by prolyl endo peptidase, followed by cleavage by endopeptidase 24.15 at the Tyr(5)-G ly(6) bond.