Endopeptidase EC 3.4.24.15 Presence in the Rat Median Eminence and Hypophysial Portal Blood and its Modulation of the Luteinizing Hormone Surge

Abstract

The endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is widely distributed in a variety of tissues, including the testes, pituitary and the central nervous system. Among its numerous roles in metabolizing and processing biologically-active peptides, the enzyme degrades gonadotropin-releasing hormone (GnRH) by cleaving the central Tyr5-Gly6 bond. The aim of the present studies was to determine whether EP24.15 can modulate the concentrations of GnRH within the hypothalamo-hypophysial portal blood and thereby play a physiological role in reproduction. Our data suggest the presence of immunoreactive EP24.15 in the perivascular space of the median eminence and that this enzyme is secreted into portal blood. We have also shown a physiological role for this enzyme in that an inhibition of its activity with a specific inhibitor augmented the steroid-induced LH increase in ovariectomized rats. The present results suggest that secretory and post-secretory mechanisms are important in shaping the GnRH signal from the central nervous system; GnRH metabolism by EP24.15 may be one such mechanism.

The Neuropeptide Processing Enzyme EC 3.4.24.15 Is Modulated by Protein Kinase A Phosphorylation

“…GnRH [1][2][3][4][5][6][7][8][9] , Bradykinin, and Neurotensin EP24.15 activity was determined under discontinuous assay conditions by quantification of substrate product peaks via high performance liquid chromatography as described previously (22) with modifications. 5-40 ng of EP24.15 (phosphorylated and non-phosphorylated) was incubated at 37°C with varying concentrations of peptide substrate (11.2 M to 1 mM for GnRH, 10 -100 M for GnRH [1][2][3][4][5][6][7][8][9] and NT, and 2-100 M for bradykinin and bradykinin-amide).…”

“…5-40 ng of EP24.15 (phosphorylated and non-phosphorylated) was incubated at 37°C with varying concentrations of peptide substrate (11.2 M to 1 mM for GnRH, 10 -100 M for GnRH [1][2][3][4][5][6][7][8][9] and NT, and 2-100 M for bradykinin and bradykinin-amide). For NT assays, both cleavage products, NT 1-8 and NT 9 -13 , were used as standards.…”

“…Hydrolyzed amino acids were mixed with phosphoamino acid standards (Ser(P), Thr(P), and Tyr(P)) (ICN Biomedicals, Aurora, OH). Samples were spotted on 20 ϫ 20-cm 2 cellulose TLC plates (EM Science, Gibbstown, NJ), dried, and run at least 15 cm in a proprionic acid, 1 M NH 4 OH, isopropyl alcohol (45:17.5:17.5) solvent system (33). Amino acid standards were visualized using a ninhydrin spray (0.25% in acetone), and experimental phosphoamino acids visualized by autoradiography.…”

“…Inhibition of EP24.15 activity has been demonstrated in vivo in rat models resulting in an increased half-life of the hormone by decreased GnRH degradation and subsequent augmentation of the luteinizing hormone surge (2)(3)(4). Other important EP24.15 substrate targets include neurotensin, where inhibition of EP24.15 in mice prolonged forepaw licking latency (5), bradykinin (6), somatostatin [1][2][3][4][5][6][7][8][9][10][11][12][13][14] (7), and nociceptin (8). EP24.15 also processes Met-and Leu-enkephalin from the enkephalin-containing peptides (9), and the specific EP24.15 inhibition increased Metenkephalin antinociception in rodents (10).…”

Crystal Structure of Human Thimet Oligopeptidase Provides Insight into Substrate Recognition, Regulation, and Localization

“…It is widely distributed in mammalian tissues with the highest expression levels in the brain, pituitary gland, and testis (5)(6)(7)(8). TOP is present in different subcellular locations depending on cell type, with reports of secreted and cytosolic forms (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), membrane association (5-7, 17, 18), and nuclear localization (7,12,14). Consistent with its broad tissue and subcellular compartment distribution, TOP appears to play a variety of physiological roles.…”