The complete amino acid sequence of rat testes metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.

Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.

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.

N-Arg dibasic convertase is a me tidase from rat brain cortex and testis that cleaves peptide substrates on the N terminu ofArg residues in dibasic s s.By using both an oinu ide and antibodies to screen a rat testis cDNA library, a fill-length cDNA (4,5). On the basis of its similarity to subtilisin and to furin (6), a human homolog of the Kex2 protein, a family of prohormone convertases (PCs) has been identified by PCR techniques. Their involvement in processing of a number of propeptides and proproteins was inferred mainly from cotransfection experiments (7-9), and for PC1, by the use of antisense mRNA (10).Characterization of putative processing endoproteases by classical biochemical techniques has led to the identification of a number of activities, selective for basic residues in precursors, that belong to the four classes of proteases (metallo-, serine, aspartyl, and thiol enzymes; for review, see ref. 11). This suggested that more than one processing endoprotease family could exist (12). To our knowledge, none of these basic-residue-specific enzymes has been cloned.Recently, a metalloendopeptidase was completely purified from rat testis and shown to cleave a number of peptide substrates on the N terminus of Arg residues in dibasic moieties (13). This enzyme was also present in rat brain cortex and its functional properties appeared undistinguishable from those of the somatostatin-28 convertase activity previously identified in this tissue (14,15). By using microsequencing of tryptic fragments of the purified enzyme to design an oligonucleotide probe and polyclonal antibodies raised against the purified protein (13) (20)].The in vitro highly restricted specificity ofNRD convertase for Arg residues in dibasic processing signals and its belonging to the M16 family, which contains other enzymes involved in maturation, suggest that this enzyme is the prototype of a distinct family of processing endoproteases. MATERIALS AND METHODSIsolatin and Cherizatin of cDNA Cones E d NRD Convertae. Four tryptic fragments were sequenced after digestion of previously purified NRD convertase following native PAGE. One fragment, GMQLIYLPPSPLLAE, was used to design the following degenerate inosine-containing oligonucleotide: 5'-GGIGGIAG(A/G)TAIATIA(G/A)(T/ C)TGCATICC-3'. Two additional peptides were obtained by endolysine C treatment (13) autoradiography of the filters, positive phage plaques were isolated and rescreened to obtain single purified phage isolates. A similar aliquot ofthe library was plated onto NZ-amine medium plates for screening using polyclonal antibodies raised Abbreviations: NRD convertase, N-arginine dibasic convertase; IDE, insulin degrading enzyme; MPP, mitochondrial matrixprocessing peptidase.