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.

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An endopeptidase was isolated from Xenopus laevis skin secretions. This enzyme, which has an apparent molecular mass of 100 kDa, performs a selective cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bond (Xaa = Ser, Phe, Tyr, His, or Gly) of a number of peptide hormones, including atrial natriuretic factor, substance P, angiotensin H, bradykinin, somatostatin, neuromedins B and C, and litorin. The peptidase exhibited optimal activity at pH 7.5 and a Km in the micromolar range. No cleavage was produced in vasopressin, ocytocin, minigastrin I, and [Leu5Jenkephalin, which include in their sequence an Xaa-Phe, Xaa-Leu, or Xaa-Ile motif. The endopeptidase activity was inhibited by divalent cation chelators and by phosphoramidon only at high concentrations (ICss = 50 jAM), whereas it was insensitive to classical inhibitors of chymotrypsin, angiotensin convertase, and serine and cysteine peptidases, as well as carboxypeptidases. It is hypothesized that this enzyme, which is distinct from neutral endopeptidase (EC 3.4.24.11), constitutes the prototype of a family of related metalloendopeptidases that inactivate peptide substrates by cleavage at the Xaa-Phe, Xaa-Leu, or Xaa-fle bond.A rather limited number of peptidases seem to be involved in the postsecretory inactivation of peptide hormone messengers. The importance of these proteolytic mechanisms in regulating hormonal action can be demonstrated by the fact that their effects can be prolonged in vivo or in vitro by selective inhibitors of these enzymes. This has been shown in the case of the enkephalin-degrading enzyme neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) and in the case of angiotensin-converting enzyme (ACE; EC 3.4.15.1). Many of these previously identified peptidases cleave with a limited selectivity and sometimes at multiple sites.The skin secretions of Xenopus laevis contain an extraordinary number of peptide hormones eliciting a broad spectrum of biological actions (reviewed in ref. 1). In addition to those substances so far identified exclusively in the batrachian skin exudate such as caeruleins, magainins, levitide, PGLa, xenopsin, etc. (1,2), the secretory glands of these amphibians can also produce mammalian peptide hormones and some of their analogs, such as substance P, corticoliberin, thyroliberin, and angiotensin I. In the course of isolating the enzyme responsible for the activation of some X. laevis skin secretion hormone precursors by cleavage at the Arg-Gly bond of a consensus Arg-Xaa-Val-Arg-Gly (RX-VRG) sequence, we have detected a contaminating proteolytic activity (3). This endopeptidase copurifies with the RXVRG-endoprotease of X. laevis skin secretions and cleaves at the Ser-Phe bond situated on the carboxyl side of the RXVRG consensus sequence in a synthetic peptide (3).Indeed the Ser-Phe dipeptide, or a related motif such as Phe-Phe, Ala-Phe, or His-Phe, is often present near the carboxyl terminus of substances from the bombesin and tachykinin families (1). Xaa-Phe, Xaa-Leu, or Xaa-Ile was also found frequently at a ...

A novel metallo-endopeptidase from human neuroblastoma NB-OK-1 cells was partially purified and characterized. This enzyme activity was detected in the culture medium and could be detached from intact cells by gentle washing, suggesting a peripheral localization of the enzyme. This endopeptidase inactivated Atrial Natriuretic Peptide (ANP) by a unique and selective cleavage of the Ser123-Phe124 bond. It also produced hydrolysis at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bonds of other peptide hormones such as bradykinin, somatostatin 14, litorin, substance P, neuromedin C and angiotensin II. The substrate selectivity and inhibition profile of the enzyme showed obvious similarities with the peptide hormone inactivating endopeptidase (PHIE) recently purified from Xenopus laevis skin secretions and indicated a thermolysin-like activity distinct from neutral endopeptidase (EC 3.4.24.11) and from angiotensin converting enzyme (EC 3.4.15.1).