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 ...

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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).

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