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D-amino acids are present in some peptides from amphibian skin. These residues are derived from the corresponding L-amino acids present in the respective precursors. From skin secretions of Bombinae, we have isolated an enzyme that catalyzes the isomerization of an L-Ile in position 2 of a model peptide to D-allo-Ile. In the course of this reaction, which proceeds without the addition of a cofactor, radioactivity from tritiated water is incorporated into the second position of the product. The amino acid sequence of this isomerase could be deduced from cloned cDNA and genomic DNA. After expression of this cDNA in oocytes of Xenopus laevis, isomerase activity could be detected. Polypeptides related to the frog skin enzyme are present in several vertebrate species, including humans.amphibia ͉ bombinin H ͉ isomerase ͉ chirality F rom amphibian skin, a wide variety of biologically active peptides have been isolated (1). One of these compounds, the heptapeptide dermorphin (present in the skin of the South American tree frog Phyllomedusa sauvagei), is a potent opiate that binds with high affinity and selectivity to -opiate receptors (2). Quite unexpectedly, dermorphin was found to contain D-Ala at position 2, and this residue is essential for binding to the receptor. Subsequently, several additional opiate peptides containing a D-amino acid were found in the skin of other Phyllomedusinae (3). The bombinins H, antibacterial and hemolytic peptides isolated from skin secretions of the frog Bombina variegata, contain either L-Ile or D-allo-Ile (D-aIle) as the second amino acid (4). Peptides with D-amino acids in their sequences were also found in different invertebrate species, such as snails (5-7), the venom of a spider (8), and crustaceans (9). The most recent addition to this group was a homologue of C-type natriuretic peptides isolated from the venom of the male platypus (10).In principle, the biosynthesis of these D-amino acids in animal peptides has been clarified. As shown for dermorphin (11) and subsequently for several cases of vertebrate and invertebrate peptides, the secreted products are derived from larger precursors encoded by mRNAs. At the positions where a D-amino acid is present in the end product, a codon for the corresponding L-isomer has been found (reviewed in refs. 12 and 13). This finding implies that, at some stage in the processing of the precursors, a conversion of certain L-amino acids to the D-form takes place. Indeed, in many cases, the L and D forms of the mature peptides coexist in the respective secretory glands and secretions, which can be interpreted as incomplete isomerization. An enzyme that catalyzes such an unusual reaction has been characterized from the venom of a funnel web spider (8). In this case, the chirality of a Ser close to the C terminus of a peptide termed -agatoxin IV is inverted. This ''isomerase'' is related to Ser proteases and functions without any added cofactors (14, 15).As mentioned above, skin secretions of Bombina species contain a group of hemolytic peptides tha...

From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascarissuum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with Ki values in the range of 0.1 to 1 pM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor. With few exceptions, these were rather small molecules containing less than 30 amino acids. As for their biological function, two main groups can be discerned. One comprises a multitude of peptides interacting with specific receptors present in the nervous system, the gastrointestinal tract and other organs of mammals. Many of these peptides are in fact similar or even identical to hormones and neurotransmitters. The second group encompasses antimicrobial peptides, at least some of which exert their function through direct binding to the phospholipid bilayer of cell membranes.Recently, a new family of peptides, the xenoxins, was isolated and characterized from skin secretions of Xenopus laevis (Kolbe et al., 1993). These 66-amino-acid peptides are related to neurotoxins and cytotoxins from snake venoms, but they are apparently devoid of toxic activity. In the course of our analysis of the skin secretion of Bombina bombina, a main fraction containing peptides with an apparent molecular mass of 8-10 kDa was isolated. In view of the earlier findings on xenoxins, we Reprint requests to: G. Kreil, Institute of Molecular Biology, Billrothstrasse 11, A-5020 Salzburg, Austria; e-mail: gkreil@oeaw.ac.at.wanted to test whether similar constituents were also present in skin secretion of this species. Here we present our analysis of one of the two main components of this fraction. As we have shown, this is a trypsin inhibitor related to the protease inhibitors from Ascaris (Grasberger et al., 1994; Huang et al., 1994).The peptide has been termed Bombina skin trypsin inhibitor, abbreviated BSTI. Results Isolation and amino acid sequence of BSTISkin secretions from B. bombina were collected and extracted with n-butanol. The aqueous phase was chromatographed over ConA-Sepharose to remove glycoproteins, proteoglycans, mucins etc. The eluate was further fractionated by molecular sieve chromatography (see the Materials and methods). One of the main peaks contained polypeptides with an apparent molecular weight of 8-10 kDa, as shown by SDS-PAGE. Separation of this fraction by reverse-phase HPLC yielded three peaks (see Fig. 1). Of these, the main constituent was investigated f...

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