melitten and temporin

Peptidomic analysis of an extract of the skin of the stream brown frog Rana sakuraii Matsui and Matsui, 1990 led to the isolation of a C-terminally alpha-amidated peptide (VR-23; VIGSILGALASGLPTLISWIKNR x NH2) with broad-spectrum antimicrobial activity that shows structural similarity to the bee venom peptide, melittin together with two peptides belonging to the temporin family (temporin-1SKa; FLPVILPVIGKLLNGIL x NH2 and temporin-1SKb; FLPVILPVIGKLLSGIL x NH2), and peptides whose primary structures identified them as belonging to the brevinin-2 (2 peptides) and ranatuerin-2 (1 peptide) families. Using a forward primer that was designed from a conserved region of the 5'-untranslated regions of Rana temporaria preprotemporins in a 3'-RACE procedure, a cDNA clone encoding preprotemporin-1SKa was prepared from R. sakuraii skin total RNA. Further preprotemporin cDNAs encoding temporin-1SKc (AVDLAKIANIAN KVLSSL F x NH2) and temporin-1SKd (FLPMLAKLLSGFL x NH2) were obtained by RT-PCR. Unexpectedly, the 3'-RACE procedure using the same primer led to amplification of a cDNA encoding a preprobradykinin whose signal peptide region was identical to that of preprotemporin-1SKa except for the substitution Ser18-->Asn. R. sakuraii bradykinin ([Arg0,Leu1,Thr6,Trp8] BK) was 28-fold less potent than mammalian BK in effecting B2 receptor-mediated relaxation of mouse trachea and the des[Arg0] derivative was only a weak partial agonist. The evolutionary history of the Japanese brown frogs is incompletely understood but a comparison of the primary structures of the R. sakuraii dermal peptides with those of Tago's brown frog Rana tagoi provides evidence for a close phylogenetic relationship between these species.

Topics: 5' Untranslated Regions; Amino Acid Sequence; Amphibian Proteins; Animals; Antimicrobial Cationic Peptides; Bradykinin; Cloning, Molecular; DNA, Complementary; Female; Gene Expression; In Vitro Techniques; Male; Melitten; Mice; Molecular Sequence Data; Muscle Relaxation; Peptides; Proteins; Ranidae; Sequence Homology, Amino Acid; Skin

The activities of two antimicrobial peptides belonging to the temporin family (temporin-1DRa from Rana draytonii and temporin-1Va from Rana virgatipes) and two peptides with structural similarity to the bee venom peptide melittin (AR-23 from Rana tagoi and RV-23 from R. draytonii) were evaluated against a range of reference strains and clinical isolates of anaerobic bacteria. These peptides were selected because they show broad-spectrum growth inhibitory activity against reference strains of several medically important aerobic microorganisms and against clinical isolates of methicillin-resistant Staphylococcus aureus. All peptides showed relatively high potency (minimum inhibitory concentration (MIC)

Topics: Amino Acid Sequence; Amphibian Proteins; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacteria, Anaerobic; Melitten; Microbial Sensitivity Tests; Molecular Sequence Data; Proteins; Ranidae; Skin

Temporins are a novel family of small (10-13 residues) cationic antimicrobial peptides recently isolated from the skin of the European red frog Rana temporaria. Although recently acquired evidence shows that temporins have the potential to kill bacteria by permeabilizing the cytoplasmic membrane, the molecular mechanisms of membrane selectivity and permeabilization are largely unknown. In this study, it was found that temporins cause the release of fluorescent markers entrapped in phosphatidylcholine liposomes in a manner that depends significantly on the size of the solute. Temporins were also shown to lack a detergent-like effect on lipid vesicles, indicating that marker leakage caused by these peptides is not due to total membrane disruption but to perturbation of bilayer organization on a local scale. Binding of temporins to liposomes did lead to a small increase in lipid hydrocarbon chain mobility, as revealed by EPR spectroscopy of nitroxide-labeled fatty acids incorporated in the bilayer. Reference experiments were conducted using the bee venom peptide melittin, whose properties and behavior in natural and model membrane systems are well known. Our findings for temporins are discussed in relation to the models proposed to date to account for the action of antimicrobial peptides on membranes.

Topics: Antimicrobial Cationic Peptides; Cell Membrane Permeability; Dextrans; Electron Spin Resonance Spectroscopy; Fluoresceins; Lipid Bilayers; Liposomes; Melitten; Models, Chemical; Particle Size; Permeability; Phosphatidylcholines; Phospholipids; Proteins