Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth-inhibitory activity against the gra m-positive bacterium Staphylococcus aureus, the gram negative bacterium Esc herichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary struct ures of these antimicrobial peptides were different from previously charact erized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previousl y characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranale xin families, first identified in the North American bullfrog, Rana catesbe iana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculent in-1, and esculentin-2 families, previously isolated from the skins of othe r species of Ranid frogs, were not identified in the extracts. The ranatuer in-1 and ranalexin peptides showed broadest spectrum of antimicrobial activ ity whereas the temporins were active only against S. aureus. Synthetic rep licates of temporin-1Gb (SILPTIVSFLSKFL.NH2) and temporin-1Gd (FILPLIASFLSK FL.NH2) produced concentration-dependent relaxation of preconstricted vascu lar rings from the rat thoracic aorta (EC50 = 2.4+/-0.1 mu M for temporin-1 Gb and 2.3+/-0.2 mu M for temporin-1Gd). The antimicrobial peptides that we re isolated in extracts of the skin R. grylio were present in the same mole cular forms in electrically-stimulated skin secretions of the animal demons trating that the peptides are stored in the granular glands of the skin in their fully processed forms. (C) 2000 Elsevier Science B.V. All rights rese rved.