Purification, Characterization, Molecular Cloning, Structures And Functions Of Bio-Active Peptides From Skin Secretions Of Amolops Loloensis

Amphibian is a biological treasure with great potentials as well as important natural resources with ecological, research, economic and cultural value. A variety of bio-active substances from amphibian skins which play an important role in defense against invading factors have attracted significant attention of the researchers all over the world. So far, proteins and polypeptides of all kinds have been isolated and characterized.Skin cDNA library of Amolops loloensis was constructed. 1×10~6 independent clones were got in the skin cDNA library.Two groups of 3 antimicrobial peptides have been isolated from the skin secretion of Amolops loloensis. The peptides in the first group, composed of 24 amino acid residues and named amolopin-1a and amolopin-1b respectively, share 87% and 79% similarity to brevinin-1E from Rana esculenta. The other peptide in the other group, being 16 amino acid residues and designated as amolopin-2a, is homologous with temporin-1F from Rana temporaria.The. molecular weights of 3 amolopins were 2665.3, 2663.7 and 1626.0 Da.14 different cDNA clones encoding the precursor of amolopins were screened and sequenced from the skin cDNA library of A. loloensis and 8 different mature amolopins of 3 kinds were got due to synonymous mutation. 3 families of amolopins were designated series of amolopin-1, 2 and 3. The amino acid sequences deduced from the cDNA sequences contain antimicrobial peptides we got previously. This is the first report of antimicrobial peptides from Amolops amphibian.A novel bradykinin related peptide, composed of 12 amino acid residues and named amolopkinin (1341.6) was purified from skin secretion of Amolops loloensis via Sephadex G-50 filtration and RP-HPLC. The primary sequence exhibits 76% identity with ranakinin R from Rana rugosa and have an unusual -APV- insertion in the N-terminal part of amolopkinin. Full length of cDNAs encoding the isolated amolopkinin has been cloned. This is the first report of a bradykinin-like peptide that comprises of bradykinin with an insertion in its N-terminal part.