Purification, Characterization, Molecular Cloning And Structures Of Cholecystokinin And Bradykinin From Skin Of Rana Nigrovittata

Amphibian is a biological treasure with great potentials. To be important natural resources, the ecological, academic, economic and cultural values of this kind of organism become more and more important. Varieties of bio-active substances in amphibian skins which play an important role in defense against invading factors have attracted the researchers all over the world. In this study, two bioactive proteins/ploypeptides were purified and identified by biochemical and molecular biological methods. By usuing a combination of gel filtration chromatography and reverse phase high performance liquid chromatography (RP-HPLC) steps, a new CCK ploypeptides were purified. Four completed precursors that encoding CCK cDNAs were cloned from Rana nigrovittata skin by molecular methods. Amino acid sequence for purified CCK was determined by using automated Edman degradation methods. The partial N-minal amino acid sequence of the purified CCK was RVDGNSDQKAVIGAMLAKDLQTRKAX. The amino acid fragment of the new purified CCK displayed significant similarity to that of CCKs from Rana catesbeiana and Xenopus laevis in the database by BLAST search. First discovery of CCK was purified and cloned cDNAs encoding CCKprecursors from R. nigrovittata skin.The predicted molecular weight (6502.26 Da) was about 80.8 Da lower than the observed molecular weight (6583.07) determinted by MALDI-TOF mass spectrometry analysis. The observed molecular weight of 6583.07 in this study may be resulted from the Tyr-SO₃ H modification of skin CCK of R. nigrovittata.A novel bradykinin-like peptide was also obtained from skin secretions of the Rana nigrovittata. The new purified bradykinin-like peptide was named as Ranakinin-N. Its primary structure (RAEAVPPGFTPFR) was also determined by Edman degradation and FAB-MS spectrometry. Sequence of Ranakinin-N is composed of 13 amino acid residues and it showed high similarity to the bradykinin purified from the skin secretions of Odorrana schmackeri, which is composed of 9 amino acid residues. Ranakinin-N was found to exert concentration -dependent contractile effects on isolated guinea pig ileum. The cDNA sequence encoding the precursor of ranakinin-N was cloned from a skin cDNA library of R. nigrovittata. The amino acid seqence deduced from the cDNA sequence match well with the result from Edman degradation. Analysis of different amphibian bradykinin cDNA structures revealed that a deficiency of an 15-nucleotide fragment (agaatgatcagacgc in the cDNA encoding bradykinin from O. schmackeri) in the peptide-coding region resulted in an absence of a di-basic site for trypsin-like proteinases and an unusual -AEAV-insertion in the N-terminal part of ranakinin-N. The-AEAV- insertion resulted in neutral net charge at the N-terminus of ranakinin-N. Ranakinin-N is the first report of bradykinin-like peptide with a neutral net charge at the N-terminus.