RESUMO
Multiple bradykinin-related peptides including a novel bradykinin structural variant, (Val(1))-bradykinin, have been identified from the defensive skin secretion of Guenther's frog, Hylarana guentheri by a tandem mass spectrometry method. Subsequently, four different preprobradykinin cDNAs, which encoded multiple bradykinin copies and its structural variants, were consistently cloned from a skin derived cDNA library. These preprobradykinin cDNAs showed little structural similarity with mammalian kininogens and the kininogens from the skin of toads, but have regions that are highly conserved in the kininogens from another ranid frog, Odorrana schmackeri. Alignment of these preprobradykinins revealed that preprobradykinin 1, 2 and 3 may derive from a single gene by alternative exon splicing.
Assuntos
Anuros/metabolismo , Bradicinina/isolamento & purificação , Peptídeos/isolamento & purificação , Pele/metabolismo , Sequência de Aminoácidos , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/isolamento & purificação , Proteínas de Anfíbios/farmacologia , Animais , Anuros/genética , Sequência de Bases , Bradicinina/genética , Bradicinina/farmacologia , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Técnicas In Vitro , Cininogênios/química , Masculino , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Peptídeos/genética , Peptídeos/farmacologia , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Ratos , Ratos Wistar , Análise de Sequência de DNA , Espectrometria de Massas em TandemRESUMO
The electrospray ionisation-ion-trap mass spectrometry (ESI-MS(n)) of selected drug compounds with amine-containing side chains has been investigated. Certain characteristic in-source fragmentations have been observed for these molecules. Sequential product ion fragmentation experiments (MS(n)) have been performed in order to elucidate the degradation pathways for the [M + H](+) ions and their predominant fragment ions. These MS(n) experiments also show certain characteristic fragmentations with respect to the amine-containing side chains. QTOF-MS/MS has been used to support the identity of the proposed fragments. The data presented in this paper therefore provides useful information on the structure of these compounds with amine-containing side chains and can be used in the characterisation of such drugs, their structurally related metabolites and unknown molecules of pharmaceutical significance extracted from animal and plant sources, for example. Amphetamine, clenbuterol, flurazepam and methadone can be identified and determined in mixtures at low ng/ml concentrations by the application of HPLC-ESI-MS which can also be used for their analysis in saliva samples.
Assuntos
Aminas/análise , Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Aminas/química , Cromatografia Líquida de Alta Pressão/métodos , Preparações Farmacêuticas/químicaRESUMO
We have isolated a novel bradykinin B(2)-receptor antagonist peptide, kinestatin, from toad (Bombina maxima) defensive skin secretion. Mass spectroscopy established a molecular mass of 931.56 Da and a provisional structure: pGlu-Leu/Ile-Pro-Gly-Leu/Ile-Gly-Pro-Leu/Ile-Arg.amide. The unmodified sequence, -QIPGLGPLRG-, was located at the C-terminus of a 116-amino-acid residue open-reading frame following interrogation of a sequenced B. maxima skin cDNA library database. This confirmed the presence of appropriate primary structural attributes for the observed post-translational modifications present on the mature peptide and established residue 2 as Ile and residues 5/8 as Leu. Kinestatin represents a prototype novel peptide from amphibian skin.
