Isolamento e caracterização de uma enzima conversora de angiotensina da peçonha do Thalassophryne nattereri

Abstract

The animal poison are complex mixtures that comprise peptides, proteins, enzymes and other elements produced by animals for predation, digestion and defense. These molecules were investigated for their molecular mechanisms associated with physiological action and possible pharmacological applications. Recently, we describe the presence of angiotensin converting activity (ACE) in fish poison Thalassophryne nattereri. ACE is a zinc dependent dipeptidyl carboxypeptidase with a wide range of effects. By removing the His-Leu dipeptide from the C-terminal, an ACE converts angiotensin I (AngI) to angiotensin II (AngII) and inactivates bradykinin by cleaving the penultimate Pro-Phe binding, thereby forming peptides regulating blood pressure and homeostasis Electrolyte. The objective of this study was to isolate and characterize an angiotensin converting enzyme contained in the poison of T. nattereri and the vasoactive role of the enzyme in upper isolated rat artery was appreciated. The fractionation of the T. nattereri poison in 56 ml CM-Sepharose FF column, indicated a peak (CM2) with angiotensin converting activity, converting AngI to AngII. A polyacrylamide gel electrophoresis (12%) revealed a 30 kDa band for CM2 of size for natterins, which are toxins with proteolytic activity found in a T. nattereri poison. Mass spectrometry indicated that the ACE protein sequence purified from T. nattereri poison corresponds to natterin 1. The isolated protein also demonstrated inhibition by captopril and EDTA and was characterized by a classic ACE, by an activity in vitro and by a model of inhibition. Despite such evidence, the molecular weight does not resembles human ACE. With this data it is seen that this poison go against other venoms, which present ACE inhibitors, which reinforces the relevance of this study.Thus, an isolated enzyme purified from T. nattereri poison is a first isolated fish ECA. The angiotensin-converting enzyme isolated from T. nattereri venom was able to promote dose-dependent vasorelaxation in the superior mesenteric artery rings of rats with intact endothelium and without endothelium pre-contracted with phenylephrine, demonstrating an antagonistic effect to the venom. The identification of this protease producing a vasoactive peptide in the venom of T. nattereri, highlights its role in the pawning and improves our general understanding of the action process of marine animal venoms.