Identificar e caracterizar a enzima conversora de angiotensina no inseto T. castaneum

Abstract

Coleoptera, which are popularly known as beetles, constitute a reasonably diverse insect order with species occupying all the ecological niches. Most insects feed on plant tissues and for this reason, consist of important grain pests and derivatives of agricultural products in the act of storage. In this context, we have the Tribolium castaneum, which is an important pest of storage. Chymotrypsin-like serine proteases are a relevant class of proteolytic enzymes present in various biological systems, vertebrates, and invertebrates. In insects, this enzyme family is a crucial component in different metabolic pathways including digestion, defense, and development. Chymase and elastase - 2 are chymotrypsin-like belong to the class of serine proteases and are responsible for several pathophysiological processes in mammals, these enzymes convert Ang I to Ang II, thereby forming a blood pressure regulating peptide and electrolyte homeostasis in mammals. This study aims to identify and characterize an angiotensin-converting enzyme in T. castaneum. Fractionation of the protein extract in gel filtration (S - 100 sephacril) produced three fractions (fractions 13, 14 and 15) with angiotensin converting activity, converting Ang I to Ang II. The 10% SDS-PAGE gel revealed two semi-purified bands in fractions 13 and 15. Fraction 15 and the crude extract also demonstrated the ability to produce Ang (1 - 7) via Ang I, allegedly revealing the presence of endopeptidase as neprilysin and timete oligopeptidase in the physiology of the insect. In the inhibition assay in the crude extract the enzyme was not inhibited by either ACE-specific inhibitor captopril or by EDTA indicating that the enzyme is not a classical ACE. However, it demonstrated an inhibitory sensitivity to PMSF, characterizing the enzyme as a serine protease of the chemosis or elastase 2 type present in mammals, by the angiotensin-converting activity in vitro and by the inhibition profile.