RESUMO
Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less sensitive to organophosphate inhibition than AChE from terrestrial animals, although this sensitivity is variable among species and has not yet been fully evaluated in fish species. In this setting, inhibition kinetic constants for progressive irreversible inhibition of brain acetylcholinesterase due to methyl-paraoxon exposure were determined in three fish species (Mugil liza, Genidens genidens and Lagocephalus laevigatus) and hen (Gallus domesticus). Enzyme extraction using a detergent was shown to be adequate, and samples presented activity inhibition in high substrate concentrations and suppression of inhibition by methyl-paraoxon in the presence of the substrate, similar to kinetic patterns from purified enzyme preparations. Catfish (G. genidens) AChE presented the highest sensitivity among the evaluated fish species (IC50 = 1031.20 nM ± 63.17) in comparison to M. liza and L. laevigatus (IC50: 2878.83 ± 421.94 and 2842.5 ± 144.63 nM respectively). The lower dissociation constant (Kd = 20.3 ± 2.95 µM) of catfish AChE showed greater enzyme affinity for methyl-paraoxon, explaining this species higher sensitivity to organophosphates. Hen AChE presented higher ki (900.57 ± 65.3 mM-1min-1) and, consequently, greater sensitivity to methyl-paraoxon, explained by a lower Kd (0.6 ± 0.13 µM). Furthermore, hen AChE did not differentiate between the propionylthiocholine and acetylthiocholine substrates, indicating easier access of methyl-paraoxon to the hen enzyme activity site. The results obtained herein indicate a suitable extraction of AChE and, despite different inhibition kinetic constants, demonstrate that fish AChE is less sensitive to methyl-paraoxon, probably due to reduced access to the catalytic center which provides greater enzyme substrate selectivity.
RESUMO
We show here that serum of piaussu, a Neotropical characin fish, has the highest butyrylcholinesterase activity so far described for humans and fish. To clarify whether this cholinesterase could protect piaussu against anticholinesterase pesticides by scavenging organophosphates, we purified it 1700-fold, with a yield of 80%. Augmenting concentrations (from 0.01 to 20 mM) of butyrylthiocholine activated it. The pure enzyme was highly inhibited by chlorpyriphos-oxon (ki=10,434x10(6) M-1 min-1) and by the specific butyrylcholinesterase inhibitor, isoOMPA (ki=45.7x10(6) M-1 min-1). Electrophoresis of total serum and 2-D electrophoresis of the purified cholinesterase showed that some enzyme molecules could circulate in piaussu serum as heterogeneously glycosylated dimers. The enzyme's N-terminal sequence was similar to sequences found for butyrylcholinesterase from sera of other vertebrates. Altogether, our data present a novel butyrylcholinesterase with the potential of protecting a fish from poisoning by organophosphates.
Assuntos
Butirilcolinesterase/sangue , Peixes/sangue , Sequência de Aminoácidos , Animais , Butirilcolinesterase/isolamento & purificação , Butirilcolinesterase/metabolismo , Butiriltiocolina/metabolismo , Clorpirifos/análogos & derivados , Clorpirifos/farmacologia , Inibidores da Colinesterase/farmacologia , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Dados de Sequência Molecular , Intoxicação por Organofosfatos , Paraoxon/análogos & derivados , Paraoxon/farmacologia , Intoxicação/prevenção & controle , Alinhamento de Sequência , Tetraisopropilpirofosfamida/farmacologiaRESUMO
O antibiotico excretado pelo Bacillus licheniformis IOC 2390 (ATCC 33632), foi analisado, em seguida a sua extracao do meio de producao con n-butanol. Concentrados aquosos finais (Fracao III), submetidos a filtracao por colunas de Sephadex G-25, mostratam tres componentes com identidades de peso molecular (i.e., mesmo volume de eluicao com picos em A252nm e atividade antibiotica) e de perfil cromatografico com o grupo das bacitracinas