Carboxilesterase na adaptação do metabolismo de lipídios do pacu à hipoxia

Abstract

Fish are vertebrates that dwell in many habitats. Physiological and biochemical adaptations of fish are studied to understand how these animals endure the challenges present in each environment. Decrease in dissolved oxygen concentrations in water is a natural cyclic phenomenon in the Pantanal and Amazon watersheds. The growing anthropic pollution of rivers in Pantanal and Amazon can, added to hypoxia, threaten the permanence of several fish species. Studying carboxylesterase (CarbE), an important enzyme for xenobiotics biotransformation, we found that its activity drops in pacu, a typical fish of Pantanal, kept in hypoxia for 42 hours. Carboxylesterases take part in several chemical reactions in the organism, including a transesterification capable of producing toxic fatty acid ethyl esters (FAEE) from ethanol and fatty acid esters. As the decrease of CarbE activity could be beneficial, since ethanol a product from glycolysis in fish under hypoxia would be less esterified, we decided to know more about the biochemistry of CarbE from plasma and liver of pacu. Pacus were submitted to hypoxia by decreasing the concentration of oxygen up to 0.5 mg/mL by bubbling nitrogen in the water. Once these animals reached hypoxia they were maintained at 0.5 mg/mL of oxygen for 42 hours. After this time, blood was withdrawn and their liver excised. The classical activity of CarbE dropped over 50% in serum and 25% in liver microsomes compared to that from fish under 6 mg O2 for 42 h. The CarbE was isolated from the serum of pacus in normoxia has a molecular relative mass of 56,000. The denaturing gel electrophoresis yielded three bands, but the native gel showed only two bands with activity upon α-naphthyl acetate. We inferred that more than one isoform of the enzyme exist in pacu plasma. The isolated CarbE from serum shows no lipase activity with Tween® 20, but liver microsomes from pacu in normoxia or hypoxia do. However, purified CarbE from serum as much as the activity present in liver microsomes can hydrolyze acyl-CoA. The enzyme purified from serum has a three-fold Vmax and a four-fold KM compared to the activity present in liver microsomes. Furthermore, liver microsomes from pacu under normoxia can hydrolyze acyl-CoA twice as faster as do the microsomes from animals in hypoxia. Activity of serum CarbE with p-nitrophenylacetate was inhibited by 4 mM 4-HNE. Hydrolysis of acyl CoA by liver microsomes was also inhibited by 4 mM 4-HME, while 2 mM 4-HNE inhibited half of this activity in serum.