Caracterização da atividade da enzima acetilcolinesterase de veneno de bungarus sindanus: estudos comparativos

Abstract

Acetylcholinesterase (AChE) belongs to a distinct family of serine hydrolases and is found in both synaptic and non-synaptic locations. At the synapses, it plays a major role in the hydrolysis of the neurotransmitter acetylcholine (ACh) while in non-synaptic tissue its function is unclear. Snake venom, particularly from the Elapidae family, is a common non-synaptic source of AChE. The venom of krait (Bungarus sindanus), an Elapidae snake, contained a high level of AChE activity. As there is no literature study about Bungarus sindanus venom AChE, it was selected as a main source of AChE activity. We characterized the venom AChE following comparative inhibitory studies with human, horse and rat cholinesterase using different ligands (tacrine, malathion, carbofuran, paraquat, antidepressants, TEMED). The enzyme of krait venom showed optimum activity at alkaline pH 8.5 with an optimal temperature of 45oC. We observed a significant reduction in substrate inhibition of krait venom AChE by using a high ionic strength buffer. With a low ionic strength buffer (10 mM PO4 pH 7.5) the enzyme was inhibited by 1.5 mM AcSCh, while with a high ionic strength buffer (62 mM PO4 pH 7.5) the enzyme was inhibited by 1mM AcSCh. Furthermore, we found that krait 5 venom acetylcholinesterase is thermally stable at 45oC. The enzyme lost only 5% of its activity after incubation at 45oC for 40 min. The Michaelis-Menten constant (Km) for the hydrolysis of acetylthiocholine iodide was found to be 0.052 mM. We noted that snake venom AChE was also inhibited by ZnCl2, CdCl2 and HgCl2 in a concentration dependent manner. In addition, this enzyme showed high sensitivity to tacrine, which is known to inhibit synaptic AChE. We observed that tacrine caused a mixed type of inhibition in krait venom as well as in human serum BChE. Snake venom AChE presents similar inhibitory behavior toward commonly used pesticides and herbicides as that of human serum BChE. The snake venom AChE exhibited a mixed type of inhibition for the pesticides malathion and carbofuran and the herbicide paraquat while human serum BChE presented a mixed inhibition for malathion and paraquat and an uncompetitive inhibition for carbofuran. The krait venom AChE was also affected by antidepressants such as paroxetine, imipramine, clomipramine and sertraline. Paroxetine and sertraline caused a mixed type of inhibition, while imipramine and clomipramine exhibited a competitive inhibition. Moreover, the well-known chemical N,N,N´,N´-tetramethylethylene diamine (TEMED) caused a mixed type of inhibition in snake (Bungarus) venom as well as in horse serum BChE. Futhermore, the inhibition of TEMED, was also confirmed from in vivo study in different structures of the brain, such as striatum, hippocampus, cortex, hypothalamus and cerebellum. Decrease in AChE activity was observed in all treated groups. The results suggest that TEMED exhibits toxic effect via inhibition of cholinesterase. Taken together the krait venom AChE showed similar behavior towards different ligands (tacrine, malathion, carbofuran, paraquat, antidepressants, TEMED) like other sources of cholinestease. Furthermore, krait venom contains large amount of acetylcholinesterase having highest catalytic activity and comparatively more stable than any other sources, making it more valuable for biochemical analysis.