Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae.
Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae.
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DOI：
10.1016/j.ibmb.2009.07.002
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发表时间：
2009-09
影响因子：
3.8
通讯作者：
Pope, Carey
中科院分区：
文献类型：
--
作者：
Jiang, Haobo;Liu, Siwei;Zhao, Picheng;Pope, Carey
来自易感和抗性昆虫的乙酰胆碱酯酶 (AChE) 及其基因已被广泛研究，以了解靶位点不敏感性的分子基础。然而，由于其他耐药机制的存在，将突变与耐药表型直接关联起来可能会存在问题。另一种方法涉及高度纯化的野生型和突变型 AChE 的重组表达和表征，这是研究结构功能关系的可靠平台。我们使用杆状病毒系统表达冈比亚按蚊 AChE1 (r-AgAChE1) 的催化结构域，并将其从条件培养基中纯化 26,000 倍至接近均质。虽然 r-AgAChE1 的 KM 对于 ATC、AβMTC、PTC 和 BTC 相当，但 Vmax 却有很大不同。 eserine、西维因、对氧磷、BW284C51、马拉氧磷和乙丙嗪的 IC50 值分别为 8.3、72.5、83.6、199、328 和 6.59×104 nM。我们确定了其中四种化合物抑制 r-AgAChE1 的动力学常数。该酶与丝氨酸或对氧磷的结合强于西维因或马拉氧磷。由于 eserine 对 r-AgAChE1 的共价修饰比其他化合物发生得更快，因此 eserine 比对氧磷、西维因和马拉氧磷更有效。此外，我们发现胆碱抑制r-AgAChE1，这种现象与高浓度乙酰胆碱下酶活性降低有关。
Acetylcholinesterases (AChEs) and their genes from susceptible and resistant insects have been extensively studied to understand the molecular basis of target site insensitivity. Due to the existence of other resistance mechanisms, however, it can be problematic to correlate directly a mutation with the resistant phenotype. An alternative approach involves recombinant expression and characterization of highly purified wild-type and mutant AChEs, which serves as a reliable platform for studying structure-function relationships. We expressed the catalytic domain of Anopheles gambiae AChE1 (r-AgAChE1) using the baculovirus system and purified it 26,000-fold from the conditioned medium to near homogeneity. While KM's of r-AgAChE1 were comparable for ATC, AβMTC, PTC, and BTC, Vmax's were substantially different. The IC50's for eserine, carbaryl, paraoxon, BW284C51, malaoxon, and ethopropazine were 8.3, 72.5, 83.6, 199, 328, and 6.59×104 nM, respectively. We determined kinetic constants for inhibition of r-AgAChE1 by four of these compounds. The enzyme bound eserine or paraoxon stronger than carbaryl or malaoxon. Because the covalent modification of r-AgAChE1 by eserine occurred faster than that by the other compounds, eserine is more potent than paraoxon, carbaryl, and malaoxon. Furthermore, we found that choline inhibited r-AgAChE1, a phenomenon related to the enzyme activity decrease at high concentrations of acetylcholine.
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DOI：
10.1016/j.cbpb.2008.03.008
发表时间：
2008-07-01
影响因子：
2.2
作者：
Alout, Haoues;Djogbenou, Luc;Weill, Mylene
通讯作者：
Weill, Mylene
影响因子：
4.1
作者：
GRAY, PJ;DUGGLEBY, RG
通讯作者：
DUGGLEBY, RG
影响因子：
5.1
作者：
Oakeshott, JG;Devonshire, AL;Russell, RJ
通讯作者：
Russell, RJ
影响因子：
2.9
作者：
HART, GJ;OBRIEN, RD
通讯作者：
OBRIEN, RD
影响因子：
8
作者：
Harel, M;Kryger, G;Sussman, JL
通讯作者：
Sussman, JL