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DNA sequence of butyrylcholinesterase from the rat: expression of the protein and characterization of the properties of rat butyrylcholinesterase.

机译:大鼠丁酰胆碱酯酶的DNA序列:蛋白质的表达和大鼠丁酰胆碱酯酶的特性表征。

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The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher K(m) for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min(-1)). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3hr. Human serum contains 5mg/L of BChE and 0.01mg/L of AChE. Male rat serum contains 0.2mg/L of BChE and approximately 0.2mg/L of AChE.
机译:大鼠是毒性研究的模型动物。丁酰胆碱酯酶(BChE)对某些有机磷和氨基甲酸酯类农药的抑制作用敏感,是中毒暴露的生物标志物。这项工作的目的是表征纯化的大鼠BChE酶。 cDNA序列显示出大鼠和人BChE的活性位点峡谷之间有八个氨基酸差异,六个聚集在酰基结合口袋周围,两个在活性位点丝氨酸下方。大鼠的一个显着差异是酰基袋中第286位的精氨酸取代了亮氨酸。野生型大鼠BChE,突变体R286L,野生型人BChE和突变体L286R在CHO细胞中表达并纯化。发现Arg286负责Triton X-100对大鼠BChE的抑制作用。用亮氨酸替代Arg286导致与Triton X-100的亲和力增加20倍,使其与人BChE一样对Triton X-100的抑制敏感。与野生型人BChE相比,野生型大鼠BChE的带正电荷的底物丁硫胆碱,乙酰硫胆碱,丙酰硫胆碱,苯甲酰胆碱和可卡因的K(m)高8至9倍。野生型大鼠BChE催化的营业额比人类BChE快2至7倍,显示丙酰硫代胆碱营业额最高(201,000 min(-1))。人BChE在被硫代硫酸盐抑制后不会自发活化,但大鼠BChE的半衰期为4.3小时。人血清中含有5mg / L的BChE和0.01mg / L的AChE。雄性大鼠血清含有0.2mg / L的BChE和约0.2mg / L的AChE。

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