Saboori A M, Newcombe D S
School of Hygiene and Public Health, Department of Environmental Health Sciences, Johns Hopkins University, Baltimore, Maryland 21205.
J Biol Chem. 1990 Nov 15;265(32):19792-9.
Human peripheral blood monocytes were isolated by density gradient centrifugation and purified by counterflow centrifugation elutriation. Membrane-localized carboxylesterase (CBE) was extracted with nonionic detergent (Triton X-100) and purified by ion exchange (DEAE-cellulose), gel filtration (Sephacryl S-300), hydroxylapatite column, and high performance liquid chromatography. The purified enzyme migrated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single protein band with a molecular weight of 60,000. Under nondenaturing conditions, monocyte CBE formed a trimer and eluted from a gel filtration column as a protein with an approximate molecular weight of 200,000. Electrophoretic patterns of the enzyme on polyacrylamide gels run a neutral pH did not vary during enzyme purification. At least four major isoenzymes of human monocyte CBE were observed with isoelectric points between 7.5 and 7.8. Pure human monocyte CBE hydrolyzed short chain alpha-naphthyl, o-nitrophenyl, and p-nitrophenyl esters. Amide esters and thioesters were not hydrolyzed by the enzyme. Short chain alcohols activated the enzyme and organophosphorus compounds, diphenyl carbonate, sodium fluoride, and phenylmethylsulfonyl fluoride inhibited the enzyme. EDTA and sulfhydryl reagents had no effect on enzyme activity. The amino acid content of the enzyme was consistent with other CBEs. Inhibitors reacted either with the active or effector site of the enzyme. Purified enzyme now permits the characterization of CBE structure and regulation.
人外周血单核细胞通过密度梯度离心分离，再经逆流离心淘析法纯化。膜定位羧酸酯酶（CBE）用非离子去污剂（Triton X - 100）提取，然后通过离子交换（DEAE - 纤维素）、凝胶过滤（Sephacryl S - 300）、羟基磷灰石柱和高效液相色谱法进行纯化。纯化后的酶在12%十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上迁移时呈现为一条分子量为60,000的单一蛋白带。在非变性条件下，单核细胞CBE形成三聚体，并从凝胶过滤柱上以近似分子量为200,000的蛋白质形式洗脱下来。在中性pH条件下进行聚丙烯酰胺凝胶电泳时，该酶的电泳图谱在酶纯化过程中没有变化。观察到至少四种人单核细胞CBE的主要同工酶，其等电点在7.5至7.8之间。纯的人单核细胞CBE能水解短链α - 萘基酯、邻硝基苯基酯和对硝基苯基酯。酰胺酯和硫酯不能被该酶水解。短链醇能激活该酶，而有机磷化合物、碳酸二苯酯、氟化钠和苯甲基磺酰氟能抑制该酶。EDTA和巯基试剂对酶活性没有影响。该酶的氨基酸含量与其他CBE一致。抑制剂与酶的活性位点或效应位点发生反应。纯化后的酶现在可以用于表征CBE的结构和调节。
