Development of a scintillation proximity assay for β-ketoacyl-acyl carrier protein synthase III
Development of a scintillation proximity assay for β-ketoacyl-acyl carrier protein synthase III
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DOI：
10.1006/abio.2000.4594
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发表时间：
2000-06-15
影响因子：
2.9
通讯作者：
Reynolds, KA
中科院分区：
文献类型：
--
作者：
He, X;Mueller, JP;Reynolds, KA
β-酮脂酰-酰基载体蛋白酶III（KASIII; FabH）是启动细菌II型脂肪酸生物合成的关键酶，其测定通常涉及放射性标记的乙酰辅酶A和丙二酰酰基载体蛋白（MACP）的孵育。放射性标记的乙酰乙酰-ACP产物在定量前沉淀并与底物分离。我们开发了一种闪烁邻近测定法（SPA），其中使用生物素化MACP（BMACP）可以生成生物素化乙酰乙酰-ACP。该产物在被链霉亲和素包被的澄清剂浸渍的微球捕获时产生SPA信号。使用该SPA和淡灰链霉菌FabH测定BMACP K-m为7.1 μ M。在沉淀试验中测定了类似的MACP K-m（6 μ M），表明BMACP是FabH的有效底物。用碘乙酰胺和S.用抗生素硫乳霉素和大肠杆菌FabH也获得了160 μ M（SPA）和125 μ M的可比IC 50值。这些观察结果表明，FabH抑制剂可以使用具有BMACP的SPA容易地检测到，并且SPA中抑制剂的有效性与使用MACP和标准TCA沉淀测定法获得的有效性相当。一个适合高通量筛选的FabH SPA将有助于发现潜在的新型抗生素。(C)北京大学出版社.
Assays of beta-ketoacyl-acyl carrier protein synthases III (KASIII; FabH), a key enzyme initiating bacterial type II fatty acid biosynthesis, usually involve incubation of radiolabeled acetyl-coenzyme A and malonylacyl carrier protein (MACP). The radiolabeled acetoacetyl-ACP product is precipitated and separated from the substrate before quantitation. We have developed a scintillation proximity assay (SPA) where use of biotinylated MACP (BMACP) allows the generation of a biotinylated acetoacetyl-ACP, This product, when captured by the streptavidin-coated scintillant-impregnated microspheres, generates an SPA signal. A BMACP K-m of 7.1 mu M was determined using this SPA with the Streptomyces glaucescens FabH. A similar MACP K-m (6 mu M) was determined in a precipitation assay, demonstrating that BMACP is an effective substrate for FabH. IC50 values of 15.2 mu M (SPA) and 24.8 mu M were obtained with iodoacetamide and the S. glaucescens FabH, Comparable IC50 values of 160 mu M (SPA) and 125 mu M were also obtained with the antibiotic thiolactomycin and the Escherichia coli FabH. These observations demonstrate that FabH inhibitors can be readily detected using a SPA with BMACP and that the effectiveness of inhibitors in the SPA is comparable to that obtained using MACP and a standard TCA precipitation assay. A FabH SPA adaptable to high-throughput screening should facilitate the discovery of potential novel antibiotics. (C) 2000 Academic Press.