文章基本信息

标题：A novel direct homogeneous assay for ATP citrate lyase
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作者：Zhengping Ma ; Ching-Hsuen Chu ; Dong Cheng 等
期刊名称：JLR Papers In Press
印刷版ISSN：0022-2275
电子版ISSN：1539-7262
出版年度：2009
卷号：50
期号：10
页码：2131-2135
DOI：10.1194/jlr.D900008-JLR200
语种：English
出版社：American Society for Biochemistry and Molecular Biology
摘要：ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg²⁺ as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [¹⁴C]acetyl-CoA without detecting [¹⁴C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1 ) incubation of ACL enzyme with [¹⁴C]citrate and other substrates/cofactors CoA, ATP, and Mg²⁺, 2 ) EDTA quench, 3 ) addition of MicroScint-O, the agent that specifically detects product [¹⁴C]acetyl-CoA, and 4 ) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.
关键词：fatty acid ; acetyl-CoA ; obesity ; dyslipidemia