A high-throughput screening assay for pyruvate carboxylase

被引：6
作者：
Wyatt, Brittney N. [1 ]
Arnold, Leggy A. [2 ,3 ]
Maurice, Martin St. [1 ]
机构：
[1] Marquette Univ, Dept Biol Sci, Milwaukee, WI 53233 USA
[2] Univ Wisconsin, Dept Chem & Biochem, Milwaukee, WI 53201 USA
[3] Univ Wisconsin, Milwaukee Inst Drug Discovery, Milwaukee, WI 53201 USA
来源：
ANALYTICAL BIOCHEMISTRY | 2018年 / 550卷
关键词：
Pyruvate carboxylase; Diazonium salt; Fast violet B; Oxaloacetate; High throughput screening; GLUTAMIC-OXALACETIC TRANSAMINASE; PANCREATIC BETA-CELLS; C-DI-AMP; INSULIN-SECRETION; LISTERIA-MONOCYTOGENES; ACETYL-COA; SERUM; ANAPLEROSIS; DEHYDROGENASE; SUPPRESSION;
D O I：
10.1016/j.ab.2018.04.012
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potent small molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobiwn etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC.
引用
收藏
页码：90 / 98
页数：9
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