Microplate filtration assay for nicotinamide release from NAD using a boronic acid resin

被引：28

作者：

McDonagh, T ^{[1
]}

Hixon, J ^{[1
]}

DiStefano, PS ^{[1
]}

Curtis, R ^{[1
]}

Napper, AD ^{[1
]}

机构：

[1] Elixir Pharmaceut, Cambridge, MA 02139 USA

来源：

METHODS | 2005年 / 36卷 / 04期

关键词：

SIRT; deacetylase; nicotinamide; NAD-hydrolyzing; boronic acid; inhibition; microplate; filtration; high-throughput screening;

D O I：

10.1016/j.ymeth.2005.03.005

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We describe a microplate-based assay for NAD-dependent Class III historic deacetylases (also known as SIRTs) that measures the enzyme-catalyzed release of nicotinamide from radiolabeled NAD, using a boronic acid resin to selectively capture the NAD. This method avoids the need for fluorogenic or radiolabeled peptides or separation of the reaction products using solvent extraction. The protocol reported here is rapid and uses commercially available materials. The use of a simple microplate filtration device allows for the simultaneous processing of 96 samples, facilitating enzyme kinetic analyses and inhibition studies. Furthermore, monitoring nicotinamide release rather than peptide deacetylation obviates the need for chemical modification of protein and peptide substrates. This assay is applicable to SIRTs and other enzymes that cleave nicotinamide from NAD. (c) 2005 Elsevier Inc. All rights reserved.

引用

页码：346 / 350

页数：5

共 31 条

[1] EVALUATION OF IMMOBILIZED BORONATES FOR STUDIES OF ADENINE AND PYRIDINE-NUCLEOTIDE METABOLISM [J].