North Brian J, Schwer Bjoern, Ahuja Nidhi, Marshall Brett, Verdin Eric
Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA, USA.
Methods. 2005 Aug;36(4):338-45. doi: 10.1016/j.ymeth.2005.03.004.
Class III histone deacetylases, or sirtuins, are homologous to the Saccharomyces cerevisiae transcriptional regulator SIR2. The class III enzymes are characterized by their dependence on nicotinamide adenine dinucleotide (NAD+). This cofactor serves as an acetyl-group acceptor in the deacetylation reaction generating O-acetyl-ADP-ribose. Enzymatic activity of sirtuin can be measured in vitro using recombinant proteins purified from mammalian cells after overexpression or after purification from Escherichia coli. This review discusses protocols for the purification of enzymatically active human sirtuin 1, 2, and 3 and their activities on histone and nonhistone substrates.
III类组蛋白去乙酰化酶，即沉默调节蛋白，与酿酒酵母转录调节因子SIR2同源。III类酶的特点是依赖烟酰胺腺嘌呤二核苷酸（NAD+）。该辅因子在脱乙酰化反应中作为乙酰基受体，生成O-乙酰-ADP-核糖。沉默调节蛋白的酶活性可以在体外通过使用从哺乳动物细胞中过表达后纯化的重组蛋白或从大肠杆菌中纯化后的重组蛋白来测量。本综述讨论了具有酶活性的人沉默调节蛋白1、2和3的纯化方案及其对组蛋白和非组蛋白底物的活性。
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