Zhu Anita, Su Xiaoyang, Lin Hening
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA.
Methods Mol Biol. 2013;1077:179-89. doi: 10.1007/978-1-62703-637-5_12.
Sirtuins are a class of enzymes with important functions in regulation aging, metabolism, and genome stability. They were originally known as nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacetylases. However, recently it has been discovered that certain sirtuins with weak deacetylase activity also hydrolyze novel acyl lysine modifications. These findings indicate that other sirtuins with weak deacetylase activity may also possess novel activities on unknown protein posttranslational modifications. Analytical methods that can help to identify new activity of sirtuins and new acyl lysine modifications are thus needed. Here we describe a sensitive method that uses (32)P-labeled NAD and thin-layer chromatography to detect sirtuin-catalyzed deacylation reactions. This method can help to discover new acyl lysine modifications that can be removed by novel sirtuin activities.
沉默调节蛋白是一类在调节衰老、新陈代谢和基因组稳定性方面具有重要功能的酶。它们最初被认为是烟酰胺腺嘌呤二核苷酸（NAD）依赖性蛋白赖氨酸脱乙酰酶。然而，最近发现某些具有弱脱乙酰酶活性的沉默调节蛋白也能水解新型酰基赖氨酸修饰。这些发现表明，其他具有弱脱乙酰酶活性的沉默调节蛋白可能在未知的蛋白质翻译后修饰上也具有新的活性。因此，需要有助于鉴定沉默调节蛋白新活性和新型酰基赖氨酸修饰的分析方法。在这里，我们描述了一种灵敏的方法，该方法使用（32）P标记的NAD和薄层色谱法来检测沉默调节蛋白催化的脱酰基反应。这种方法有助于发现可被新型沉默调节蛋白活性去除的新型酰基赖氨酸修饰。
