Andres H H, Klem A J, Szabo S M, Weber W W
Anal Biochem. 1985 Mar;145(2):367-75. doi: 10.1016/0003-2697(85)90376-8.
Simple and sensitive spectrophotometric and radiochemical procedures are described for the assay of acetyl-CoA:arylamine N-acetyltransferase (NAT; EC 2.3.1.5), which catalyzes the reaction acetyl-CoA + arylamine----N-acetylated arylamine + CoASH. The methods are applicable to crude tissue homogenates and blood lysates. The spectrophotometric assay is characterized by two features: (i) NAT activity is measured by quantifying the disappearance of the arylamine substrate as reflected by decreasing Schiff's base formation with dimethylaminobenzaldehyde. (ii) During the enzymatic reaction, the inhibitory product CoASH is recycled by the system acetyl phosphate/phosphotransacetylase to the substrate acetyl-CoA. The radiochemical procedure depends on enzymatic synthesis of [3H]acetyl-CoA in the assay using [3H]acetate, ATP, CoASH, and acetyl-CoA synthetase. NAT activity is measured by quantifying N-[3H]acetylarylamine after separation from [3H]acetate by extraction. Product inhibition by CoASH is prevented in this system by the use of acetyl-CoA synthetase.
本文描述了用于测定乙酰辅酶A：芳胺N - 乙酰转移酶（NAT；EC 2.3.1.5）的简单且灵敏的分光光度法和放射化学法，该酶催化乙酰辅酶A + 芳胺→N - 乙酰化芳胺 + 辅酶A的反应。这些方法适用于粗制组织匀浆和血液裂解物。分光光度法具有两个特点：（i）通过用二甲基氨基苯甲醛定量席夫碱形成减少所反映的芳胺底物消失来测量NAT活性。（ii）在酶促反应过程中，抑制性产物辅酶A通过乙酰磷酸/磷酸转乙酰酶系统循环回底物乙酰辅酶A。放射化学法依赖于在测定中使用[³H]乙酸盐、ATP、辅酶A和乙酰辅酶A合成酶酶促合成[³H]乙酰辅酶A。通过萃取从[³H]乙酸盐中分离后，通过定量N - [³H]乙酰芳胺来测量NAT活性。在该系统中，通过使用乙酰辅酶A合成酶可防止辅酶A的产物抑制。
