Dihydrolipoyl transacetylase of Escherichia coli. Formation of 8-S-acetyldihydrolipoamide.
Dihydrolipoyl transacetylase of Escherichia coli. Formation of 8-S-acetyldihydrolipoamide.
复制标题
大肠杆菌的二氢硫辛酰转乙酰酶。
DOI：
10.1021/bi00373a008
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Frey,PA
中科院分区：
文献类型：
--
作者：
Yang,YS;Frey,PA
材料和方法酶。磷酸转乙酰酶购自 Sigma。丙酮酸脱氢酶复合物通过 Reed 和 Mukherjee (1969) 的方法纯化，并由 Speckhard 和 Frey (1975) 修改和扩展，包括通过磷酸钙凝胶-纤维素柱的色谱法。将酶以约20mg/mL的浓度冷冻在液N2中后储存于-70℃。通过将 2-2.5 mg/mL 的复合物与 TPP (0.2 mM)、NADH (0.7 mM)、NAD+(2 mM) 和 NEM (1 mM) 在磷酸钾缓冲液 (0.1 M，pH 7) 中室温孵育，丙酮酸脱氢酶复合物的硫辛酰部分被还原并烷基化 (Danson 等人，1981)。孵育 10 分钟后，丙酮酸脱氢酶复合物活性 (eq 6) 约为初始活性的 2%。二氢硫辛酰转乙酰酶活性（eq 7）不受烷基化的影响。然后将NEM烷基化酶针对用于后续酶促反应的相同缓冲液进行透析。如Reed和Willms (1966)所述，在E已被pH 9.5的乙醇胺-磷酸盐缓冲液洗脱后，衍生自丙酮酸脱氢酶复合物的E、E2亚复合物被8%(NH 4 ) 2 SO 4 从磷酸钙凝胶-纤维素柱洗脱。将含有 E2E3 亚复合物的溶液在 pH 6.5 的 0.1 M PIPES 缓冲液中透析，并通过超滤浓缩。
Materials and MethodsEnzymes. Phosphotransacetylase was purchased from Sigma. The pyruvate dehydrogenase complex was purified by the method of Reed and Mukherjee (1969) as modified and extended by Speckhard and Frey (1975) to include chroma-tography through a column of calcium phosphate gel-cellulose. The enzyme was stored at-70 C after freezing in liquid N2 at a concentration of about 20 mg/mL. The lipoyl moieties of the pyruvate dehydrogenase complex were reduced and alkylated (Danson et al., 1981) by incubation of 2-2.5 mg/mL of complex with TPP (0.2 mM), NADH (0.7 mM), NAD+(2 mM), and NEM (1 mM) in potassium phosphate buffer (0.1 M, pH 7) at room temperature. The pyruvate dehydrogenase complex activity (eq 6) after 10 min of incubation was about 2% of the initial activity. The dihydrolipoyl transacetylase activity (eq 7) was unaffected by alkylation. The NEM-alkylated enzyme was then dialyzed against the same buffer used for thesubsequent enzymatic reaction. The E, E2 subcomplex derived from the pyruvatedehydrogenase complex was eluted from a calcium phosphate gel-cellulose column by 8%(NH4) 2S04 after E, had been eluted by ethanolamine-phosphate buffer at pH 9.5 as described by Reed and Willms (1966). The solution containing E2E3 subcomplex was dialyzed against 0.1 M PIPES buffer at pH 6.5 and concentrated by ultrafiltration.