Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 1. Identification of acyl-CoA:cholesterol O-acyltransferase subtypes by differential diethyl pyrocarbonate sensitivity.
Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 1. Identification of acyl-CoA:cholesterol O-acyltransferase subtypes by differential diethyl pyrocarbonate sensitivity.
复制标题
酰基辅酶A的化学修饰：胆固醇O-酰基转移酶。
DOI：
10.1021/bi00419a025
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Lange,LG
中科院分区：
文献类型：
--
作者：
Kinnunen,PM;DeMichele,A;Lange,LG
材料和方法材料。使用的所有试剂均为市售最高等级。[1 - 14 C]油酸（50 mCi/mmol）和[3 H]油酸（2 Ci/mmol）得自阿默舍姆。Aquasol是从新英格兰核公司购买的。蛋白质修饰试剂苯甲磺酰氟、水溶性碳二亚胺、乙酰亚胺酸甲酯、乙酸酐、苯基乙二醛、焦碳酸二乙酯、四硝基甲烷和对-（羟基汞）苯甲酸酯得自Sigma Chemical Co.，密苏里州圣路易斯辅酶A、油酸乙酯、ATP、二硫苏糖醇、BME和草酰氯也从Sigma获得。缺乏脂肪酸的BSA购自Calbiochem。甲醇、氯仿、无水乙醇、四氢呋喃、DMSO和乙醚得自Sigma，不经进一步纯化即使用。通过[3H]油酸在乙醇中的酸催化酯化来合成[3H]油酸乙酯（Lange等人，1981年）。将总共5 mCi的[3H]油酸溶解在100 mL乙醇中，并将溶液用HCl气体鼓泡20 min。将乙醇在减压下蒸发，并将残余脂质溶解在丙酮中。然后通过在石油醚/乙醚/乙酸（75：5：1）中展开的硅胶H板上的制备型薄层色谱分离[3H]油酸乙酯（Lange等人，1981年）。在用作内标物之前，用油酸乙酯将[3H]油酸乙酯稀释至最终比放射性为40 dpm/nmol。
Materials and Methods Materials. All reagents used were of the highest com-mercially available grade.[l-14C] 01eic acid (50 mCi/mmol) and [3H] oleic acid (2 Ci/mmol) were obtained from Amersham. Aquasol was purchased from New England Nuclear. The protein modification reagents phenylmethanesulfonyl fluoride, water-soluble carbodiimide, methyl acetimidate, acetic anhydride, phenylglyoxal, diethyl pyrocarbonate, tetranitro-methane, and p-(hydroxymercuri) benzoate were obtained from Sigma Chemical Co., St. Louis, MO. Coenzyme A, ethyl oleate, ATP, dithiothreitol, BME, and oxalyl chloride were also obtained from Sigma. Fatty acid poor BSA was purchased from Calbiochem. Methanol, chloroform, punctilious ethanol, tetrahydrofuran, DMSO, and diethyl ether were obtained from Sigma and used without further purification.Synthesis of Ethyl Oleate. Ethyl [3H] oleate was synthesized by acid-catalyzed esterification of [3H] oleic acid in ethanol (Lange et al., 1981). A total of 5 mCi of [3H] oleic acid was dissolved in 100 mL of ethanol, and the solution was bubbled with HC1 gas for 20 min. The ethanol was evaporated under reduced pressure, and residual lipids were dissolved in acetone. Ethyl [3H] oleate was then isolated by preparative thin-layer chromatography on silica gel H plates developed in petroleum ether/diethyl ether/acetic acid (75: 5: 1)(Lange et al., 1981). Prior to its use as an internal standard, the [3H] ethyl oleate was diluted with ethyl oleate to a final specific radioactivity of 40 dpm/nmol.