Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase.
Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase.
复制标题
兔心肌中的非氧化乙醇代谢：脂肪酰乙酯合酶的同质纯化。
DOI：
10.1021/bi00313a010
复制
发表时间：
1984
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：
Mogelson,S;Lange,LG
[14C]油酸和0.2M乙醇为31nmol/(Gh)，在48400g上清液中全部回收。可溶性乙酯合成酶活性在pH=8时与DEAE-纤维素结合，用氯化钠梯度(0-0.25M)洗脱，分离出两种酶活性，分别占回收合酶活性的13%和87%。通过连续凝胶渗透、疏水相互作用和抗蛋白亲和层析，主要酶活性纯化了5000倍以上，总收率为40%。每克心肌中含有高达45毫克的酶。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测得单一多肽的相对分子质量为26000，非变性条件下凝胶渗透层析法测得该酶的相对分子质量为50000。用纯化后的产物进行动力学分析。酒精引起的心脏病的特征是心肌收缩功能和能量代谢异常，即使在没有营养剥夺的情况下也是如此(Lochner等人，1969；西格尔等人，1979)。慢性酒精摄入引起的心肌脂质代谢的改变，包括甘油三酯稳态异常和脂肪酸氧化，已经得到了很好的描述(Regan等人，1966；Kikuchi&Kako，1970；Kako等人，1973)，但乙醇引起这些扰动的生化机制在很大程度上是未知的，因为还没有心肌乙醇代谢的描述。在最近的研究中，乙醇掺入脂肪酰乙酯家族被证明发生在用乙醇灌流的离体兔心脏，在兔心脏
[14C] oleic acid and 0.2 M ethanol, was 31 nmol/(gh), and all of it was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elutionwith a NaCl gradient (0-0.25 M) separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was then purified over 5000-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-albuminaffinity chromatographies with an overall yield of 40%. Up to 45 Mg of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mt 26 000, and gel permeationchromatog-raphy under nondenaturing conditions indicated a Mr of 50000 for the active enzyme. Kinetic analyses using the purified.^^. lcohol-induced heart disease is characterized by abnormal myocardial contractile function and energy metabolism, even in theabsence of nutrient deprivation (Lochner et al., 1969; Segal et al., 1979). Alterations in myocardial lipid metabolism pursuant to chronic ethanol ingestion, including abnormal triglyceride homeostasis and fatty acid oxidation, were well described (Regan et al., 1966; Kikuchi & Kako, 1970; Kako et al., 1973), but the biochemical mechanisms by which these perturbations are engendered by ethanol have been largely unknown since no myocardial ethanol metabolism hadbeen described. In recent studies, ethanolincorporation into a family of fatty acyl ethyl esters was documented to occur in isolated rabbit hearts perfused with ethanol, in rabbit heart