Mogelson S, Lange L G
Biochemistry. 1984 Aug 28;23(18):4075-81. doi: 10.1021/bi00313a010.
Fatty acyl ethyl esters, previously identified in our laboratory as metabolites of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to isolate and purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of rabbit myocardium, as assayed by the rate of synthesis of ethyl [14C]oleate from 0.4 mM [14C]oleic acid and 0.2 M ethanol, was 31 nmol/(g.h), and all of it was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with 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-albumin affinity chromatographies with an overall yield of 40%. Up to 45 micrograms of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mr 26 000, and gel permeation chromatography under nondenaturing conditions indicated a Mr of 50 000 for the active enzyme. Kinetic analyses using the purified enzyme indicated that greatest rates of ethyl ester synthesis were observed with unsaturated octadecanoic fatty acid substrates [Vmax = 1.9 and 1.5 nmol/(mg.s) for linoleate and oleate, respectively], with lesser rates associated with palmitate, stearate, and arachidonate substrates [0.14, 0.03, and 0.35 nmol/(mg.s), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)
脂肪酸乙酯，此前在我们实验室中被鉴定为人和兔心肌中乙醇的代谢产物，它是在没有ATP和辅酶A的情况下，由游离脂肪酸与乙醇酯化反应产生的。本研究旨在分离和纯化兔心肌中催化此反应的酶。用0.4 mM [14C]油酸和0.2 M乙醇合成乙基[14C]油酸酯的速率来测定兔心肌匀浆中的酶活性，为31 nmol/(g·h)，且所有活性都在48400g的上清液中被回收。这种可溶性乙酯合成酶活性在pH 8时结合到DEAE -纤维素上，用NaCl梯度（0 - 0.25 M）洗脱分离出两种酶活性，分别占回收的合成酶活性的13%和87%。然后通过连续的凝胶渗透、疏水相互作用和抗白蛋白亲和层析将主要的酶活性纯化至超过5000倍的纯度，总产率为40%。每克心肌中存在多达45微克的酶。十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳显示一条分子量为26000的单条多肽，非变性条件下的凝胶渗透色谱表明活性酶的分子量为50000。使用纯化酶的动力学分析表明，不饱和十八碳脂肪酸底物的乙酯合成速率最高[亚油酸和油酸的Vmax分别为1.9和1.5 nmol/(mg·s)]，棕榈酸、硬脂酸和花生四烯酸底物的速率较低[分别为0.14、0.03和0.35 nmol/(mg·s)]。（摘要截短至250字）
