Kaphalia B S, Fritz R R, Ansari G A
Department of Pathology, University of Texas Medical Branch, Galveston 77555, USA.
Chem Res Toxicol. 1997 Feb;10(2):211-8. doi: 10.1021/tx960079e.
Previous studies have shown that fatty acid ethyl ester synthase (FAEES) which catalyzes the formation of ethyl or 2-chloroethyl esters of long-chain fatty acids is localized in the microsomal fraction of rat liver. A recent study suggests that rat adipose tissue FAEES is similar to rat liver microsomal carboxylesterase (CE) [Tsujita and Okuda (1992) J. Biol. Chem. 267, 23489-23494]. Since the interrelationships among FAEES, 2-chloroethyl ester synthase (FACEES), and cholesterol esterase (ChE) are also not clear at present, we purified and characterized FAEES and FACEES from rat hepatic microsomes and studied their functional and structural relationships with CE and ChE. The results of these studies showed that CE, FAEES, and FACEES activities copurified during each step of purification. Although gel-filtration column chromatography of DEAE-Sephacel purified microsomal protein resolved into two peaks with an estimated molecular weight of 180 (major) and 60 kDa (minor, this paper describes characterization of only the 180 kDa protein. CE, FAEES, and FACEES activities associated with homogeneous 180 kDa protein could be inhibited by a beta-esterase inhibitor (diisopropyl fluorophosphate) in an identical manner. This protein, however, showed only the hydrolytic activity, but not the synthetic activity for cholesterol oleate, indicating that it is distinct from ChE. The purified protein could be immunoprecipitated with the antibodies raised against rat adipose tissue FAEES, but not with antibodies against rat pancreatic ChE, demonstrating again that the purified protein is distinct from ChE. A single band corresponding to 60 kDa upon SDS-PAGE, under reduced denaturing conditions, indicates that the purified protein is a trimer. N-terminal amino acid sequence of the first 27 residues were identical to that of rat hepatic microsomal CE [Robbi et al. (1990) Biochem. J., 451-458] which suggests structural similarity of the purified protein with rat hepatic microsomal CE. Therefore, the functional and structural properties of the purified protein demonstrate that FAEES, FACEES, as well as CE activities are expressed by the same protein, purified in this study, which exists as a trimer (180 kDa) and is involved in biosynthesis of long-chain fatty acid esters of xenobiotic alcohols. Further studies on purification and characterization of the enzymes responsible for the esterification of xenobiotic alcohols with endogenous fatty acids from various target organs need to be conducted to determine their functional and structural interrelationships. Inhibition and induction studies of these enzyme(s) and the extent of observed toxicity could be important in understanding their role in etiology of chronic diseases induced by alcohol abuse.
先前的研究表明，催化长链脂肪酸乙酯或2 - 氯乙酯形成的脂肪酸乙酯合酶（FAEES）定位于大鼠肝脏的微粒体部分。最近的一项研究表明，大鼠脂肪组织中的FAEES与大鼠肝脏微粒体羧酸酯酶（CE）相似[辻田和奥田（1992年）《生物化学杂志》267卷，23489 - 23494页]。由于目前FAEES、2 - 氯乙酯合酶（FACEES）和胆固醇酯酶（ChE）之间的相互关系也不清楚，我们从大鼠肝脏微粒体中纯化并鉴定了FAEES和FACEES，并研究了它们与CE和ChE的功能及结构关系。这些研究结果表明，在纯化的每个步骤中，CE、FAEES和FACEES的活性都一起被纯化出来。尽管用DEAE - Sephacel纯化的微粒体蛋白进行凝胶过滤柱层析可分离出两个峰，估计分子量分别为180 kDa（主要峰）和60 kDa（次要峰，本文仅描述180 kDa蛋白的特性）。与均一的180 kDa蛋白相关的CE、FAEES和FACEES活性可被β - 酯酶抑制剂（二异丙基氟磷酸酯）以相同方式抑制。然而，该蛋白仅表现出水解活性，而对油酸胆固醇没有合成活性，这表明它与ChE不同。纯化的蛋白可用针对大鼠脂肪组织FAEES产生的抗体进行免疫沉淀，但不能用针对大鼠胰腺ChE的抗体进行免疫沉淀，这再次证明纯化的蛋白与ChE不同。在还原变性条件下，SDS - PAGE上对应60 kDa的单一条带表明纯化的蛋白是三聚体。前27个残基的N端氨基酸序列与大鼠肝脏微粒体CE的相同[罗比等人（1990年）《生物化学杂志》，451 - 458页]，这表明纯化的蛋白与大鼠肝脏微粒体CE在结构上相似。因此，纯化蛋白的功能和结构特性表明，在本研究中纯化的同一蛋白表达了FAEES、FACEES以及CE的活性，该蛋白以三聚体（180 kDa）形式存在，并参与外源醇类长链脂肪酸酯的生物合成。需要对负责将外源醇类与来自各种靶器官的内源性脂肪酸进行酯化反应的酶进行进一步的纯化和鉴定研究，以确定它们的功能和结构相互关系。对这些酶的抑制和诱导研究以及观察到的毒性程度，对于理解它们在酒精滥用所致慢性疾病病因学中的作用可能很重要。
