Bora P S, Bora N S, Wu X L, Lange L G
Department of Medicine, Jewish Hospital of St. Louis, Missouri.
J Biol Chem. 1991 Sep 5;266(25):16774-7.
Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.
脂肪酸乙酯合酶III（FAEES-III）先前已从人心脏中纯化至同质，可通过非氧化方式代谢乙醇。利用推导得到的部分氨基酸序列和相应的寡核苷酸探针，已从人心脏λgtll文库中克隆出该酶的cDNA。在获得的五个阳性克隆中，有一个包含完整的编码区（630个碱基对）和整个3'-非编码区（41个碱基对）。根据该核苷酸序列，报道了FAEES-III完整的210个氨基酸序列（Mr 23,307）。将其氨基酸序列与谷胱甘肽S-转移酶pi-1的氨基酸序列进行比较，发现它们仅在六个核苷酸和四个氨基酸上存在差异，表明它们属于同一基因家族。还将FAEES-III的序列与胎盘谷胱甘肽S-转移酶和碱性谷胱甘肽S-转移酶的序列进行了比较。FAEES-III与胎盘谷胱甘肽S-转移酶的同源性为84%，但与碱性谷胱甘肽S-转移酶的同源性不到10%。Northern印迹显示FAEES-III mRNA在正常人肝脏、胎盘和心脏中表达。在所有情况下，该酶的mRNA大小均为0.7千碱基。与对照细胞相比，转染了FAEES-III cDNA的MCF-7细胞的合酶活性增加了14倍，谷胱甘肽S-转移酶（GST）活性增加了12倍。与对照细胞相比，转染了GST pi-1 cDNA的MCF-7细胞的GST活性增加了13倍，但合酶活性没有增加。当用兔FAEES-III抗体对转染了FAEES-III cDNA的COS-7细胞的上清液进行免疫印迹时，在24千道尔顿处出现了一条带。因此，我们获得了首个具有显著GST活性的人FAEES-III的cDNA和氨基酸序列，并且我们确定了4个可能赋予GST乙醇识别能力的残基。
