Abstract
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.
Highlights
Fatty acid ethyelster synthase-I11 (FAEES-111),pre- No mechanism has been proposed which accounts for the viously purified to homogeneity from human heart, propensity of certain individuals to drink toexcess, to develop metabolizes ethanol nonoxidatively
We report the totalamino acid sequence deduced from the nucleotide sequence, a comparison of the sequences with known GSTs, the tissue distribution of the FAEES-111 mRNA, andthe transfection of cDNA into theMCF-7 and COS-7 cells
Nucleotide Sequence of FAEES-111-The complete nucleotide sequences of both strands were obtained by sequencing the cDNAs obtained from different clones
Summary
The costsof publication of this article were defrayed in partby the payment of page charges. Thisarticlemusttherefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely t o indicate this fact. The nucleotide sequence(s)reported in thispaperhas been submitted totheGenBankTM/EMBLDataBankwith accession number(s)
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