PubMed · 7039261
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S H Mudd. 1982. Vitamin-responsive genetic abnormalities.. https://doi.org/10.1007/978-1-4613-9934-6_1
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Chronic ethanol intake selectively increases concentrations of branched-chain amino acids (BCAA) in the liver. To determine whether a reduced oxidation plays a role in this effect, we measured substrate flux through branched-chain keto-acid (BCKA) dehydrogenase in livers of rats pair-fed liquid diets containing either 0% or 36% of total calories as ethanol for 21 days. Substrate (1.0 mmol/L ketoisocaproate [KIC]) fluxes in the liver of ethanol-fed and control rats were 225 +/- 18 and 319 +/- 27 mumol/h per whole liver (P < .05), respectively. We then studied whether this effect was due to either ethanol or the products of its metabolism, or to an alteration in the activity of BCKA dehydrogenase. Addition of ethanol (25 to 200 mmol/L) to the perfusion medium had no significant effect on the flux through BCKA dehydrogenase in the liver of control rats. Ethanol-fed rats had lower (P < .01) basal activity (0.84 +/- 0.11 v 1.39 +/- 0.12 U/g liver) and total activity (0.94 +/- 0.11 v 1.42 +/- 0.11 U/g liver) than control rats, but a similar activity state (90% +/- 4% v 99% +/- 4%) of BCKA dehydrogenase. In conclusion, chronic ethanol intake reduces the flux through liver BCKA dehydrogenase by decreasing the basal and total activity of BCKA dehydrogenase and not increasing the conversion of the enzyme to its inactive form.
We have determined the structural organization of the dihydrolipoyl transacylase (E2) gene of the human branched chain alpha-keto acid dehydrogenase complex. The single copy E2 gene spans approximately 68 kilobases of genomic DNA. The complete coding region consisting of the 5'- and 3'-untranslated regions, the mitochondrial targeting sequence (61 amino acids), and the mature E2 sequence (421 amino acids) are encoded by 11 exons ranging from 62 to 2239 base pairs. All the donor and acceptor splice sites conform to the gt-ag rule. Sequence analysis of the promoter-regulatory region showed the presence of a "CAAT box"-like sequence 537 bases upstream of the transcription initiation site. The "TATA box"-like sequence is absent. Also located in this region are sequences resembling glucocorticoid-responsive and cAMP-responsive elements, fat-specific elements, and Sp1- and AP-2-binding sites. Several sets of direct and inverted repeats are also present. Promoter assays using human hepatoma cells (Hep-G2) and Swiss mouse preadipocytes (3T3-L1) showed that a 4.1-kilobase PstI fragment upstream of the transcription start site confers high expression of the luciferase reporter gene. Moreover, an intronless E2 pseudogene was isolated. It corresponds to the complete mitochondrial presequence and the lipoyl-bearing domain that are encoded by exons I through IV of the functional E2 gene. However, the E2 pseudogene contains multiple base changes, deletions, and insertions, and is flanked by short direct repeats. The data indicate that the E2 pseudogene is a retroposon.
The 5' end including the start AUG codon has been defined for the human E1 alpha subunit of the branched chain alpha-ketoacid dehydrogenase complex by rapid amplification of cDNA ends. Considering conservative substitutions the amino acid sequence in the mitochondrial targeting sequence for the human clone is 73% identical to this sequence in rat and 84% identical to the bovine sequence. This peptide also shows similarity to the targeting sequence for the human beta subunit but not with targeting sequence for the other subunits of the complex.