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The pyruvate dehydrogenase complex: cloning of the rat somatic E1 alpha subunit and its coordinate expression with
T E Cullingford1, J B Clark, I R Phillips
1Department of Biochemistry, Queen Mary and Westfield College, London, England.
Insights
Researchers isolated rat pyruvate dehydrogenase complex E1 alpha subunit cDNA. mRNA levels increase significantly in developing rat brains, suggesting translational control of enzyme complex and citrate synthase expression.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- The pyruvate dehydrogenase complex (PDC) is crucial for cellular energy metabolism.
- Understanding the regulation of PDC subunits, like E1 alpha, is vital for comprehending metabolic control during development.
- Previous studies reported discrepancies in rat E1 alpha mRNA and protein sequences.
Purpose of the Study:
- To isolate and characterize cDNA clones for the somatic form of the rat pyruvate dehydrogenase complex E1 alpha subunit.
- To investigate discrepancies in reported E1 alpha mRNA sizes and amino acid sequences.
- To analyze the developmental expression of PDC subunit mRNAs and citrate synthase mRNA in the rat brain.
Main Methods:
- Isolation of cDNA clones encoding the rat E1 alpha subunit.
- Sequence analysis and comparison with orthologous proteins.
- Polymerase Chain Reaction (PCR) to investigate sequence differences.
- Northern blot hybridization and RNase protection assays to analyze mRNA.
- Developmental expression analysis using rat whole brain RNA from fetal to postnatal stages.
Main Results:
- High amino acid sequence identity was found between rat E1 alpha and orthologous proteins from other species (mouse, human, pig).
- Discrepancies in E1 alpha mRNA size and sequence were investigated using molecular techniques.
- RNase protection assays revealed a 2.5-fold increase in PDC subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late fetal stage to 5 days postpartum.
- mRNA levels stabilized after 5 days postpartum.
Conclusions:
- The study successfully isolated cDNA for the rat somatic E1 alpha subunit, clarifying sequence information.
- Developmental increases in pyruvate dehydrogenase complex and citrate synthase in the rat brain are primarily regulated at the translational and/or posttranslational levels, not transcriptional.
- These findings provide insights into the metabolic adaptations occurring during early brain development.
Abstract:
We report the isolation of cDNA clones encoding the somatic form of the E1 alpha subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1 alpha sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1 alpha subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1 alpha cDNA, in conjunction with cDNA probes to the E1 beta, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5-fold increase in the concentration of each of the subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six- and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.
