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United States Department of Agriculture

Agricultural Research Service

Title: The Dihydrolipoamide S-Acetyltransferase Subunit to the Mitochondrial Pyruvate Dehydrogenase Complex from Maize Contains a Single Lipoyl Domain

item Thelen, J - U OF MO, COLUMBIA, MO
item Miernyk, Jan
item Randall, D - U OF MO, COLUMBIA, MO

Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only
Publication Acceptance Date: July 16, 1999
Publication Date: N/A

Technical Abstract: The dihydrolipoamide acetyltransferase (E2) subunit to the maize mitochondrial pyruvate dehydrogenase complex (mPDC) was previously postulated to contain a single lipoyl domain based upon molecular mass (52 kDa) and N-terminal protein sequence. Using the N-terminal protein sequence as a search query, a maize cDNA was identified from an expressed sequence tag database. The primary amino acid sequence was 30-37% identical to other E2s and contained a single lipoyl domain. Since the only other reported plant mitochondrial E2 contains two lipoyl domains, the activity and assembly properties of this putative maize E2 were investigated. The purified recombinant protein exhibited acetyltransferase activity and assembled into a complex of approximately 2.7 MDa based upon size-exclusion chromatography. The size of this complex was close to the predicted size of 2.8 MDa for a 60-mer pentagonal dodecahedron, which was confirmed by electron microscopy. To determine if a single lipoyl-domain E2 is present in all plants, a monoclonal antibody raised against native maize E2 was developed for immunoblot analyses. The antibody recognized recombinant maize E2 and the epitope it detected was mapped to the lipoyl domain. Immunoanalysis with mitochondrial protein purified from various plant sources revealed 50-54 kDa cross-reacting polypeptides in all plants assayed. A larger protein was also recognized from enriched mPDC from pea (76 kDa), indicating two distinct E2s in these mitochondria. The presence of a single lipoyl-domain E2 in A.thaliana was confirmed by identifying a gene encoding a protein with 62% amino acid identity to the maize homologue.

Last Modified: 5/5/2015
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