Plant mitochondrial 2-oxoglutarate dehydrogenase complex: purification and characterization in potato.
The 2-oxoglutarate dehydrogenase complex (OGDC) in potato (Solanum tuberosum cv. Romano) tuber mitochondria is largely associated with the membrane fraction of osmotically ruptured organelles, whereas most of the other tricarboxylic acid cycle enzymes are found in the soluble matrix fraction. The purification of OGDC from either membrane or soluble matrix fractions resulted in the increasing dependence of its activity on the addition of dihydrolipoamide dehydrogenase (E3). A 30-fold purification of OGDC to apparent homogeneity and with a specific activity of 4.6 micromol/min per mg of protein in the presence of exogenously added E3 was obtained. SDS/PAGE revealed that the purified complex consisted of three major polypeptides with apparent molecular masses of 48, 50 and 105 kDa. Before the gel-filtration purification step, E3 polypeptides of 57 and 58 kDa were identified by immunoreaction as minor proteins associated with OGDC. The N-terminal sequence of the 57 kDa protein was identical with that previously purified as the E3 component of the pyruvate dehydrogenase complex from potato. The 105 kDa protein was identified as the 2-oxoglutarate dehydrogenase subunit of OGDC by N-terminal sequencing. The N-terminal sequences of the 50 and 48 kDa proteins shared 90-95% identity over 20 residues and were identified by sequence similarity as dihydrolipoamide succinyltransferases (OGDC-E2). The incubation of OGDC with [U-(14)C]2-oxoglutarate resulted in the reversible succinylation of both the 48 and the 50 kDa protein bands. Proteins previously reported as subunits of complex I of the respiratory chain from Vicia faba and Solanum tuberosum are proposed to be OGDC-E2 and the possible basis of this association is discussed.
Millar
A H
AH
Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, U.K. harvey.millar@plants.ox.ac.uk
Hill
S A
SA
Leaver
C J
CJ
D000217:Acyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D015153:Blotting, Western; D019069:Cell Respiration; D002850:Chromatography, Gel; D002952:Citric Acid Cycle; D006863:Hydrogen-Ion Concentration; D007655:Ketoglutarate Dehydrogenase Complex / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000201:enzymology; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000166:cytology* / Q000201:enzymology*; D019802:Succinic Acid / Q000378:metabolism
D007656:Ketoglutaric Acids; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D019289:Pyruvic Acid; D019802:Succinic Acid; D007655:Ketoglutarate Dehydrogenase Complex; D000217:Acyltransferases; C021515:dihydrolipoamide succinyltransferase