Plant mitochondrial 2-oxoglutarate dehydrogenase complex: purification andcharacterization in potato

The 2-oxoglutarate dehydrogenase complex (OGDC) in potato (Solanum tuberosu m cv. Romano) tuber mitochondria is largely associated with the membrane fr action of osmotically ruptured organelles, whereas most of the other tricar boxylic acid cycle enzymes are found in the soluble matrix fraction. The pu rification of OGDC from either membrane or soluble matrix fractions resulte d in the increasing dependence of its activity on the addition of dihydroli poamide dehydrogenase (E3). A 30-fold purification of OGDC to apparent homo geneity and with a specific activity of 4.6 mu mol/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 mo lecular masses of 48, 50 and 105 kDa, Before the gel-filtration purificatio n 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 k Da 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-termina l sequencing. The N-terminal sequences of the 50 and 48 kDa proteins shared 90-95% identity over 20 residues and were identified by sequence similarit y as dihydrolipoamide succinyltransferases (OGDC-E2). The incubation of OGD C with [U-C-14]2-oxoglutarate resulted in the reversible succinylation of b oth the 48 and the 50 kDa protein bands. Proteins previously reported as su bunits of complex I of the respiratory chain from Vicia faba and Solanum tu berosum are proposed to be OGDC-E2 and the possible basis of this associati on is discussed.