IDENTIFICATION AND PURIFICATION OF A DISTINCT DIHYDROLIPOAMIDE DEHYDROGENASE FROM PEA-CHLOROPLASTS

Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have be en detected in pea (Pisum sativum L. cv. Little Marvel) leaf extracts and purified to at or near homogeneity. The major enzyme, a homodimer with an apparent subunit M(r) value 56000 (80-90% of overall activity) , corresponded to the mitochondrial isoform studied previously, as con firmed by electrospray mass spectrometry and N-terminal sequence analy sis. The minor activity (10-20%), which also behaved as a homodimer, c opurified with chloroplasts, and displayed a lower subunit M(r) value of 52 000 which was close to the M(r) value of 52 614 +/- 9.89 Da dete rmined by electrospray mass spectrometry. The plastidic enzyme was als o present at low levels in root extracts where it represented only 1-2 % of total E3 activity. The specific activity of the chloroplast enzym e was three- to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD(+) and was m ore sensitive to product inhibition by NADH. It exhibited no activity with NADP(+) as cofactor nor was it inhibited by the presence of high concentrations of NADP(+) or NADPH. Antibodies to the mitochondrial en zyme displayed little or no cross-reactivity with its plastidic counte rpart and available amino acid sequence data were also suggestive of o nly limited sequence similarity between the two enzymes. In view of th e dual location of the pyruvate dehydrogenase multienzyme complex (PDC ) in plant mitochondria and chloroplasts, it is likely that the distin ct chloroplastic E3 is an integral component of plastidic PDC, thus re presenting the first component of this complex to be isolated and char acterised to date.