caa a22 4500 477100341 CHVBK 20180405111539.0 cr unu---uuuuu 170330e19961001xx s 000 0 eng 10.1007/BF00208309 doi (NATIONALLICENCE)springer-10.1007/BF00208309 Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts [Elektronische Daten] [Mark Conner, Tino Krell, J. Lindsay] Two distinct dihydrolipoamide dehydrogenases (E3s, EC 1.8.1.4) have been 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 Mr value 56 000 (80-90% of overall activity), corresponded to the mitochondrial isoform studied previously, as confirmed by electrospray mass spectrometry and N-terminal sequence analysis. The minor activity (10-20%), which also behaved as a homodimer, copurified with chloroplasts, and displayed a lower subunit Mr value of 52 000 which was close to the Mr value of 52 614±9.89 Da determined by electrospray mass spectrometry. The plastidic enzyme was also present at low levels in root extracts where it represented only 1-2% of total E3 activity. The specific activity of the chloroplast enzyme was three-to fourfold lower than its mitochondrial counterpart. In addition, it displayed a markedly higher affinity for NAD+ and was more 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 enzyme displayed little or no cross-reactivity with its plastidic counterpart and available amino acid sequence data were also suggestive of only limited sequence similarity between the two enzymes. In view of the dual location of the pyruvate dehydrogenase multienzyme complex (PDC) in plant mitochondria and chloroplasts, it is likely that the distinct chloroplastic E3 is an integral component of plastidic PDC, thus representing the first component of this complex to be isolated and characterised to date. Springer-Verlag, 1996 Pisum nationallicence Chloroplasts (enzyme complex) nationallicence Dihydrolipoamide dehydrogenase nationallicence Mitochondrion (enzyme complex) nationallicence Pyruvate dehydrogenase complex nationallicence E1 : pyruvate dehydrogenase nationallicence E2 : dihydrolipoamide acetyltransferase nationallicence E3 : dihydrolipoamide dehydrogenase nationallicence PDC : pyruvate dehydrogenase complex nationallicence OGDC : 2-oxoglutarate dehydrogenase complex nationallicence GDC : glycine decarboxylase complex nationallicence SDS-PAGE : sodium dodecyl sulphate/polyacrylamide gel electrophoresis nationallicence TDP : thiamine diphosphate nationallicence Mr : relative molecular mass nationallicence Conner Mark Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, G128QQ, Glasgow, UK aut Krell Tino Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, G128QQ, Glasgow, UK aut Lindsay J. Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, G128QQ, Glasgow, UK aut Planta Springer-Verlag 200/2(1996-10-01), 195-202 0032-0935 200:2<195 1996 200 425 https://doi.org/10.1007/BF00208309 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00208309 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Conner Mark Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, G128QQ, Glasgow, UK aut NATIONALLICENCE 700 1- Krell Tino Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, G128QQ, Glasgow, UK aut NATIONALLICENCE 700 1- Lindsay J. Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, G128QQ, Glasgow, UK aut NATIONALLICENCE 773 0- Planta Springer-Verlag 200/2(1996-10-01), 195-202 0032-0935 200:2<195 1996 200 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer