Analysis of wheat mitochondrial complex I purified by a one-step immunoaffinity chromatography.
In order to isolate the mitochondrial respiratory chain complex I (NADH:ubiquinone oxidoreductase EC 1.6.99.3) from wheat, we developed a one-step immunoaffinity procedure using antibodies raised against the NAD9 subunit. By native electrophoresis we showed that the antibodies are able to recognize the NAD9 subunit on the complex in its native form, therefore allowing the immunoaffinity chromatography. The complex retained on the column proved to be a functional complex I, since the preparation showed NADH:duroquinone and NADH:FeK3(CN)6 reductase activities which were inhibited by rotenone. The pattern of the protein subunits (about 30) eluted from the purified complex showed a high level of similarities with complex I purified from potato and broad bean by conventional techniques. Twelve subunits were identified by cross-reactions with antibodies against heterologous complex I subunits including mitochondrial- and nuclear-encoded proteins. In order to study the genetic origin of the subunits, we purified wheat complex I after in organello labelling of mitochondrial-encoded polypeptides. We found that no other complex I subunit than those corresponding to the nine mitochondrial nad genes sequenced so far, is encoded in the mitochondria of wheat.
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Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis-Pasteur, Strasbourg, France.
Grienenberger
J M
JM
D000818:Animals; D002846:Chromatography, Affinity; D042967:Electron Transport Complex I; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes; D008928:Mitochondria / Q000737:chemistry*; D024101:Mitochondrial Proteins; D016660:NAD(P)H Dehydrogenase (Quinone); D009245:NADH Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D010940:Plant Proteins / Q000032:analysis*; D011817:Rabbits; D014908:Triticum
D024101:Mitochondrial Proteins; D010940:Plant Proteins; D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D009245:NADH Dehydrogenase; D042967:Electron Transport Complex I