Analysis of wheat mitochondrial complex I purified by a one-step immunoaffinity chromatography

In order to isolate the mitochondrial respiratory chain complex I (NADH:ubi quinone oxidoreductase EC 1.6.99.3) from wheat, we developed a one-step imm unoaffinity 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 a nd NADH:FeK3(CN)(6) reductase activities which were inhibited by rotenone. The pattern of the protein subunits (about 30) eluted from the purified com plex showed a high level of similarities with complex I purified from potat o and broad bean by conventional techniques. Twelve subunits were identifie d by cross-reactions with antibodies against heterologous complex I subunit s including mitochondrial- and nuclear-encoded proteins. In order to study the genetic origin of the subunits, we purified wheat complex I after in or ganello labelling of mitochondrial-encoded polypeptides. We found that no o ther complex I subunit than those corresponding to the nine mitochondrial n ad genes sequenced so far, is encoded in the mitochondria of wheat. (C) Soc iete francaise de biochimie et biologie moleculaire / Elsevier, Paris.