Analysis of wheat mitochondrial complex I purified by a one-step immunoaffinity chromatography
| Autor: | Jean-Michel Grienenberger, Bruno Combettes |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Protein subunit
Mitochondrion Biology Reductase Biochemistry Chromatography Affinity Mitochondrial Proteins Duroquinone chemistry.chemical_compound Affinity chromatography Oxidoreductase NAD(P)H Dehydrogenase (Quinone) Animals NADH NADPH Oxidoreductases Mitochondrial respiratory chain complex I Triticum Plant Proteins chemistry.chemical_classification Electron Transport Complex I NADH Dehydrogenase Intracellular Membranes General Medicine Molecular biology Mitochondria chemistry Electrophoresis Polyacrylamide Gel Rabbits NAD+ kinase |
| Zdroj: | Biochimie. 81:645-653 |
| ISSN: | 0300-9084 |
| DOI: | 10.1016/s0300-9084(99)80121-7 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect