Open AccessImmunochemical identification of a two‐subunit NADH‐ubiquinone oxidoreductase from Paracoccus denitrificans
Author(s) -
GEORGE Christina L.,
FERGUSON Stuart J.
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb08407.x
Subject(s) - paracoccus denitrificans , nadh dehydrogenase , biochemistry , oxidoreductase , respiratory chain , polyacrylamide gel electrophoresis , chemistry , protein subunit , gel electrophoresis , size exclusion chromatography , dehydrogenase , flavoprotein , biology , enzyme , chromatography , microbiology and biotechnology , gene
Analysis by crossed‐immunoelectrophoresis of Paracoccus denitrificans membrane vesicles has shown that only one antigen stains for NADH dehydrogenase activity. This activity could be partially purified by a combination of gel filtration and ion‐exchange chromatography of membrane vesicles that had been solubilised in the non‐ionic detergent Nonidet P‐40. From the limited number of precipitates observed after crossed immunoelectrophoresis of this partially purified preparation of NADH dehydrogenase it was possible to excise specifically part of the precipitate that stained for NADH dehydrogenase. Excised precipitates containing NADH dehydrogenase that had been radiolabelled by growth of cells in the presence of [ 35 S]SO 4 2‐ allowed the polypeptide composition of the enzyme to be determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by fluorography. Two subunits were identified with estimated relative molecular masses of 48000 and 25000. Subunits of similar molecular weight are found in the flavoprotein fragment of the NADH dehydrogenase of the mammalian mitochondrial respiratory chain. The latter has general similarities with the respiratory chain in the plasma membrane of P. denitrificans.