Separation by blue-native PAGE and identification of the whole NAD(P)H dehydrogenase complex from barley stroma thylakoids

Chloroplasts contain a NAD(P)H dehydrogenase (NAD(P)H DH) complex preferentially located in the stroma thylakoids, which is homologous to the mitochondrial complex I (EC 1.6.5.3). Until now, the separation of this complex by native PAGE has led to its dissociation into several enzymatic activity bands. In this work, we have separated by blue-native PAGE (BN-PAGE) the NAD(P)H DH complex from stroma thylakoids, obtained by sonication of barley (Hordeum vulgare L.), revealing only one band with NAD(P)H-nitroblue tetrazolium oxidoreductase activity and with a molecular mass higher than that of photosystem I. Immunoblotting revealed the presence in the complex of the polypeptide encoded by ndhA chloroplast gene (NdhA), the polypeptide encoded by ndhH chloroplast gene (NdhH), and the 56-kDa NADH-binding polypeptide, which are subunits of the membrane hydrophobic subcomplex, the connecting fragment, and the peripheral subcomplex, respectively, these three subcomplexes being those already reported for the plastidial complex. Additional immunoblotting revealed the association of the complex with ferredoxin-NADP oxidoreductase enzyme (EC 1.18.1.2). The complex electroeluted from the activity band represents 0.9 % of stroma thylakoid protein.