Isolation of an NADH dehydrogenase complex not associated to ferredoxin-NADP oxidoreductase from oat stroma thylakoids

Abstract

Summary With the aim of isolating for further characterisation the chloroplast NADH dehydrogenase (NADH DH) complex of low abundance, we investigated a plant species of comparatively high complex activity. The chloroplast NAD(P)H-ferricyanide oxidoreductase (NAD(P)H-FeCNR) activities of barley ( Hordeum vulgare L.), wheat ( Triticum aestivum L.), oat ( Avena sativa L.) and pea ( Pisum sativum L.) were studied. Oat chloroplasts have approximately 50 percnt; higher NADH-FeCNR activity than barley and pea chloroplasts, and 70 percnt; higher than wheat chloroplasts, and the highest NADH-FeCNR/NADPH-FeCNR activity ratio. The NADH DH complex isolated from the stroma thylakoids of oat is NADH-specific and its NADH-FeCNR specific activity is double than that found in the barley chloro plast complex. Immunoblotting revealed the presence of the NdhA (35 kDa), NdhH (50 kDa), NdhK (29 kDa) and NADH-binding (56 kDa) polypeptides, which are four subunits of the three subcomplexes into which the plastidial complex can be fractionated. The oat complex has a molecular mass of 587 kDa and is composed of at least 15 different polypeptides with molecular masses ranging from 12 to 80 kDa. Additional immunoblotting revealed that the isolated complex is not associated with ferredoxin-NADP oxidoreductase enzyme (EC 1.18.1.2). The results presented here might be the starting point for further purification and characterisation of the chloroplast NADH DH complex.