Comparison of the thylakoidal NAD(P)H dehydrogenase complex and the mitochondrial complex I separated from barley leaves by blue-native PAGE

Abstract

Plastids contain an 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). However, until now, the two complexes have not been simultaneously studied in the same plant species. In this work we initiate a comparative study between both complexes from Hordeum vulgare L. The mitochondrial complex I and the stroma thylakoid NAD(P)H DH complex from barley, when separated by blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by NADH nitroblue tetrazolium staining, showed different sizes and electrophoretic mobilities. Complex I had an apparent molecular mass of 740 kDa and the thylakoidal NAD(P)H DH complex a mass of 690 kDa. The two complexes were identified by immunoblotting assays, since antibodies raised against the NdhA, NdhH and NdhK thylakoidal polypeptides recognized three polypeptides of the barley thylakoidal complex, and antibodies raised against the B12 and B15 bovine complex I subunits reacted with two polypeptides of the barley mitochondrial complex I. Additional immunoblotting revealed that the thylakoidal NAD(P)H DH complex contains the three polypeptides (24, 56, 76 kDa) from the NADH-oxidizing unit of the complex, similar in size and antigenicity to the mitochondrial subunits. Both complexes have at least one more homologous subunit (29 kDa) of the complex minimal structure, which was recognized by the anti-TYKY antibody.