The relation between the soluble factor associated with H +-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli

Abstract

Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H +-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Th 2) and a membrane-bound component are together required for activity. Th s was selectively removed from chromatophore membranes of Rhs. rubrum and was purified to homogeneity by precipitation with (NH 4) 2SO 4 and ion-exchange, affinity dye and gel exclusion chromatography. The latter indicated an M r of approx. 74 000 under non-denaturing conditions but analysis of the pure proteins by SDS PAGE revealed a single polypeptide, M r 43 000. Antibodies against this polypeptide inhibited transhydrogenase activity of chromatographores and decreased the capacity of Th s to restore activity to depleted membranes. They reacted with a polypeptide of M r 43 000 in crude cell extract, chromatophore membranes and chromatophore washing but not with transhydrogenase polypeptides from the membranes of E. coli, Rb. capsulatus or animal mitochondria. The N-terminal amino acid sequence of the 43 000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the α subunit of the E. coli protein. The break between the α and β polypeptides of E. coli transhydrogenase is such that both components are membrane associated. In contrast, these results suggest that in the Rhs. rubrum enzyme Th s has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble. There is a predicted similarity between Th s and the reported sequence of alanine dehydrogenase from Bacillus but Th s did not have any alanine dehydrogenase activity.