Purification and properties of the H+-nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus

Timothy M Lever, Tracy Palmer, Ian J. Cunningham, Nicholas P. J. Cotton, J. Baz Jackson

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    Abstract

    1. H+-transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water-soluble component for activity.

    2. The enzyme from Rb. capsulatus was solubilised in Triton X-100 and subjected to ion-exchange, hydroxyapatite and then gel-exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent M(r) 53 000 and 48 000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants.

    3. Antibodies raised to the 53-kDa and 48-kDa polypeptides cross-reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York] that the soluble component associated with H+-transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48-kDa and 53-kDa polypeptides of the Rb. capsulatus enzyme cross-reacted with equivalent polypeptides in solubilised membranes of Escherichia coli.

    4. The dependence of the rate of H- transfer by purified H+-transhydrogenase on the nucleotide substrate concentrations under steady-state conditions, the effects of inhibition by nucleotide products and the inhibition by 2'-AMP and by 5-'AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex.

    5. In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD+) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279-324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H+-transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive.

    Original languageEnglish
    Pages (from-to)247-255
    Number of pages9
    JournalEuropean Journal of Biochemistry
    Volume197
    Issue number1
    DOIs
    Publication statusPublished - 10 Apr 1991

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