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Eur J Biochem. 1976 Aug 16;67(2):357-65.

Reconstitution of reduced nicotinamide adenine dinucleotide oxidase activity with menadione in membrane vesicles from the menaquinone-deficient Bacillus subtilis aro D. Relation between electron transfer and active transport.


Membrane vesicles from the menaquinone-deficient Bacillus subtilis aro D contain a low content of menaquinone and consequently oxidaze reduced nicotinamide adenine dinucleotide (NADH) at low rate. Supplementation of the membrane vesicles suspension with the menaquinone-analogue menadione, results in an incorporation of menadione in the membranes. The incorporated menadione increases with the external menadione concentration up to a maximum of 7 nmol of menadione bound per mg membrane protein. The NADH oxidase activity of the membrane vesicles increases linearly with the menadione content and a 35-fold stimulation is obtained in fully reconstituted membrane vesicles; this maximal NADH oxidase activity is about two-fold higher than the NADH oxidase activity in membrane vesicles from wild-type B.subtilis W23. Supplementation of membrane vesicles from B.subtilis W23 with menadione also results in a stimulation of the NADH oxidase activity but only a stimulation of 1.6-fold is maximally obtained. The NADH oxidase activities in reconstituted B.subtilis aro D and B.subtilis W23 membrane vesicles are similarly affected by respiratory chain inhibitors, indicating that menadione occupies physiological sites of menaquinone. NADH and the non-physiological electron donor ascorbate + phenazine methosulphate are the best energy sources for active amino acid transport in membrane vesicles from B.subtilis W23. Membrane vesicles from B.subtilis aro D accumulate amino acids in the presence of acorbate + phenazine methosulphate, but not with NADH. However, membrane vesicles from this mutant, reconstituted with menadione, demonstrate NADH-driven transport activity. This activity increases linearly with the NADH oxidase activity, but maximal transprt activities are reached under conditions where the NADH oxidase activity is not yet maximal. These results indicate that the rate of energy supply is the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor for transport at low NADH oxidase activities and that the transport system itself becomes the limiting factor under conditions of high NADH oxidase activities. Under energy-limiting conditions 135-235 molecules of NADH have to be oxidized in order to transport one molecule of amino acid. At all levels of energy supply a competition by the different amino acid transport systems for the available energy could not be observed. These observations indicate that only a fraction of the energy, generated by the respiratory chain, is used for the transport of an amino acid and that the bulk of the energy dissipates via other channels in the membrane vesicles.

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