Multiple Sites for Coupling of Glucose Transport to the Restkatorv Chain of Membrane Vesicles from Am~obacttk- vinelandii*

Glucose transport by membrane vesicles isolated from Azotobacter uinelandii is coupled primarily to malate oxidation via a flavin-linked L-malate dehydrogenase. The addition of flavin adenine dinucleotide is required for malatedependent glucose transport but is not obligatory for malate oxidation. Both NADH and NADPH are oxidized by the vesicles at rates nearly identical with that of malate yet are only 17 % as effective in support of the rate of glucose uptake. Succinate and D-lactate are oxidized at 55 and 20% of the malate rate, respectively. Succinate oxidation does not stimulate glucose transport while D-lactate is 18% as effective as L-malate. However, diEerence spectra reveal that each of these electron donors is able to reduce almost quantitatively all of the cytochrome components of the membrane preparations. These findings indicate that malate dehydrogenase is coupled to glucose transport at a low potential site which is proximal to ubiquinone. Ascorbate-reduced tetramethylphenylenediamine is oxidized more rapidly by the vesicles than malate and is about as effective as malate in support of the glucose transport rate. This artificial reductant is oxidized via a branch of the respiratory system containing cytochrome c that is blocked by 2 PM cyanide but not by 2-heptyl-4-hydroxyquinoline-N-oxide. Glucose transport that is dependent on this artificial system is inhibited in a similar fashion. However, malate-dependent transport is blocked by the quinoline compound but not by 2 pM cyanide. This reveals a second site of higher potential in the respiratory chain that is linked to glucose transport.