Abstract
A method is described for preparation of membrane vesicles (diameter 80nm) capable of respiration-linked ATP synthesis. Vesicles prepared from succinate-grown bacteria oxidized NADH, succinate and ascorbate plus NNN′N′-tetramethylphenylenediamine; vesicles prepared from methanol-grown bacteria also oxidized methanol and formaldehyde, but they were otherwise identical. The uncoupling agent carbonyl cyanide chlorophenylhydrazone and the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide both inhibited ATP synthesis, whereas they had no effect on the rate of respiration. Rotenone inhibited ATP synthesis and respiration with NADH as substrate; antimycin A inhibited with succinate as substrate, and cyanide inhibited with all substrates. P/O ratios were usually 0.7–1.3 with NADH, 0.6–1.0 with succinate and 0.2–0.6 with reduced NNN′N′-tetramethylphenylenediamine or methanol as respiratory substrate. When 2,6-dichlorophenol-indophenol was used as an alternative electron acceptor to O2 (NADH as donor) the P/2e ratio was 1.65. Although these P/O ratios are minimum values, because they do not take into account unknown amounts of uncoupled O2 consumption, they are consistent with previous proposals [O'Keeffe & Anthony (1978) Biochem, J. 170, 561–567] based on measurements of proton translocation in whole cells. The results also confirm that methanol dehydrogenase and cytochromes c and a/a3 are arranged so that the first step in methanol oxidation is coupled to synthesis of ATP.
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Selected References
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