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. 1974 Mar;117(3):1043–1054. doi: 10.1128/jb.117.3.1043-1054.1974

Nature of the Specificity of Alcohol Coupling to l-Alanine Transport into Isolated Membrane Vesicles of a Marine Pseudomonad

G Dennis Sprott a,1, Robert A MacLeod b
PMCID: PMC246583  PMID: 4360536

Abstract

Ethanol stimulated the uptake of l-alanine into isolated membrane vesicles of a marine pseudomonad at a rate and to an extent comparable with that obtained with reduced nicotinamide adenine dinucleotide (NADH) or the artificial electron donor ascorbate-N, N, N′, N′-tetramethyl-p-phenylenediamine (ascorbate-TMPD). Methanol and branched-chain alcohols had little or no capacity to energize transport. No quantitative relationship was found between the ability of a compound to induce oxygen uptake and to energize transport, since with ethanol initial rates of oxygen uptake were approximately 4% of that obtained with NADH or ascorbate-TMPD. Cytochrome analysis revealed that NADH and ethanol reduced cytochromes b and c, whereas ascorbate-TMPD coupled primarily at the level of cytochrome c. Approximately 25% of the cytochromes reduced by dithionite were reducible by ethanol. Ethanol reduction of both cytochromes b and c was prevented by 2-heptyl-4-hydroxyquinoline-N-oxide, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. The ethanol- and NADH-energized transport systems for l-alanine were subject to quantitatively similar inhibition by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, 2, 4-dinitrophenol, and the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetate. In contrast, for ascorbate-TMPD-driven transport, only cyanide and 2, 4-dinitrophenol remained fully effective as inhibitors, p-chloromercuribenzoate was only half as effective, and the other compounds stimulated transport. Inhibition of ethanol oxidation strikingly paralleled the inhibition of ethanol-driven transport for each of the inhibitors, including 2, 4-dinitrophenol. Marked differences between inhibition of oxygen uptake and inhibition of transport were observed when NADH or ascorbate-TMPD were the electron donors. The data indicate that only a small proportion of the respiratory chain complexes in the membrane vesicles are involved in transport and these are efficiently coupled to ethanol oxidation. The results also suggest that when 2, 4-dinitrophenol inhibits transport it is not acting as an uncoupling agent.

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Selected References

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  1. Barnes E. M., Jr, Kaback H. R. Beta-galactoside transport in bacterial membrane preparations: energy coupling via membrane-bounded D-lactic dehydrogenase. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1190–1198. doi: 10.1073/pnas.66.4.1190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnes E. M., Jr, Kaback H. R. Mechanisms of active transport in isolated membrane vesicles. I. The site of energy coupling between D-lactic dehydrogenase and beta-galactoside transport in Escherichia coli membrane vesicles. J Biol Chem. 1971 Sep 10;246(17):5518–5522. [PubMed] [Google Scholar]
  3. Barnes E. M., Jr Respiration-coupled glucose transport in membrane vesicles from Azotobacter vinelandii. Arch Biochem Biophys. 1972 Oct;152(2):795–799. doi: 10.1016/0003-9861(72)90275-5. [DOI] [PubMed] [Google Scholar]
  4. Bhattacharyya P., Epstein W., Silver S. Valinomycin-induced uptake of potassium in membrane vesicles from Escherichia coli. Proc Natl Acad Sci U S A. 1971 Jul;68(7):1488–1492. doi: 10.1073/pnas.68.7.1488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. CASTOR L. N., CHANCE B. Photochemical determinations of the oxidases of bacteria. J Biol Chem. 1959 Jun;234(6):1587–1592. [PubMed] [Google Scholar]
  6. CHANCE B. The carbon monoxide compounds of the cytochrome oxidases. I. Difference spectra. J Biol Chem. 1953 May;202(1):383–396. [PubMed] [Google Scholar]
  7. CHANCE B., WILLIAMS G. R. Respiratory enzymes in oxidative phosphorylation. II. Difference spectra. J Biol Chem. 1955 Nov;217(1):395–407. [PubMed] [Google Scholar]
  8. Forsberg C. W., Costerton J. W., Macleod R. A. Separation and localization of cell wall layers of a gram-negative bacterium. J Bacteriol. 1970 Dec;104(3):1338–1353. doi: 10.1128/jb.104.3.1338-1353.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gow J. A., DeVoe U. W., MacLeod R. A. Dissociation in a marine pseudomonad. Can J Microbiol. 1973 Jun;19(6):695–701. doi: 10.1139/m73-113. [DOI] [PubMed] [Google Scholar]
  10. Harold F. M. Conservation and transformation of energy by bacterial membranes. Bacteriol Rev. 1972 Jun;36(2):172–230. doi: 10.1128/br.36.2.172-230.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirata H., Asano A., Brodie A. F. Respiration dependent transport of proline by electron transport particles from mycobacterium phlei. Biochem Biophys Res Commun. 1971 Jul 16;44(2):368–374. doi: 10.1016/0006-291x(71)90609-7. [DOI] [PubMed] [Google Scholar]
  12. Hong J. S., Kaback H. R. Mutants of Salmonella typhimurium and Escherichia coli pleiotropically defective in active transport. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3336–3340. doi: 10.1073/pnas.69.11.3336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hunter G. R., Brierley G. P. Ion transport by heart mitochondria. XIV. The mannitol-impermeable compartment of the mitochondrion and its relation to ion uptake. Biochim Biophys Acta. 1969 May;180(1):68–80. doi: 10.1016/0005-2728(69)90195-9. [DOI] [PubMed] [Google Scholar]
  14. Jones K., Heathcote J. G. The rapid resolution of naturally occurring amino acids by thin-layer chromatography. J Chromatogr. 1966 Sep;24(1):106–111. doi: 10.1016/s0021-9673(01)98107-5. [DOI] [PubMed] [Google Scholar]
  15. Kaback H. R., Barnes E. M., Jr Mechanisms of active transport in isolated membrane vesicles. II. The mechanism of energy coupling between D-lactic dehydrogenase and beta-galactoside transport in membrane preparations from Escherichia coli. J Biol Chem. 1971 Sep 10;246(17):5523–5531. [PubMed] [Google Scholar]
  16. Kaback H. R., Milner L. S. Relationship of a membrane-bound D-(-)-lactic dehydrogenase to amino acid transport in isolated bacterial membrane preparations. Proc Natl Acad Sci U S A. 1970 Jul;66(3):1008–1015. doi: 10.1073/pnas.66.3.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klein W. L., Boyer P. D. Energization of active transport by Escherichia coli. J Biol Chem. 1972 Nov 25;247(22):7257–7265. [PubMed] [Google Scholar]
  18. Konings W. N., Barnes E. M., Jr, Kaback H. R. Mechanisms of active transport in isolated membrane vesicles. 2. The coupling of reduced phenazine methosulfate to the concentrative uptake of beta-galactosides and amino acids. J Biol Chem. 1971 Oct 10;246(19):5857–5861. [PubMed] [Google Scholar]
  19. Konings W. N., Freese E. Amino acid transport in membrane vesicles of Bacillus subtilis. J Biol Chem. 1972 Apr 25;247(8):2408–2418. [PubMed] [Google Scholar]
  20. LOVELOCK J. E., BISHOP M. W. Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature. 1959 May 16;183(4672):1394–1395. doi: 10.1038/1831394a0. [DOI] [PubMed] [Google Scholar]
  21. MacLeod R. A., Thurman P., Rogers H. J. Comparative transport activity of intact cells, membrane vesicles, and mesosomes of Bacillus licheniformis. J Bacteriol. 1973 Jan;113(1):329–340. doi: 10.1128/jb.113.1.329-340.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Martin E. L., MacLeod R. A. Isolation and chemical composition of the cytoplasmic membrane of a gram-negative bacterium. J Bacteriol. 1971 Mar;105(3):1160–1167. doi: 10.1128/jb.105.3.1160-1167.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol Rev Camb Philos Soc. 1966 Aug;41(3):445–502. doi: 10.1111/j.1469-185x.1966.tb01501.x. [DOI] [PubMed] [Google Scholar]
  24. Short S. A., White D. C., Kaback H. R. Active transport in isolated bacterial membrane vesicles. V. The transport of amino acids by membrane vesicles prepared from Staphylococcus aureus. J Biol Chem. 1972 Jan 10;247(1):298–304. [PubMed] [Google Scholar]
  25. Sprott G. D., MacLeod R. A. Na + -dependent amino acid transport in isolated membrane vesicles of a marine pseudomonad energized by electron donors. Biochem Biophys Res Commun. 1972 May 26;47(4):838–845. doi: 10.1016/0006-291x(72)90569-4. [DOI] [PubMed] [Google Scholar]
  26. Thompson J., Costerton J. W., MacLeon R. A. K plus-dependent deplasmolysis of a marine pseudomonad plasmolyzed in a hypotonic solution. J Bacteriol. 1970 Jun;102(3):843–854. doi: 10.1128/jb.102.3.843-854.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Thompson J., MacLeod R. A. Functions of Na+ and K+ in the active transport of -aminoisobutyric acid in a marine pseudomonad. J Biol Chem. 1971 Jun 25;246(12):4066–4074. [PubMed] [Google Scholar]
  28. Thompson J., MacLeod R. A. Specific electron donor-energized transport of alpha-aminoisobutyric acid and K+ into intact cells of a marine pseudomonad. J Bacteriol. 1974 Mar;117(3):1055–1064. doi: 10.1128/jb.117.3.1055-1064.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wong P. T., Thompson J., MacLeod R. A. Nutrition and metabolism of marine bacteria. XVII. Ion-dependent retention of alpha-aminoisobutyric acid and its relation to Na+ dependent transport in a marine pseudomonad. J Biol Chem. 1969 Feb 10;244(3):1016–1025. [PubMed] [Google Scholar]

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