Nature of the specificity of alcohol coupling to L-alanine transport into isolated membrane vesicles of a marine pseudomonad.
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.
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000409:Alanine / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000438:Alcohols / Q000378:metabolism*; D001205:Ascorbic Acid / Q000378:metabolism; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism*; D003486:Cyanides / Q000494:pharmacology; D003580:Cytochromes / Q000378:metabolism; D004140:Dinitrophenols / Q000494:pharmacology; D004579:Electron Transport; D000431:Ethanol / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010655:Phenylenediamines / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology / Q000378:metabolism*; D012623:Seawater; D013237:Stereoisomerism; D014316:Tritium; D014871:Water Microbiology
D000438:Alcohols; D002250:Carbon Radioisotopes; D003486:Cyanides; D003580:Cytochromes; D004140:Dinitrophenols; D010655:Phenylenediamines; D009243:NAD; D014316:Tritium; D000431:Ethanol; D000255:Adenosine Triphosphate; D000429:Alcohol Oxidoreductases; D000409:Alanine; D001205:Ascorbic Acid; D011188:Potassium