Energy-linked reduction of nicotinamide--adenine dinucleotide in membranes derived from normal and various respiratory-deficient mutant strains of Escherichia coli K12.
1. Assay conditions are described for the ATP-dependent, uncoupler-sensitive, energy-linked reduction of NAD(+) by succinate, dl-alpha-glycerophosphate or d-lactate in membranes from aerobically grown Escherichia coli. 2. The reaction may be demonstrated in electron-transport particles (ET particles) from cells grown in glycerol, but not in depleted particles washed in low-ionic-strength buffer, or in ET particles from cells grown in glucose. 3. The latter two classes of particles have low specific activities of ATPase (adenosine triphosphatase), succinate dehydrogenase, dl-alpha-glycerophosphate dehydrogenase and d-lactate dehydrogenase relative to undepleted ET particles from cells grown in glycerol. 4. Reconstitution of energy-linked NAD(+) reduction in particles from cells grown in glucose was done by: (a) addition of the high-speed supernatant fraction from sonicates of the same cells; (b) addition of a protein fraction, precipitated by (NH(4))(2)SO(4) from this supernatant, or (c) addition of an (NH(4))(2)SO(4)-precipitated fraction from the low-ionic-strength wash of particles from cells grown in glycerol. 5. The use of (NH(4))(2)SO(4)-precipitated fractions from ATPase- or succinate dehydrogenase-deficient mutants grown in glycerol in the above reconstitution indicated that failure to demonstrate the reaction in particles from cells grown in glucose was a result of inadequate activities of appropriate dehydrogenases, rather than of ATPase. 6. Energy-linked NAD(+) reduction could be demonstrated in particles from a ubiquinone-deficient mutant only after restoration of NADH oxidase activity by adding ubiquinone-1. 7. The measured rate of the energy-linked reaction in particles from a haem-deficient mutant, however, was not stimulated after the ATP- and haematin-dependent acquisition of functional cytochromes. 8. Results are interpreted as evidence of the ubiquinone-dependent, but cytochrome-independent, nature of the site I region of the respiratory chain in E. coli.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002458:Cell Fractionation; D011232:Chemical Precipitation; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism*; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D008566:Membranes / Q000378:metabolism*; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013386:Succinates / Q000378:metabolism; D014451:Ubiquinone / Q000172:deficiency
D005994:Glycerophosphates; D007773:Lactates; D013386:Succinates; D009243:NAD; D014451:Ubiquinone; D005993:Glycerolphosphate Dehydrogenase; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D000251:Adenosine Triphosphatases