Assay of metabolic superoxide production in Escherichia coli.
Superoxide production has been measured in subcellular fractions of SOD-deficient Escherichia coli provided with physiological reductants. Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain. At 37 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate 3 O2-./10,000 electrons transferred. This corresponds to intracellular O2-. production, in glucose-fed cells, of 5 microM/s. The high SOD content of normal cells restricts O2-. accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell. SOD-deficient mutants achieve a much higher steady-state content of O2-.. Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2-. imposes a significant oxidative stress.
Imlay
J A
JA
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Fridovich
I
I
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011190:Potassium Cyanide / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*
D009243:NAD; D013481:Superoxides; D013482:Superoxide Dismutase; D009245:NADH Dehydrogenase; D011190:Potassium Cyanide