Study of a time lag in the assay of Escherichia coli membrane-bound dehydrogenases based on tetrazolium salt reduction.
The Escherichia coli membrane-bound D-lactate dehydrogenase and succinate dehydrogenase were assayed on the basis of the phenazine methosulfate- (PMS-) mediated reduction of the tetrazolium salt, MTT. An initial slower phase (lag) in the time-course of the reaction was observed and analyzed. The results were as follows. (1) The time lag in the assay of the D-lactate dehydrogenase was eliminated by preincubating the membranes with PMS plus D-lactate, with PMS plus succinate, or with PMS plus NADH (conditions which implicated PMS reduction). (2) When the D-lactate dehydrogenase was assayed by another method based on the measurement of the pyruvate formed, neither was a time lag observed nor was the enzyme activity affected by membrane preincubation with PMS plus D-lactate. (3) Although the superoxide radical was involved in MTT reduction, this radical seemed not to participate in the generation of the time lag. (4) Membranes whose D-lactate dehydrogenase activity had previously been destroyed by heating at 80 degrees C for 1 min, were able to prolong the time lag in MTT reduction when added to the assay medium for the D-lactate dehydrogenase from untreated membranes, whereas membranes previously heated at 100 degrees C instead of 80 degrees C did not have this effect. It was concluded that the E. coli membranes interfered in the dehydrogenase assay based on the PMS-mediated reduction of MTT. The time lag was interpreted as a period during which the interfering substance reacted with reduced PMS inhibiting the reduction of MTT.
D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology; D013385:Succinate Dehydrogenase / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism*; D013844:Thiazoles / Q000378:metabolism*
D013778:Tetrazolium Salts; D013844:Thiazoles; D007770:L-Lactate Dehydrogenase; D013482:Superoxide Dismutase; D013385:Succinate Dehydrogenase; C022616:thiazolyl blue