Immobilized respiratory chain activities from Escherichia coli utilized to measure D- and L-lactate, succinate, L-malate, 3-glycerophosphate, pyruvate, or NAD(P)H.
The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.
D004566:Electrodes; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005994:Glycerophosphates / Q000032:analysis*; D007773:Lactates / Q000032:analysis*; D008293:Malates / Q000032:analysis*; D009097:Multienzyme Complexes / Q000378:metabolism; D009243:NAD / Q000032:analysis*; D009249:NADP / Q000032:analysis*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011773:Pyruvates / Q000032:analysis*; D013386:Succinates / Q000032:analysis*
D004800:Enzymes, Immobilized; D005994:Glycerophosphates; D007773:Lactates; D008293:Malates; D009097:Multienzyme Complexes; D011773:Pyruvates; D013386:Succinates; D009243:NAD; D009249:NADP; C029620:alpha-glycerophosphoric acid