Stopped-flow spectrophotometric assay of glycerol permeation in Escherichia coli: applicability and limitations.
The passive permeation and facilitated diffusion of glycerol in various strains of Escherichia coli have been studied by stopped-flow spectrophotometry. Contrary to the prediction for glycerol entry by simple diffusion, the reciprocal relaxation time (1/tau, s-1) for the passive permeation of glycerol in cells grown in the presence of glucose was not constant but decreased as the glycerol concentration increased above 100 mM. This anomaly was not due to refractive index differences or to the presence of residual levels of the glycerol facilitator protein in non-induced cells. Although reciprocal relaxation times for glycerol-induced E. coli exhibited the expected elevation relative to non-induced cells, a similar anomalous decrease 1/tar (s-1) with increasing glycerol concentration was observed. In addition, at early times after suspension in dilute buffer, the 1/tau (s-1) values obtained for induced or non-induced E. coli swelling in glycerol were considerably greater than for organisms incubated in dilute buffer for longer times. We concluded that either this spectrophotometric technique was not monitoring solely the permeation of glycerol into E. coli, or concentrations of glycerol above 100 mM significantly perturbed the structure of the E. coli cell envelope.
D002452:Cell Count; D002463:Cell Membrane Permeability; D004926:Escherichia coli / Q000166:cytology / Q000502:physiology*; D005990:Glycerol; D007700:Kinetics; D055095:Optics and Photonics; D013053:Spectrophotometry
D005990:Glycerol