The indirect nature of interaction of glucose transport with the system transporting galactosides

The membrane transport systems for galactosides and glucose derivatives interact in enteric microorganisms. Stop-flow experiments with a double wavelength spectrophotometer and a flow-through cuvette (designed to minimize light-scattering effects) were used to measure the speed of interaction in Escherichia coli. The in vivo hydrolysis of ortho-nitrophenol-beta-D-galactopyranoside was measured by comparing the light transmitted by cell suspensions at 420 nm with that at 500 nm. Measurements at the latter wavelength corrected for residual scattering effects. The stop-flow experiment allowed the study of the early kinetics of transport and hydrolysis. It was found with strain ML308 that there was a significant lag in the achievement of steady-state inhibition by glucose and its derivative methyl-alpha-D-glucopyranoside (alpha MG). This strain constitutively produces high levels of permease and beta-galactosidase. The absorbancy increases at 420 nm are limited by transport because the beta-galactosidase is present inside the cells in excess. From earlier results, it was not surprising that inhibition is delayed with low concentrations of the glucose compounds, but the new double wavelength technique showed no kinetic component of rapid inhibition. This result therefore excludes competition for some membrane-bound component and is consistent with the production of the dephosphorylated form of the soluble Enzyme IIIglu that binds and inhibits the permease system in the membrane.