Galactose Transport in Escherichia coli THE MECHANIS1\1 UKDERLYING THE RETENTION OF INTRACELLULAR GALACTOSE* BORIS ROTMAN AND JASNA RADOJKOVIC

The intracellular concentration of nonmetabolixable compounds in bacteria can be depicted as the result of a difference between the rate of entry and the rate of exit (l-3). The entry mechanism, originally envisioned as the only active process in the system, has been termed “permease” because of its enzymic behavior (1). The exit mechanism, previously regarded as passive diffusion, is now also considered as an active process (3-5). Met,abolic inhibitors, such as azide or 2,4-dinitrophenol, prevent the intracellular accumulation of nonmetabolizable substrates (1). The mechanism of action of these inhibitors has been the subject of several studies. Two alternative hypotheses have been proposed: (a) the exit mechanism is the main target of the inhibitors (6), and (b) the entry mechanism is blocked by the inhibitors (7). Current experiment,al evidence favors the former theory (4, 8). In connection with these studies on the effect of 2,4-dinitrophenol, a retention mechanism functionally distinct from the entry process has been proposed by Osborn, McLellan, and Horecker (8) in order to explain the fact that intracellular galactose in a galactokinaseless mut,ant is not removed from the cells by washing or prolonged incubation in a galactose-free medium, but can be released by adding 2,4-dinitrophenol or by increasing the extracellular concentration of galactose. It is the purpose of this investigation to present evidence indicating that the postulated retention mechanism is in fact a transport system which accumulat,es galactose with high efficiency. Osborn et al. (8) ruled out this mechanism because they found an apparent K, of 3 X 1OV M for galactose accumulation, whereas the external concentration of galactose in the culture, after resuspension in galactose-free medium, was estimated at 6.9 x lo-’ M. They concluded that, under these conditions, the uptake of galactose would be reduced to about 10% of the maximum. However, these conclusions involve the implicit assumption that the K, value is the same at lower concentrations of galactose. We tested this assumption by measuring the K, with galactose of high specific radioactivity and found a different value. The K, at lower concentrations of substrate is 10 times lower than that obtained with higher galactose concentrations. Thus, there is no discrepancy between the mechanism proposed here and previous results. Another corollary from our esperiments is that the galactose