The Km value for 6-deoxyglucose influx into Chlorella vulgaris was found to be 0.21 mM and for efflux it was 21 mM. The sugar was accumulated up to 1500-fold, an accumulation which cannot be explained by the difference in K , values.Using these Km values together with estimated relative translocation constants for carrier and carrier-substrate it was possible to determine Ks values. The Ks value thus obtained for the carrier a t the inner side of the membrane was 70 times larger than that on the outer side.Although different K , values to the ones given above for influx as well as for efflux were obtained when the measurements were carried out in the presence of high sugar concentrations at the opposite side of the membrane, the calculations yielded almost identical K s values ( Table 3).The experimentally observed accumulation of 6-deoxyglucose can be well explained when the Werent K s values and the estimated difference of 25-fold between the translocation constants for outward as compared to inward "movement" of free carrier is considered.Most transport systems for ions and nonelectrolytes of bacteria [1,2], yeasts and other fungi [3-51, plants [6,7] and animals [8,9] can be described as "steady-state" uptake systems. During the time course of uptake a constant level of substrate is reached in the cells as a result of a n efflux counteracting the influx of the substrate in question.I n organisms possessing an active transport system for a certain substrate the steady-state concentration of substrate-analogues in the cell can be considerably higher than the outside concentration. The accumulation could be brought about in three different ways [lo]: (a) the affinity of the carrier to the substrate is much higher outside than inside the cell, (b) the velocity constants for carrier-substrate flux into, or for carrier flux out of, the cells or both are larger than the corresponding constants for the fluxes in opposite directions; (c) both (a) and (b) are contributing to accumulation. Energetically these differences are brought about either in a transient way in a counterflow or overshoot situation [ill (then the accumulation also is transient) or the differences are sustained continuously by metabolic energy. I n this latter case a steady-state accumulation is observed.The accumulation factor can be expressed by kinetic parameters (see Results). So far it has been tried only for the 8-galactoside transport of Escherichia coli to test such a relationship experimentally. I n this case it had been assumed that the first alternative (a) mentioned above was the only reason for accumulation [12]. Although there was a fair agreement between the accumulation factor and the ratio of the Km values for 0-nitrophenyl-p-galactoside, there was only a poor one for lactose.In the following paper this question has been studied with the inducible, active hexose transport of Chlorella vulgaris [7,13]. For this system it has been postulated that translocational steps are affected by energy corresponding to the second case (b) above ...
Of nine species of unicellular algae tested, Chlorella vulgaris showed the highest inducibility for an active hexose transport system. Whereas the rate of uptake in all other species was increased by induction less than 5-fold, it was increased more than 400-fold in one strain of C. vulgaris. With glucose as inducer, the minimum time necessary to synthesize inducible proteins of the transport system was 15 minutes. The Km for induction with glucose is 5 AM and with 6-deoxyglucose 1 mM.The inducing sugars have to penetrate the cells to be effective.Evidence indicating that regulation of induction occurs at the transcriptional level was obtained. The induction was inhibited by 6-methylpurine. When cells were exposed to induce in the presence of actidione no increase in transport activity could be measured. After removal of actidione as well as the inducer, an increased uptake activity was observed after 30 to 60 minutes. The induced uptake system showed a turnover with a half-life of 4 to 6 hours at 26 C under nongrowing conditions; at 0 C turnover was negligible. Turnover was partly inhibited by anaerobic condition and by actidione; it was accelerated under growing conditions. Chlorella vulgaris possesses an inducible active hexose transport system (21,23). The induction occurs in the presence of glucose or can be brought about by glucose analogues such as 3-OMG2 or 6-dG, which are not metabolized (13, 21). The induction is energy-dependent and probably involves the synthesis of one or several proteins (23).In this paper the occurrence of the inducible hexose transport system among unicellular algae and the characteristics of induction and turnover in C. vulgaris have been examined in greater detail. It has been found that the total process of induction takes about 15 min and that the induced activity shows a turnover with a half-life of 4 to 6 hr. Evidence for an induction from within the cells and for a regulation at the transcriptional level has been obtained. MATERIALS AND METHODSThe strain of Chlorella vulgaris used and the conditions of autotrophic culture were the same as previously described (23 Adaptation and Uptake Experiments. To induce sugar uptake, approximately 300 al of autotrophically grown packed cells were incubated in 10 ml of 32 mm sodium phosphate buffer, pH 6.5, with 7.8 mm glucose present and shaken in the dark in air at 26 C in a 100-ml Erlenmeyer flask while the control was shaken without sugar. The glucose was completely consumed in about 2 to 3 hr. All strains, which showed a constitutive uptake system, were tested once more for inducibility after being starved in 32 mm sodium phosphate buffer, pH 6.5, for 24 or 48 hr. To measure sugar uptake, the algae were incubated with the sugar analogue (0.2 mm "4C-3-OMG or 3H-6-dG) in 32 mm sodium phosphate buffer, pH 6.5. The reaction mixture was shaken in air in an Erlenmeyer flask in the dark at 26 C; where not otherwise indicated samples were withdrawn at 1-min intervals for 4 min and filtered rapidly through membrane filters. The filt...
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