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Assays carried out over a range of substrate concentrations showed that transport by Ap_ST3 could be saturated, giving hyperbolic curves of rate against glucose or fructose concentrations (Fig.?6A, C). Analysis by Michaelis-Menten kinetics gave estimates for Km of 66?��?12?mM http://www.selleck.cn/products/LY294002.html for glucose and 47?��?6?mM for fructose. Varying the extracellular pH from 5.5 to 8 led to only a small increase in glucose uptake (approx. 10%) by cells transformed with pDR195-Ap_ST3 (Fig.?7A), suggesting that transport was not dependent on a pH gradient across the yeast plasma membrane. In confirmation, pre-treating cells with the proton gradient uncouplers CCCP and DNP decreased transport by http://www.selleckchem.com/products/obeticholic-acid.html respectively, in good agreement with the theoretical values calculated from the determined Km values (due to the increase in transport at higher sugar concentration being offset by the lower specific activity). Under the conditions used, fructose inhibited glucose transport to a greater extent than any other sugar tested, decreasing measured glucose uptake by 64?��?2%, while mannose gave a decrease of 30?��?3%. Trehalose and galactose did not decrease ( http://www.selleckchem.com/products/Adriamycin.html only glucose (28?��?0.3% decrease), xylose (27?��?0.4% decrease), mannose (17?��?1% decrease), sucrose (14?��?2% decrease) and myo-inositol (11?��?2% decrease) giving >10% decrease in fructose transport (Fig.?6D). Other sugars tested (arabinose, trehalose, mannitol, galactose and sorbitol) all gave