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The Role of the Phosphoenolpyruvate-phosphotransferase System in the Transport of Sugars by Isolated Membrane Preparations of Escherichia coli

H. R. Kaback 1

From the 1 From the Laboratories of Biochemistry, National Heart Institute, National Institutes of Health, Bethesda Maryland 20014

The phosphoenolpyruvate-phosphotransferase system provides a mechanism for the passage of certain sugars through the bacterial cell membrane and their accumulation as phosphorylated derivatives. These conclusions are based on the following observations.

1. Isolated bacterial membrane preparations specifically require P-enolpyruvate for uptake of certain sugars which accumulate almost completely as phosphorylated derivatives.

2. Membranes prepared from Escherichia coli GN-2, a mutant lacking Enzyme I of the phosphotransferase system, are unable to take up significant quantities of agr-methylglucoside.

3. 3H-Glucose added to the incubation medium is phosphorylated more rapidly than free 14C-glucose in the intramembranal pool, suggesting that the P-transferase system is a mechanism by which sugars penetrate the membrane.

4. There is a stoichiometric relation between 32P loss from P-enolpyruvate and appearance of 32P in agr-methylglucoside-P.

5. The uptake and phosphorylation of agr-methylglucoside exhibit saturation kinetics with an apparent Km of about 4 x 10-6 m, whereas the appearance of free agr-methylglucoside in the intramembranal pool shows no saturation. The initial rate of uptake of free agr-methylglucoside is independent of the presence of P-enolpyruvate.

6. Under steady state conditions, intramembranal free agr-methylglucoside and the external pool do not equilibrate regardless of the presence of P-enolpyruvate. Furthermore, agr-methylglucoside in the external pool does not exchange with the agr-methylglucoside previously taken up by the membranes.

Membranes prepared from glucose-grown cells take up and phosphorylate agr- and ß-methylglucoside, glucose, 2-deoxyglucose, fructose, galactose, and 3-O-methylglucose in the presence of P-enolpyruvate. Ribose, arabinose, mannitol and sorbitol are not taken up or phosphorylated.

Submitted on January 31, 1968


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