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Volume 271, Number 25, Issue of June 21, 1996 pp. 14819-14824
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Purification by Ni²⁺ Affinity Chromatography, and Functional Reconstitution of the Transporter for N-Acetylglucosamine of Escherichia coli

(Received for publication, January 31, 1996)

From the Institute of Biochemistry, University of Bern, CH-3012 Bern, Switzerland

The N-acetyl-D-glucosamine transporter (II^GlcNAc) of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported GlcNAc. II^GlcNAc of Escherichia coli containing a carboxyl-terminal affinity tag of six histidines was purified by Ni²⁺ chelate affinity chromatography. 4 mg of purified protein was obtained from 10 g (wet weight) of cells. Purified II^GlcNAc was reconstituted into phospholipid vesicles by detergent dialysis and freeze/thaw sonication. II^GlcNAc was oriented randomly in the vesicles as inferred from protein phosphorylation studies. Import and subsequent phosphorylation of GlcNAc were measured with proteoliposomes preloaded with enzyme I, histidine-containing phosphocarrier protein, and phosphoenolpyruvate. Uptake and phosphorylation occurred in a 1:1 ratio. Active extrusion of GlcNAc entrapped in vesicles was also measured by the addition of enzyme I, histidine-containing phosphocarrier protein, and phosphoenolpyruvate to the outside of the vesicles. The K_m for vectorial phosphorylation and non-vectorial phosphorylation were 66.6 ± 8.2 µM and 750 ± 19.6 µM, respectively. Non-vectorial phosphorylation was faster than vectorial phosphorylation with k_cat 15.8 ± 0.9 s¹ and 6.2 ± 0.7 s¹, respectively. Using exactly the same conditions, the purified transporters for mannose (IIAB^Man, IIC^Man, IID^Man) and glucose (IICB^Glc, IIA^Glc) were also reconstituted for comparison. Although the vectorial transport activities of IICBA^GlcNAc and IICB^Glc· IIA^Glc are inhibited by non-vectorial phosphorylation, no such effect was observed with the IIAB^Man· IIC^Man·IID^Man complex. This suggests that the molecular mechanisms underlying solute transport and phosphorylation are different for different transporters of the phosphotransferase system.

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