PURIFICATION BY NI2-CHROMATOGRAPHY, AND FUNCTIONAL RECONSTITUTION OF THE TRANSPORTER FOR N-ACETYLGLUCOSAMINE OF ESCHERICHIA-COLI( AFFINITY)

The N-acetyl-D-glucosamine transporter (IIGlcNAc) Of the bacterial pho sphotransferase system couples vectorial translocation to phosphorylat ion of the transported GlcNAc, IIGlcNAc of Escherichia coli containing a carboxyl-terminal affinity tag of six histidines was purified by Ni 2+ chelate affinity chromatography. 4 mg of purified protein was obtai ned from 10 g (wet weight) of cells. Purified IIGlcNAc was reconstitut ed into phospholipid vesicles by detergent dialysis and freeze/thaw so nication. IIGlcNAc was Oriented randomly in the vesicles as inferred f rom protein phosphorylation studies. Import and subsequent phosphoryla tion of GlcNAc were measured with proteoliposomes preloaded with enzym e I, histidine-containing phosphocarrier protein, and phosphoenolpyruv ate. Uptake and phosphorylation occurred in a 1:1 ratio, Active extrus ion of GlcNAc entrapped in vesicles was also measured by the addition of enzyme I, histidine-containing phosphocarrier protein, and phosphoe nolpyruvate to the outside of the vesicles. The K-m for vectorial phos phorylation and non-vectorial phosphorylation were 66.6 +/- 8.2 mu m a nd 750 +/- 19.6 mu m, respectively, Non-vectorial phosphorylation was faster than vectorial phosphorylation with k(cat) 15.8 +/- 0.9 s(-1) a nd 6.2 +/- 0.7 s(-1), respectively. Using exactly the same conditions, the purified transporters for mannose (IIAB(Man), IICMan, IIDMan) and glucose (IICBGlc, IIA(Glc)) were also reconstituted for comparison. A lthough the vectorial transport activities of IICBA(GlcNAc) and IICBGl c IIA(Glc) are inhibited by non vectorial phosphorylation, no such eff ect was observed with the IIAB(Man) IICMan IIDMan complex, This sugges ts that the molecular mechanisms underlying solute transport and phosp horylation are different for different transporters of the phosphotran sferase system.