We have purified proline permease to homogeneity from using an -proline-linked agarose matrix as an affinity column. The eluted protein produced two bands of 64 and 67 kDa by SDS-PAGE, whereas it produced a single band of 67 kDa by native PAGE and Western blotting. The apparent for -proline binding to the purified protein was 153 μM. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of -proline transport in reconstituted systems, viz. specificity and sensitivity to -ethylmaIeimide, were very similar to those of intact cells. The antifungal cispentacin, which enters cells via an inducible proline permease, competitively inhibited the -proline binding and translocation in reconstituted proteoliposomes. However, the uptake of -proline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent of 40 μM.


Article metrics loading...

Loading full text...

Full text loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error