1887

Abstract

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 �M. 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 �M.

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1997-02-01
2021-07-27
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