Jethwaney Deepa, H Fer Milan, Khaware Raj K, Prasad Rajendra
School of Life Sciences, Jawaharlal Nehru University,New Delhi-110067,India.
Botanisches Institut, Universit�t Bonn,Kirschallee 1, D-53115 Bonn,Germany.
Microbiology (Reading). 1997 Feb;143 ( Pt 2):397-404. doi: 10.1099/00221287-143-2-397.
We have purified proline permease to homogeneity from Candida albicans using an L-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 Km for L-proline binding to the purified protein was 153 microM. The purified permease was reconstituted into proteoliposomes and its functionality was tested by imposing a valinomycin-induced membrane potential. The main features of L-proline transport in reconstituted systems, viz. specificity and sensitivity to N-ethylmaleimide, were very similar to those of intact cells, The antifungal cispentacin, which enters C. albicans cells via an inducible proline permease, competitively inhibited the L-proline binding and translocation in reconstituted proteoliposomes. However, the uptake of L-proline in proteoliposomes reconstituted with the purified protein displayed monophasic kinetics with an apparent Km of 40 microM.
