Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process.
A triethylammonium-sensitive electrode was constructed using sodium tetrakis[3,5-bis(2-methoxyhexafluoro-2-propyl)phenyl]borate as an ion-exchanger and benzyl 2-nitrophenyl ether as a solvent mediator in a poly(vinylchloride) membrane matrix and was used to determine the pH difference across a cell membrane. The method is based on monitoring of the pH gradient-induced uptake of triethylammonium in situ. The triethylammonium electrode exhibited a near-Nernstian response to triethylammonium in the concentration range of 5 x 10(-6)-1 x 10(-2) M with a slope of 58.5 mV per concentration decade in a buffer solution composed of 150 mM NaCl and 10 mM NaH2PO4/Na2HPO4 (pH 7.5). The limit of detection was 1 microM. In experiments using liposomes, the uptake of triethylammonium into liposomes was quantitatively induced according to the pH difference across the liposomal membrane. The transmembrane pH differences in Escherichia coli cells and the light-induced pH differences across the envelope vesicles of Halobacterium halobium were successfully determined by the present method.
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