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Characterization of a Ca2+ Uniporter from Bacillus Subtilis by Partial Purification and Reconstitution into Phospholipid Vesicles
Abstract
Ca2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin. Uptake was dependent on the magnitude of the membrane potential. This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+. The system showed saturation kinetics with an apparent K m for Ca2+ of about 250 μm. Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed. A fraction at pH 5·28–5·33 contained the activity. The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+). In addition, uptake was not influenced by a pH gradient imposed on the vesicles. The apparent K m for Ca2+ in the reconstituted system was about 260 μm. The specific activity was increased about 50-fold by purification with isoelectric focusing.
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© Society for General Microbiology 1987