Highly diverged novel subunit composition of apicomplexan F-type ATP synthase identified from Toxoplasma gondii
Fig 4
Partial purification of dimer and monomer forms of T. gondii F-type ATP synthase by chromatography.
(A) Ion exchange (DEAE sepharose) separation of mitochondrial lysates prepared from TgATPβ-YFP-HA expressing transgenic parasites. Absorbance at 280 nm (filled circles) and NaCl concentration (open circles) are plotted for each fraction. Fractions 5 and 18 are marked with arrows. Bottom panel shows SDS-PAGE western blotting for fractions 5–18 to find out the elution profile of TgATPβ-YFP-HA. (B) Size exclusion profile of the pooled fractions from ion exchange chromatography. The absorbance at 280 nm is plotted for each fraction. The size exclusion column was calibrated with the following native markers: thyroglobulin (labeled “T,” 660 kDa), ferritin (labeled “F,” 440 kDa), conalbumin (labeled “C,” 75 kDa), Ovalbumin (labeled “O,” 45 kDa). Peak elution volume for each marker is indicated by arrow. Fractions 1–3, 4–6, and 7–9 were pooled, concentrated, and subject to SDS-PAGE western blotting to detect TgATPβ-YFP-HA, as shown in bottom panel. DEAE, diethylaminoethanol; OD, optical density; TgATPβ, T. gondii ATP synthase β subunit; YFP-HA, yellow fluorescent protein plus hemagglutinin.