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1.
Fig. 7

Fig. 7. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

Proton pumping of F1Fo with ε-GFP fusion. The ATP synthase was purified from the strains expressing wild type, ε-cytochrome b562, and the ε-GFP enzymes and reconstituted into liposomes. Proton pumping was monitored by fluorescence quenching of 9ACMA coupled with ATP hydrolysis. Oligomycin (5 μg) was added as indicated by the arrow

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.
2.
Fig. 4

Fig. 4. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

Effectiveness of detergents on the coupling capacity of the ATP synthase. The ATP synthase was purified and the detergents were exchanged as described in Material and Methods. The resulting preparations were reconstituted into liposomes and proton pumping was measured by fluorescence decrease of 9ACMA coupled to ATP hydrolysis. Oligomycin (5 μg) was added as indicated. The bottom panel shows a tabular form of the data with the detergent and percentage fluorescence quenching coupled to ATP hydrolysis

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.
3.
Fig. 5

Fig. 5. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

Growth phenotype of yeast with the ε-fusion constructs. The yeast strain containing null mutations in ATP2 and ATP15 (encodes the β- and ε-subunits, respectively) were transformed with vectors that contained the ATP2 gene and the ε-fusion construct. The cells were grown on rich medium containing glucose (YPD) or glycerol (YPG) in 3 dilutions, at 30 °C

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.
4.
Fig. 2

Fig. 2. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

Effectiveness of lipids on the oligomycin sensitive ATPase activity with purified yeast ATP synthase. The oligomycin sensitive ATPase activity was measured in buffer containing 0.05% DDM an in the presence of specified lipids at the indicated concentrations. The circles represent the values of the ATPase (μmoles/min/mg protein) in the absence of oligomycin and the squares in the presence of 5 mg oligomycin. The error bars represent 1 standard error

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.
5.
Fig. 3

Fig. 3. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

Cardiolipin dependence by the ATP synthase for oligomycin sensitive ATPase activity. a The ATP synthase was purified in the absence of lipids, cardiolipin (BH CL) was bound (200 molar excess) and assayed in buffer containing 0.006% DDM (I) in the absence of additional added lipid and in buffer containing 0.05% DDM and 0.03% lecithin (II). b The ATP synthase was purified in the absence of lipids and assayed in the presence and absence of oligomycin (5 μg) in buffer containing 0.005% DDM with the indicated amount (molar excess) of TM CL

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.
6.
Fig. 6

Fig. 6. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

Analysis of ε-GFP fusion integrity. F1Fo ATP synthase from the strain containing the ε-GFP fusion (20 μg) was separated by SDS PAGE and analyzed by Western blot analysis with antibodies directed against the ε- and γ-subunits. For comparison, F1 ATPase from a wild type strain is shown (F1). a Coomassie Blue stain of the peptides. b Western blot analysis c Same as b, but at a longer exposure. The pertinent peptides are indicated: ε-GFPd is the ε-GFP with GFP in a denatured form, ε-GFPn is the fusion protein with GFP in more folded form. (Mass spectrometric analysis of these bands confirmed their identity: ε-GFPn is green while ε-GFPd is colorless)

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.
7.
Fig. 1

Fig. 1. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

Mono-disperse preparation of coupled yeast ATP synthase. a The elution profile as analyzed by SDS PAGE from a Superose 6 size exclusion column in the presence of 0.05% DDM. The first lane shows the peptides from the enzyme purified from the Ni-Sepharose column. The fractions peak of at 42 min (0.3 ml/min) is consistent with a molecular weight of 650,000 Da. b The peptide profile as analyzed by SDS PAGE of the enzyme purified in the absence and presence of 0.03% lecithin. c The enzyme purified in the absence and presence of lecithin were incorporated into liposomes and tested for proton pumping coupled to ATP hydrolysis as described in Materials and Methods. The acidification of the internal liposome space was monitored by the fluorescence quenching of 9ACMA. The error bars show 1 standard error in this and all of the figures

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.
8.
Fig. 8

Fig. 8. From: Characterization of the mitochondrial ATP synthase from yeast Saccharomyces cerevisae.

a The crystal of putative F1Fo with approximate dimensions (0.6×0.2×0.05 mm). b, c Typical diffraction image of the crystal as recorded at APS beamline 23-ID-C. The resolution is indicated by the rings. d Electron density (gold) in c10 region after solvent flipping and phase combination. The Cα model of Stock et al., (in magenta) is overlaid on the electron density map (Stock et al. 1999). A mask of F1Fo generated from electron microscopy data (Rubinstein et al. 2003) was manually aligned with the molecular replacement solution, which indicated that only C10 could be accommodated without serious overlaps in the crystal packing. Thus the crystal likely consists of F1C10 and not an intact F1Fo. The crystallization was done at 21 °C at 4 mg/ml F1Fo, in 12.25% PEG 400, 60 mM NaF, 25 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 2.5 mM paminobenzamidine, 2.5 mM ε-aminoacaproic acid, 0.05 mM thymol, and 100 molar excess of cardiolipin (0.67 mM)

Vijayakanth Pagadala, et al. J Bioenerg Biomembr. 2011 August;43(4):333-347.

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