J Physiol. 2006 March 1; 571(Pt 2): 287–302.
Figure 5 Location of Kir2.1 mutations used in the study A and B, crystal structure of C terminus of Kir2.1 channel (Pegan et al. 2005) was visualized using Swiss-PDB Viewer software. Two opposing subunits were removed for clarity and side chains for the mutants used in this study displayed. A represents a side view, and B shows a top view of the pore region. C, sequence alignment of Kir2 channels in the region around the residues critical for inward rectification. Arrowhead points to the highly conserved F254 residue and asterisk indicates the least conserved site in this region. J Physiol. 2006 March 1; 571(Pt 2): 287–302.
Plant Physiol. 2002 February; 128(2): 714–725.
Figure 3 Affinity chromatography on the ABA-linked EAH-Sepharose 4B column of the crude ABA-binding proteins. The crude ABA-binding proteins for affinity chromatography were solubilized only with Triton X-100 contained in the buffer described in “Materials and Methods” (method 1). After equilibration, the column was first eluted with a total volume of 1,000 mL MES/NaCl (pH 6.5) buffer containing NaCl in a step concentration gradient from 100 to 300 mm, and then the ABA-binding protein was eluted with 300 mL of the same buffer containing 1 mm (±)ABA and 150 mm NaCl. NaCl concentrations (dashed line). Elution profile expressed on the optical density at 280 nm (solid line). Relative 3H-ABA-binding activity of the eluted protein (○), taking the maximum binding activity obtained at the point of about 1,150 mL of the elution volume, as 100%. Points for the relative 3H-ABA-binding activity are means ± sd of three determinations. Plant Physiol. 2002 February; 128(2): 714–725.
Plant Physiol. 2002 February; 128(2): 714–725.
Figure 4 Commassie blue-stained SDS-PAGE (A), silver-stained native IEF (B), and silver-stained IEF/SDS-PAGE (C) of the purified ABA-binding protein. In A: a, molecular mass standards; b, purified ABA-binding proteins (3 μg), of which the calculated molecular mass was 42 kD; c, proteins in the crude extract (15 μg). In B: a, the purified ABA-binding protein, of which the measured pI was 4.86; b, the protein standards. C, The purified ABA-binding protein (2 μg) was resolved by IEF in the first dimension followed by SDS-PAGE; a, molecular mass markers; b, the purified ABA-binding protein. The measured molecular mass and pI were, respectively, 42 kD and 4.86. Plant Physiol. 2002 February; 128(2): 714–725.
Plant Physiol. 2002 February; 128(2): 714–725.
Figure 2 Effects of NaCl on ABA-binding activity in the crude extracts. A range of NaCl concentrations from 0 to 500 mm was tested. ABA-binding activity of the control (without NaCl) was taken as 100%. Points are means ± sd of three determinations. Plant Physiol. 2002 February; 128(2): 714–725.
Plant Physiol. 2002 February; 128(2): 714–725.
Figure 6 Scatchard plots of ABA-binding activities to the crude (a) and purified (b) proteins. B, 3H-ABA bound; F, 3H-ABA free. For the crude proteins (a), all points except for the first two were fitted with a linear relationship (r2 = 0.98), and according to the Scatchard plot, the maximum binding activity (Bmax) and the equilibrium dissociation constant (Kd) were calculated: Bmax = 68 nmol g−1 protein and Kd = 19 nm. The fitted relationship of the purified protein (b) was also linear (r2 = 0.99) with Bmax = 0.87 mol mol −1 protein and Kd = 21 nm. Points represent the means ± sd of five determinations. Plant Physiol. 2002 February; 128(2): 714–725.
