Article Title: Isozyme Specific Allosteric Regulation of Human Sulfotransferase 1A1
Figure Lengend Snippet: EGCG binding to the E·(PAP)2 Complex. A.) The Binding-Reaction Progress Curve . Binding-induced fluorescence changes were monitored using a stopped-flow fluorimeter (λ ex = 290 nm, λ em ≥ 325 nm (cutoff filter). Reactions were initiated by rapidly mixing (1:1 v/v) a solution containing SULT1A1 (0.25μM, active sites), PAP (0.50 mM), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.5, 25 ± 2°C with a solution that was identical except that it lacked SULT1A1 and contained EGCG. The curve through the data (red line) is the behavior predicted by the best fit to a double-exponential model. The insert shows the residuals from a single- (black) and doubleexponential (red) fit. The single-exponential model deviates substantially from the data, while the bi-phasic model provides an excellent fit. B.) k obs vs [EGCG] for the First (Fast) Phase . kobs values were obtained at the four indicated EGCG concentrations under the conditions given in Panel A. Each value is the average of three independent determinations. The reactions were pseudo-first-order in EGCG in all cases. C.) EGCG Dissociation from E·(PAP) 2 ·(EGCG) 2 Complex . A pulse solution containing SULT1A1 (20 μM, monomer), EGCG (24 μM (4.0 μM free, 100 × Kd)), and PAP (500 μM, 16 ▪ K d low affinity site ), MgCl2 (5.0 mM), NaPO4 (50 mM), pH 7.5, 25 ± 2°C, was diluted 200:1 into a chase solution containing 75 μM 4-hydroxytamoxifen (TAM, 100 ▪ K d cap open form ), PAP (0.50 mM, 16 ▪ K d low affinity site ), NaPO4 (50 mM), MgCl2 (5.0 mM), pH 7.5, 25 ± 2°C. The final EGCG concentration is 0.12 μM (0.20 ▪ K d cap open form ). Dissociation was monitored by a change in SULT1A1 fluorescence (λ ex = 290 nm, λ em = 370 nm). Three progress curves (black dots) are superposed. The curve through the data is the behavior predicted by a best-fit, single-exponential model.
Article Snippet: Ampicillin, HEPES, KCl, KOH, MgCl2 , NaCl and phenylmethylsulfonyl fluoride (PMSF) were purchased from Fisher Scientific.
Techniques: Binding Assay, Fluorescence, Flow Cytometry, Concentration Assay