Fig 1.
The effect of piceatannol on the activity of GAPDH, measured during 2 hours of incubation of GAPDH (2 μM) with piceatannol at concentrations 0, 3.2, 16, 32 and 64 μM (continuous lines).
Similarly, the effect of piceatannol at the concentration 64 μM on the activity of LDH was determined (black dashed line). Catalytic activity of enzymes are expressed as percentages referring to the activity of untreated samples (control). Data are means ± SD of n = 4–8 independent measurements.
Fig 2.
A decrease in the relative absorbance (λ = 412 nm) of the colored solution formed in the reaction of free SH groups present in GAPDH with DTNB.
Data are means ± SD of n = 3–4 independent measurements, * p < 0.05.
Fig 3.
(A) Thermal effects of the titration 0.5 mM piceatannol solution (in the syringe) into 5 μM GAPDH protein solution (in the cell) (■) and corresponded to them thermal effects of dilution of the piceatannol in pure water (○). (B) Thermal effect of the interaction between protein GAPDH and piceatannol corrected with the piceatannol dilution effects and calculated per one mole of the ligand. Solid line is approximation of ITC date of GAPDH by piceatannol using the model of one set of binding sites.
Table 1.
Binding parameters of the interaction of GAPDH protein with piceatannol in water solution at 25°C determined by isothermal titration calorimetry.
Fig 4.
Far-UV CD spectra of native GAPDH and GAPDH treated with 32 μM of piceatannol at room temperature for 50 and 100 min of incubation.
The data are expressed as molar residue ellipticity.
Fig 5.
Changes of zeta potential of GAPDH in the presence of piceatannol.
Fig 6.
(A) The largest accessible region in GAPDH tetramer for piceatannol binding is located inside the molecule, where the subunits come into contact. (B) Interactions of piceatannol inside the molecule with amino acid residues from subunit O and Q.
Table 2.
Most frequently occurring interactions of piceatannol at the active site of the O subunit of GAPDH.
Number in parentheses represents how many times the residue was involved in interaction/30 best poses.
Fig 7.
Most frequently interactions of piceatannol with amino acid residues (the catalytic region of enzyme) active site of the O subunit of GAPDH.
Fig 8.
The intensity of thioflavin-T binding-dependent fluorescence of native GAPDH, GAPDH treated with piceatannol (50 μM), GAPDH treated with hydrogen peroxide (3 mM) and GAPDH preincubated for 30 minutes with piceatannol or resveratrol (50 μM) and afterwards treated with H2O2 at 37°C for the indicated times is shown.
Data are means ± SD of n = 4–8 independent measurements.
Fig 9.
The micrographs of (A) GAPDH, (B) GAPDH and H2O2, (C) GAPDH with piceatannol and H2O2, (D) GAPDH with resveratrol and H2O2, from light microscope and transmission electron microscope. Bright field—left panel, dark field—middle panel and electron micrographs-right panel.
Fig 10.
Stereo view of piceatannol bound in GAPDH active center.
Covalent binding between nucleophilic thiol group in Cys149 and piceatannol at the 2-C position of the aromatic ring B forms the protein-piceatannol adduct.
Fig 11.
Proposed mechanism for the conjugation of catechol moiety of piceatannol to a nucleophilic thiol group (Cys149) in GAPDH molecule.