Fig. 7.

PIO interacts with and inhibits purified GAPDH.

(A) Western blot analysis of purified GAPDH in the absence (left panel) or presence (right panel) of added dithiothreitol (DTT). Noticeably, no higher aggregation forms were detected in the stacking gel. (B) Quantities of different aggregation forms (tetra-, tri-, di-, and monomeric) in the absence of DTT (open bars: initial GAPDH suspension, GAPDH plus DMSO, GAPDH plus DMSO-solubilized PIO, or in the presence of DTT (green filled bars) added before or after PIO). (C) GAPDH (2.1 mIU/ml) fluorescence in the absence (blue dots) or presence (green dots) of 12.5 μM PIO against UV (280 nm) irradiation. The presence of 1.25‰ DMSO had per se a partial protecting effect against UV (grey dots). No changes in fluorescence were observed when the enzyme was kept away from UV irradiation (black dots). Noticeably initial fluorescence in the presence or absence of PIO, or DMSO was not different ruling out potential interference of these compounds with fluorescence assay conditions (excitation: 280 nm, 10 nm bandpass; emission: 330 nm, 10 nm bandpass). (D) Initial activity of rabbit skeletal muscle GAPDH (0.05 mIU/ml) measured in the forward direction (NADH accumulation) (Velick, 1955) and of increasing amounts of PIO. (E) Activity measured in the backward direction (NADH oxidation) in the absence (blue open bar) or presence (green open bar) of 200 μM PIO, and in the presence of 1.7 mM cysteine and absence (blue filled bar) or presence (green filled bar) of 200 μM PIO. (F) Lineweaver-Burk plots of forward GAPDH activity (0.075 mIU/ml) in the absence (black line) or presence (green line) of 62.5 μM PIO. GAP, d-glyceraldehyde 3-phosphate.