Interaction of Alcohol Dehydrogenase with tert-Butylhydroperoxide: Stimulation of the Horse Liver and Inhibition of the Yeast Enzymes

Pyrene Excimer Fluorescence of Yeast Alcohol Dehydrogenase: A Sensitive Probe to Investigate Ligand Binding and Unfolding Pathway of the Enzyme

“…Cheng et al . have shown that thiol compounds profoundly inhibited HLADH but exerted no effect on YADH, whereas Tkachenko and Winston have shown that tertiary butyl hydroperoxide oxidized the SH groups of YADH inhibiting the activity of the enzyme completely but stimulating HLADH (9, 10). These investigations have attributed this differential behavior of alcohol dehydrogenase to the subtle selectivity of the zinc‐thiolate center of alcohol dehydrogenase to different compounds.…”

“…First, the reaction mixture was passed through a Sephadex G‐25 size‐exclusion column (Steinheim, Germany) (15 × 1 cm), which was previously equilibrated with 50 mM sodium phosphate buffer and subsequently equilibrated by dialyzing it against the same buffer for 8 h at 4°C. The concentration of YADH‐bound PM was determined using a molar extinction coefficient, 45 000 M ‐1 cm ‐1 at 337 nm (10). After subtracting the contribution of the absorbance of PM at 280 nm, the concentration of YADH in the labeled protein was determined by measuring absorbance at 280 nm using a molar extinction coefficient of 45 000 M ‐1 cm ‐l per YADH monomer (10).…”

“…The concentration of YADH‐bound PM was determined using a molar extinction coefficient, 45 000 M ‐1 cm ‐1 at 337 nm (10). After subtracting the contribution of the absorbance of PM at 280 nm, the concentration of YADH in the labeled protein was determined by measuring absorbance at 280 nm using a molar extinction coefficient of 45 000 M ‐1 cm ‐l per YADH monomer (10). The concentration of YADH was also measured by the Bradford method using bovine serum albumin as a standard (21) and calibrated with respect to the absorption of unlabeled YADH at 280 nm.…”

“…Thiol reagents have also been shown to differentially affect the activity of horse liver alcohol dehydrogenase (HLADH) and YADH probably because of different structural arrangements of the enzyme's active site (9). It has also been suggested that the subtle selectivity of the zinc‐thiolate center of the alcohol dehydrogenases may be because of distinct differences in enzyme function (10).…”

Inhibition of alcohol dehydrogenase by bismuth☆

“…Enzyme activity was determined by the changes of initial rate of absorbance at 340 nm corresponding to the reduction of NAD þ to NADH as previously reported [21]. The concentrations of both NAD þ and NADH were determined spectrophotometrically using the extinction coefficients of 1.8 Â 10 4 and 6.22 Â 10 3 M À1 cm À1 at 260 and 340 nm, respectively [22]. All UV-vis spectra were recorded on a computer controlled Varian Cary50 spectrometer with thermostated cuvette holders at 298 K.…”