Dye affinity labelling of yeast alcohol dehydrogenase.
The interaction of yeast alcohol dehydrogenase (ADH) with the reactive chlorotriazine dye Vilmafix Blue A-R (VBAR) was studied. VBAR was purified to homogeneity on lipophilic Sephadex LH-20 and characterised by reverse phase HPLC and analytical TLC. Incubation of ADH with purified VBAR at pH 8.0 and 37 degrees C resulted in a time-dependent inactivation of the enzyme. The observed rate of enzyme inactivation (kobs) exhibited a non-linear dependence on VBAR concentration from 22 to 106 nmol, with a maximum rate of inactivation (k3) of 0.134 min-1 and kD of 141.7 microM. The inhibition was irreversible and activity could not be recovered by gel-filtration chromatography. The inactivation of ADH by VBAR was competitively inhibited by the nucleotides NADH and NAD+. These results suggest that VBAR acts as an affinity label at the nucleotide binding site of yeast ADH.
Labrou
N E
NE
Department of Agricultural Biotechnology, Agricultural University of Athens, Greece. lambrou@auadec.aua.gr
D000345:Affinity Labels / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000880:Anthraquinones / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004396:Coloring Agents / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012440:Saccharomyces / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014227:Triazines / Q000378:metabolism*
D000345:Affinity Labels; D000880:Anthraquinones; D004396:Coloring Agents; D014227:Triazines; C091762:Vilmafix Blue A-R; D009243:NAD; D000426:Alcohol Dehydrogenase