抄録
It was found that highly purified crystalline yeast alcohol dehydrogenase (YADH) was still contaminated with a small amount of thiolesterase. Separation of these two enzymes from each other was performed by DEAE-cellulose column chromatography. YADH preparation resulted from this treatment was found to be homogeneous by disc electrophoresis and chromatography on hydroxylapatite column.
Treatment of pure YADH with O, S-diacetylthiamine (DAT) in 0.05M phosphate buffer at pH 6.0 and 30° resulted in 50% inactivation of the enzyme with concomitant loss of two SH groups per mole of the protein. Reaction of the DAT-inactivated YADH with hydroxylamine resulted in full reactivation accompanied by restoration of SH groups to the level of the native enzyme.
These results supply evidence that the change in enzymatic activity that occurs upon treatment of YADH with DAT is the consequence of acetylation of two SH groups essential for activity per molecule of the enzyme.
