New simple method for purification of class I alcohol dehydrogenase.
OBJECTIVE
The purpose of this study was to develop a new simple method for purification of rat class I alcohol dehydrogenase (ADH, EC 1.1.1.1).


RESULTS
Immobilized p-hydroxyacetophenone was used as a ligand for affinity chromatography for the initial purification step after ammonium sulfate precipitation of the cytosolic fraction of rat liver. Then the eluant was separated by using ion-exchange chromatography, and homogenous class I ADH, as judged by the results of SDS-PAGE and confirmed by the results of the amino-acid sequence of peptides degraded from a 39 kDa protein, was obtained with a high yield (57%). The purified ADH showed kinetic constants of 1.3 mmol/l for Km and 62.4 per min for Kcat with ethanol as a substrate. ADH was also successfully purified from yeast by a similar method using p-hydroxyacetophenone affinity chromatography.


CONCLUSIONS
This simple method involving only two chromatographic procedures may be very useful for purification of ADH.
Negoro
M
M
Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata, Japan.
Wakabayashi
I
I
D000426:Alcohol Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000737:chemistry* / Q000201:enzymology*; D008297:Male; D051381:Rats; D017208:Rats, Wistar
D000426:Alcohol Dehydrogenase