Interactions of alcohol dehydrogenase to p-hydroxyacetophenone-sepharose and p-acetamidophenol-sepharose.
BACKGROUND
p-Hydroxyacetophenone (HAP)-sepharose is known to be an effective ligand for isolation of aldehyde dehydrogenase and chloramphenicol acetyltransferase. In this study, we investigated ligand specificities to alcohol dehydrogenase (ADH) using p-HAP-sepharose and p-acetamidophenol (AAP)-sepharose.


METHODS
p-HAP and p-AAP were coupled to epoxy-activated sepharose and used as ligands for affinity chromatography to detect binding proteins. Fractions of affinity chromatography were collected and separated by SDS-PAGE. Commassie brilliant blue staining was carried out for detecting proteins. For determining protein sequences, polypeptide was separated by HPLC after cleavage of Lys-specific protease digestion.


RESULTS
p-HAP ligands have the ability to bind to seven proteins, including class I ADH extracted from a rat cytosolic fraction as well as HLADH (horse liver ADH). p-HAP, p-AAP and benzaldehyde (10 mM each) could elute HLADH from the complex of HLADH-pHAP-sepharose column. On the other hand, p-AAP-sepharose has no ability to bind to HLADH, although three proteins from a rat liver cytosolic fraction were found to be able to bind to p-AAP-sepharose.


CONCLUSIONS
p-HAP binds to ADH in a structure-specific manner and this binding is nonspecific to species of origin of ADH.
Negoro
Munetaka
M
Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Japan.
Wakabayashi
Ichiro
I
D000082:Acetaminophen / Q000737:chemistry*; D000098:Acetophenones / Q000737:chemistry*; D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D008099:Liver / Q000201:enzymology; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D012685:Sepharose / Q000737:chemistry*; D020539:Sequence Analysis, Protein
D000098:Acetophenones; D000082:Acetaminophen; D012685:Sepharose; D000426:Alcohol Dehydrogenase; C031335:4-hydroxyacetophenone