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Purification of Human Liver Aldehyde Dehydrogenase by High‐Performance Liquid Chromatography and Identification of Isoenzymes by Immunoblotting
Author(s) -
Johnson Charles T,
Bosron William F,
Harden Colleen A,
Li TingKai
Publication year - 1987
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/j.1530-0277.1987.tb01264.x
Subject(s) - aldehyde dehydrogenase , acetaldehyde , chemistry , isozyme , affinity chromatography , biochemistry , chromatography , polyclonal antibodies , enzyme , antibody , ethanol , biology , immunology
Human liver aldehyde dehydrogenase (ALDH) exists in multiple molecular forms. Two different isoenzymes of ALDH have been purified which will oxidize acetaldehyde to acetate. ALDH 1 is localized principally in hepatocyte cytosol and exhibits a K m for acetaldehyde of about 0.1 mM at pH 9.5. ALDH 2 is mitochondrial in origin and exhibits low K m for acetaldehyde, about 1 μM. We have developed rapid purification procedures for ALDH 1 and ALDH 2 by use of agarose‐AMP affinity chromatography and high‐performance anion‐exchange liquid chromatography (HPLC). The method takes less time and affords higher yields of the labile ALDH isoenzymes than conventional column chromatography methods. A previously uncharacterized ALDH form has been identified by anion‐exchange HPLC which exhibits high K m for acetaldehyde, about 1 mM, and is very labile. Polyclonal antibodies to the purified ALDH 1 and ALDH 2 isoenzymes have been prepared. As evidenced by immunoblotting of starch gels containing the purified isoenzymes, anti‐ALDH 1 does not crossreact with ALDH 2 and anti‐ALDH 2 does not crossreact with ALDH 1 . The anti‐ALDH 2 antibody identifies the “inactive” variant of ALDH 2 in Japanese livers exhibiting the “deficient” ALDH 2 phenotype. The sensitivity of detection of ALDH isoenzymes in liver homogenate‐supernatants by immunoblotting of starch gels is about 10‐fold greater than that by activity staining.