Purification of human liver aldehyde dehydrogenase by high-performance liquid chromatography and identification of isoenzymes by immunoblotting.
Human liver aldehyde dehydrogenase (ALDH) exists in multiple molecular forms. Two different isoenzymes of ALDH have been purified which will oxidize acetaldehyde to acetate. ALDH1 is localized principally in hepatocyte cytosol and exhibits a Km for acetaldehyde of about 0.1 mM at pH 9.5. ALDH2 is mitochondrial in origin and exhibits low Km for acetaldehyde, about 1 microM. We have developed rapid purification procedures for ALDH1 and ALDH2 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 Km for acetaldehyde, about 1 mM, and is very labile. Polyclonal antibodies to the purified ALDH1 and ALDH2 isoenzymes have been prepared. As evidenced by immunoblotting of starch gels containing the purified isoenzymes, anti-ALDH1 does not crossreact with ALDH2 and anti-ALDH2 does not crossreact with ALDH1. The anti-ALDH2 antibody identifies the "inactive" variant of ALDH2 in Japanese livers exhibiting the "deficient" ALDH2 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.
D000444:Aldehyde Dehydrogenase / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D004592:Electrophoresis, Starch Gel; D006801:Humans; D007124:Immunoenzyme Techniques; D007527:Isoenzymes / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*
D007527:Isoenzymes; D000444:Aldehyde Dehydrogenase