Purification of liver aldehyde dehydrogenase by p-hydroxyacetophenone-sepharose affinity matrix and the coelution of chloramphenicol acetyl transferase from the same matrix with recombinantly expressed aldehyde dehydrogenase.
p-Hydroxyacetophenone was coupled to epoxy-activated Sepharose 6B to generate an affinity chromatographic matrix to purify aldehyde dehydrogenase. Purified beef liver mitochondrial aldehyde dehydrogenase specifically bound to the support and could be eluted with p-hydroxyacetophenone. A post-ammonium sulfate (30-55%) fraction of bovine liver was applied to the affinity gel column and aldehyde dehydrogenase was effectively purified, although not to complete homogeneity, indicating the potential selectivity of the matrix. Both beef liver cytosolic and mitochondrial aldehyde dehydrogenase bound to the column. A post-Cibacron blue Sepharose Cl-6B affinity-fractionated liver mitochondrial aldehyde dehydrogenase was purified to complete homogeneity by p-hydroxyacetophenone-Sepharose, thus eliminating the need for the isoelectric focusing step often employed. p-Hydroxyacetophenone was found to be a competitive inhibitor against propionaldehyde and noncompetitive against NAD. Escherichia coli lysates of recombinantly expressed aldehyde dehydrogenase were purified from E. coli lysates with one major 25-kDa protein contaminant also binding to the column, as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 25-kDa contaminant was found to be chloramphenicol acetyl transferase from sequence analysis and binding studies.
Ghenbot
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Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Weiner
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D000098:Acetophenones / Q000378:metabolism* / Q000494:pharmacology; D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D015500:Chloramphenicol O-Acetyltransferase / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification; D012685:Sepharose
D000098:Acetophenones; D011994:Recombinant Proteins; D012685:Sepharose; D000444:Aldehyde Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase; C031335:4-hydroxyacetophenone