Purification of alcohol dehydrogenase from Entamoeba histolytica and Saccharomyces cerevisiae using zinc-affinity chromatography.
We have developed a single-step method for the purification of NADP(+)-dependent alcohol dehydrogenase from Entamoeba histolytica and NAD(+)-dependent alcohol dehydrogenase from Saccharomyces cerevisiae. It is based on the affinity for zinc of both enzymes. The amebic enzyme was purified almost 800 times with a recovery of 54% and the yeast enzyme was purified 30 times with a recovery of 100%. The kinetic constants of the purified enzymes were similar to those reported for other purification methods. With mammalian alcohol dehydrogenase, we obtained a 40-kDa band suggestive of purified alcohol dehydrogenase, but we failed to retain enzymatic activity in this preparation. Our results suggest that the described method is more applicable to the purification of tetrameric alcohol dehydrogenases.
Cabrera
N
N
Departamento de Microbiologia, Instituto de Fisiología Celular, U.N.A.M., México D.F., México.
Rangel
P
P
Hernández-Muñoz
R
R
Pérez-Montfort
R
R
D000426:Alcohol Dehydrogenase / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology*; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D010446:Peptide Fragments; D009249:NADP; D000426:Alcohol Dehydrogenase; D014357:Trypsin; D015032:Zinc