title: A reinvestigation of the purity, isoelectric points and some kinetic properties of the aldehyde dehydrogenases from sheep liver.
abstract: 1. Cytoplasmic aldehyde dehydrogenase was shown to be free of contamination by the mitochondrial enzyme by isoelectric focusing. 2. Both enzymes showed multiple banding in activity stains. The cytoplasmic enzyme gave two very close bands pI = 5.22 +/- 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pI = 5.22 and five further bands of pI 5.48 +/- 0.09, 5.56 +/- 0.07, 5.65 +/- 0.06, 5.70 +/- 0.03 and 5.76 +/- 0.02. Possible origins of the isoenzymes are discussed. 3. Disulfiram in a fourfold excess reduced the activity of the cytoplasmic enzyme to 9% of the initial value. The residual activity represents the activity of the disulfiram-modified enzyme and is not due to mitochondrial contamination. This casts doubt on the role of an essential thiol group. 4. The mitochondrial enzyme shows a low amplitude (22%) burst in the production of 4-nitrophenoxide ion during the hydrolysis of 4-nitrophenyl acetate at pH 7.6. The burst rate constant was 7.3 +/- 1 s-1 and the steady-state rate constant was 0.2 s-1, values similar to those previously reported for the cytoplasmic enzyme. 5. The mitochondrial enzyme shows a burst in the release of protons during the oxidation of propionaldehyde at pH 7.6. The burst rate constant was 6 s-1 and the amplitude was equal to half the formal enzyme concentration. The significance of these results for the steady-state mechanism is discussed.
mesh_terms: D000444:Aldehyde Dehydrogenase; D000445:Aldehyde Oxidoreductases / Q000302:isolation & purification*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003593:Cytoplasm / Q000201:enzymology; D004221:Disulfiram / Q000494:pharmacology; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D012756:Sheep
chemical_list: D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase; D004221:Disulfiram