Open AccessA Reinvestigation of the Purity, Isoelectric Points and Some Kinetic Properties of the Aldehyde Dehydrogenases from Sheep Liver
Author(s) -
AGNEW Kim E. M.,
BENNETT Adrian F.,
CROW Kathryn E.,
GREENWAY Robert M.,
BLACKWELL Leonard F.,
BUCKLEY Paul D.
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb05579.x
Subject(s) - isoelectric point , enzyme , disulfiram , chemistry , isoelectric focusing , aldehyde dehydrogenase , enzyme assay , steady state (chemistry) , biochemistry , cytoplasm , stereochemistry , organic chemistry
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 pl = 5.22 ± 0.03 whereas the mitochondrial enzyme showed seven bands, a pair at pl = 5.22 and five further bands of pl 5.48 ± 0.09, 5.56 ± 0.07, 5.56 ± 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.