scholarly journals Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase

1982 ◽  
Vol 205 (2) ◽  
pp. 443-448 ◽  
Author(s):  
F M Dickinson ◽  
G J Hart
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Metal Ion ◽  
Stopped Flow ◽  
Maximal Effect ◽  
Ion Concentrations ◽  
Sheep Liver ◽  
High Concentrations ◽  
Flow Experiments ◽  
Active Centres

Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.

scholarly journals The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme

1986 ◽  
Vol 233 (3) ◽  
pp. 877-883 ◽  
Author(s):  
F M Dickinson ◽  
G W Haywood
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Enzyme Hydrolysis ◽  
Stopped Flow ◽  
Sheep Liver ◽  
Dehydrogenase Reaction ◽  
Liver Aldehyde Dehydrogenase ◽  
Hydrolysis Of ◽  
Flow Experiments ◽  
Esterase Activities

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.

scholarly journals Kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase

1982 ◽  
Vol 203 (3) ◽  
pp. 617-627 ◽  
Author(s):  
G J Hart ◽  
F M Dickinson
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Properties ◽  
Stopped Flow ◽  
Flow Measurements ◽  
Sheep Liver ◽  
Low Concentrations ◽  
Mechanism Of Reaction ◽  
High Concentrations ◽  
Variable Substrate

The kinetic properties of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase (preparations that had been shown to be free from contamination with the corresponding mitochondrial enzyme) were investigated with both propionaldehyde and butyraldehyde as substrates. At low aldehyde concentrations, double-reciprocal plots with aldehyde as the variable substrate are linear, and the mechanism appears to be ordered, with NAD+ as the first substrate to bind. Stopped-flow experiments following absorbance and fluorescence changes show bursts of NADH production in the pre-steady state, but the observed course of reaction depends on the pre-mixing conditions. Pre-mixing enzyme with NAD+ activates the enzyme in the pre-steady state and we suggest that the reaction mechanism may involve isomeric enzyme-NAD+ complexes. High concentrations of aldehyde in steady-state experiments produce significant activation (about 3-fold) at high concentrations of NAD+, but inhibition at low concentrations of NAD+. Such behaviour may be explained by postulating the participation of an abortive complex in product release. Stopped-flow measurements at high aldehyde concentrations indicate that the mechanism of reaction under these conditions is complex.

scholarly journals Kinetics of Alkaline Phosphatase from Pig Kidney. Influence of complexing agents on stability and activity

1976 ◽  
Vol 153 (2) ◽  
pp. 151-157 ◽  
Author(s):  
B P Ackermann ◽  
J Ahlers
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ion ◽  
Complexing Agents ◽  
Pig Kidney ◽  
Low Concentrations ◽  
Sequential Addition ◽  
Quantitative Examination ◽  
High Concentrations ◽  
Kinetics Of ◽  
Active Centres

Metal ion-complexing agents, like KCN, EDTA etc., inactivate alkaline phosphatase of pig kidney. This inactivation is reversible at low concentrations of the complexing agents and irreversible at high concentrations. The reversible inhibition is probably due to removal of Zn2+ ions from the active site, where they are necessary for catalytic action, whereas the irreversible inhibition results from the removal of Zn2+ ions necessary for preservation of the structure. The inactivation is pseudo-first order. It depends on the concentration, size and charge of the complexing agents. β-Glycerophosphate and Mg2+ ions protect the enzyme from inactivation by complexing agents. Quantitative examination of the effect of substrate leads to a model that is similar to the “sequential model” proposed by D.E. Koshland, G. Nemethy & D. Filmer (1966) (Biochemistry 5, 365-385) to explain allosteric behavior of enzymes. It describes the sequential addition of two substrate molecules at two active centres of the dimer enzyme. The binding of the substrate molecules is accompanied by changes in the conformation, which lead to stabilization of the enzyme against attack by complexing agents.

scholarly journals Effect of disulfiram on the pre-steady-state burst in the reactions of sheep liver cytoplasmic aldehyde dehydrogenase

1987 ◽  
Vol 248 (3) ◽  
pp. 989-991 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Stopped Flow ◽  
Sheep Liver ◽  
Esterase Activities

Stopped-flow spectrophotometric experiments show that modification by disulfiram not only lowers the steady-state rates but also decreases the size of bursts seen in both dehydrogenase and esterase reactions catalysed by sheep liver cytoplasmic aldehyde dehydrogenase. This observation is consistent with the proposal that a catalytically essential group is modified by disulfiram and that this group mediates both dehydrogenase and esterase activities.

Metal-ion oxidations in solution. Part XIX. Redox pathways in the oxidation of penicillamine and glutathione by chromium(VI)

1977 ◽  
Vol 55 (18) ◽  
pp. 3335-3340 ◽  
Author(s):  
Alexander McAuley ◽  
M. Adegboyega Olatunji
Keyword(s):  
Ion Exchange ◽  
Redox Reaction ◽  
Metal Ion ◽  
Hydrogen Ion ◽  
Stopped Flow ◽  
Ion Concentrations ◽  
Flow Method ◽  
Chromium Vi ◽  
Reaction Proceeds ◽  
Transient Intermediate

Three moles of penicillamine or glutathione are required to reduce chromium(VI) to chromium(III). The kinetics and mechanism of the redox reaction have been studied using the stopped-flow method. The reaction proceeds via the formation of a transient intermediate (K1) which decomposes either in a proton-catalyzed pathway or by reaction with a second mole of thiol. The rate law[Formula: see text]where n = 1 for penicillamine and 2 for glutathione has been shown to hold over a range of thiol and hydrogen-ion concentrations. At 25 °C k2 = 14.3 ± 1.0 M−1 s−1 for penicillamine (ΔH≠ = 9 ± 2 kcal mol−1, ΔS≠ = −33 ± 6 cal K−1 mol−1) and 12.1 ± 0.4 M−1 s−1 for glutathione (ΔH≠ = 7 ± 2 kcal mol−1, ΔS≠ = −40 ± 5 cal K−1 mol−1). Several chromium(III) products have been identified by ion-exchange methods. The significance of the second-order pathways in these reactions is discussed.

scholarly journals Studies on the interaction between disulfiram and sheep liver cytoplasmic aldehyde dehydrogenase

1978 ◽  
Vol 175 (1) ◽  
pp. 83-90 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Enzyme Activity ◽  
Linear Relationship ◽  
Dehydrogenase Activity ◽  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Competitive Inhibitor ◽  
Enzyme Molecule ◽  
Covalent Interaction ◽  
Sheep Liver ◽  
Non Linear

The effect of disulfiram, [1-14C]disulfiram and some other thiol reagents on the activity of cytoplasmic aldehyde dehydrogenase from sheep liver was studied. The results are consistent with a rapid covalent interaction between disulfiram and the enzyme, and inconsistent with the notion that disulfiram is a reversible competitive inhibitor of cytoplasmic aldehyde dehydrogenase. There is a non-linear relationship between loss of about 90% of the enzyme activity and amount of disulfiram added; possible reasons for this are discussed. The remaining approx. 10% of activity is relatively insensitive to disulfiram. It is found that modification of only a small number of groups (one to two) per tetrameric enzyme molecule is responsible for the observed loss of activity. The dehydrogenase activity of the enzyme is affected more severely by disulfiram than is the esterase activity. Negatively charged thiol reagents have little or no effect on cytoplasmic aldehyde dehydrogenase. 2,2′-Dithiodipyridine is an activator of the enzyme.

scholarly journals Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase

1991 ◽  
Vol 275 (1) ◽  
pp. 207-210 ◽  
Author(s):  
T M Kitson ◽  
J P Hill ◽  
G G Midwinter
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Tryptic Digestion ◽  
Sheep Liver ◽  
Unequivocal Identification ◽  
Peptide Fractionation

Sheep liver cytoplasmic aldehyde dehydrogenase was labelled by reaction with the substrate p-nitrophenyl di[14C]methylcarbamate. After tryptic digestion and peptide fractionation the labelled residue was identified as Cys-302. This is the first unequivocal identification of the essential enzymic nucleophile in the esterase activity of aldehyde dehydrogenase. By implication, Cys-302 is probably also the residue that is acylated by aldehyde substrates and the first residue that is modified by disulfiram.

scholarly journals Effect of some thiocarbamate compounds on aldehyde dehydrogenase and implications for the disulfiram ethanol reaction

1991 ◽  
Vol 278 (1) ◽  
pp. 189-192 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Dehydrogenase Activity ◽  
Aldehyde Dehydrogenase ◽  
Esterase Activity ◽  
Negligible Effect ◽  
Weak Inhibitor ◽  
Low Concentration ◽  
The Third ◽  
Sheep Liver ◽  
Physiological Phenomenon

The effects of S-methyl diethyldithiocarbamate, S-methyl diethylmonothiocarbamate and bis(diethylcarbamoyl) disulphide on sheep liver cytoplasmic aldehyde dehydrogenase were investigated in vitro. The first compound has negligible effect. The second one is a weak inhibitor of the esterase activity of the enzyme and a weaker inhibitor of the dehydrogenase activity. A very low concentration of the third compound, however, acts as a potent inactivator of aldehyde dehydrogenase, similar in this respect to disulfiram, although somewhat slower to react. The possible involvement of these compounds in the physiological phenomenon known as the disulfiram ethanol reaction is discussed.

Prediction of Soluble Nickel Removal by Activated Sludge Using Free Metal Ion Concentrations

1987 ◽  
Vol 19 (3-4) ◽  
pp. 439-448 ◽  
Author(s):  
Jeppe S. Nielsen ◽  
Steve E. Hrudey ◽  
Frederick F. Cantwell
Keyword(s):  
Activated Sludge ◽  
Metal Ion ◽  
Municipal Sewage ◽  
Nickel Ion ◽  
Ion Concentrations ◽  
Untreated Sewage ◽  
Nickel Uptake ◽  
Nickel Removal ◽  
Free Metal ◽  
Free Metal Ion

Batch isotherm studies using spiked sewage samples containing a range of total soluble nickel concentrations typical of municipal sewage strongly suggested that it is the free (i.e. uncomplexed) nickel ion that is sorbed by activated sludge. Equations relating nickel uptake by activated sludge to free nickel ion concentrations and the extent of complexation in untreated sewage were developed and applied. Predicted and measured nickel removals generally agreed to within ± 30%.

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