scholarly journals Effect of ethanol on the redox state of the coenzyme bound to alcohol dehydrogenase studied in isolated hepatocytes

1987 ◽  
Vol 248 (2) ◽  
pp. 567-572 ◽  
Author(s):  
T Cronholm
Keyword(s):  
Alcohol Dehydrogenase ◽  
Redox State ◽  
Ethanol Oxidation ◽  
Equilibrium Composition ◽  
Isolated Hepatocytes ◽  
Indicator System ◽  
Female Rats ◽  
Equilibrium System ◽  
Efficient Removal ◽  
Redox Indicator

Hepatocytes were isolated from fed female rats and incubated with a redox indicator system consisting of cyclohexanone and unlabelled or perdeuterated cyclohexanol. The concentrations and deuterium contents of these were measured by g.l.c. and g.l.c.-m.s. of oxime t-butyldimethylsilyl derivatives. The equilibrium composition represented the redox state of the coenzyme bound to alcohol dehydrogenase, since 4-methylpyrazole inhibited the interconversion. Reduction appeared to be catalysed to a small extent also by an NADPH-dependent aldehyde reductase. The NADH/NAD+ ratio on alcohol dehydrogenase was 3 orders of magnitude higher in the presence of ethanol than in its absence. This redox shift has the degree expected from reported kinetic constants. The shift was due both to a decreased rate of oxidation and to an increased rate of reduction in the indicator system. The results indicate that the redox effect of ethanol on the free NAD system is due to efficient removal of acetaldehyde from a near-equilibrium system consisting of ethanol, acetaldehyde and bound coenzymes, together with dissociation of NADH from the enzyme. The effect on the redox state of the bound coenzyme was less marked when the ethanol was deuterated at C-1, indicating an isotope effect. The 2H excess in the cyclohexanol formed was about 70% of that in the [1,1-2H2]ethanol. This dilution, which is caused by binding of free NADH to the enzyme, indicates that reoxidation of cytosolic NADH partly limits the rate of ethanol oxidation.

scholarly journals Heterogeneity of the sn-glycerol 3-phosphate pool in isolated hepatocytes, demonstrated by the use of deuterated glycerols and ethanol

1984 ◽  
Vol 224 (3) ◽  
pp. 731-739 ◽  
Author(s):  
T Cronholm ◽  
T Curstedt
Keyword(s):  
Mass Spectrometry ◽  
Alcohol Dehydrogenase ◽  
Organic Acids ◽  
Ethanol Oxidation ◽  
Isolated Hepatocytes ◽  
Female Rats ◽  
Dihydroxyacetone Phosphate ◽  
Deuterium Excess ◽  
Relative Contribution ◽  
Glycerol Uptake

Hepatocytes were isolated from female rats and incubated with [1,1,3,3-2H4]glycerol or [2-2H]glycerol. The deuterium excess in phosphatidylcholines, sn-glycerol 3-phosphate and other organic acids was determined by g.l.c./mass spectrometry. The unlabelled fraction of the major phosphatidylcholines decreased exponentially, and the turnover was not changed by the presence of ethanol. The relative contribution of the two deuterated glycerols was about the same in the major phosphatidylcholine as in sn-glycerol 3-phosphate, indicating that formation by acylation of dihydroxyacetone phosphate is insignificant. [1,1,3,3-2H4]Glycerol had lost deuterium to a larger extent when it was incorporated in the phosphatidylcholine than when it was incorporated in sn-glycerol-3-phosphate, indicating that the phosphatidylcholines are formed from a separate pool of sn-glycerol 3-phosphate. Deuterium at C-2 was transferred between sn-glycerol 3-phosphate molecules to about 25%. Ethanol decreased the extent of deuterium transfer, the extent of glycerol uptake and the loss of deuterium at C-1 and C-3 in sn-glycerol 3-phosphate. The results indicate that the oxidation to dihydroxyacetone phosphate was inhibited by the NADH formed during ethanol oxidation. [2-2H]Glycerol also labelled an alcohol dehydrogenase substrate, malate and lactate, indicating oxidation of sn-glycerol 3-phosphate in the cytosol. The two acids appeared to be formed in reductions with different pools of NADH.

scholarly journals The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose

1984 ◽  
Vol 222 (2) ◽  
pp. 437-446 ◽  
Author(s):  
C Vind ◽  
N Grunnet
Keyword(s):  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Maximal Activity ◽  
Isolated Hepatocytes ◽  
Single Compartment ◽  
Dehydrogenase Reaction ◽  
Order Of Magnitude ◽  
Rate Limiting

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.

scholarly journals Rapid and delayed effects of epidermal growth factor on gluconeogenesis

1993 ◽  
Vol 294 (3) ◽  
pp. 865-872 ◽  
Author(s):  
C Soler ◽  
M Soley
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Redox State ◽  
Metabolic Flux ◽  
Isolated Hepatocytes ◽  
Delayed Effect ◽  
Delayed Effects ◽  
Mitochondrial Redox State ◽  
Glucose Synthesis ◽  
Epidermal Growth

Most reports on the effects of epidermal growth factor (EGF) on gluconeogenesis have indicated that such effects depend on the substrate used and are only observable after a lag time of 30-40 min. Recently, an immediate and transient effect of EGF on glucose synthesis was described in a perfused liver system. Here we extend the study of the effect of EGF on gluconeogenesis to isolated hepatocytes from fasted rats. The delayed effect of EGF on gluconeogenesis was studied by adding the substrate 40 min after the peptide. Under these conditions EGF increased glucose synthesis from pyruvate, decreased it when the substrate was lactate or glycerol and did not modify gluconeogensis from fructose or dihydroxyacetone. EGF did not affect the metabolic flux through glycolysis, determined as the production of lactate+pyruvate from 30 mM glucose. Furthermore, EGF did not modify the metabolic flux through pyruvate kinase, determined as the production of lactate+pyruvate from 1 mM dihydroxyacetone. The differing effects of EGF on gluconeogenesis depending on the substrate used can be explained by the effects of EGF on the cytosolic redox state (measured as the lactate/pyruvate ratio). About 20 min after the addition of EGF, the mitochondrial redox state (measured as the 3-hydroxybutyrate/acetoacetate ratio) decreased. This effect of EGF was blocked by ammonium, which also abolished the effect of the peptide on gluconeogenesis. Thus the effect of EGF at the mitochondrial level appears to be necessary for its effects on gluconeogenesis. Taken together, our results indicate that the delayed effects of EGF on gluconeogenesis are secondary to the effects of the peptide at both the mitochondrial and cytosolic levels. In addition to these delayed effects, we observed that EGF rapidly and transiently stimulated glucose synthesis from lactate, decreased the cytosolic redox state and increased oxygen consumption. All of these rapid effects required the presence of extracellular calcium and disappeared in the presence of rotenone, suggesting that this rapid effect of EGF on gluconeogenesis is secondary to the stimulation of mitochondrial respiration.

INTERACTION OF ETHANOL OXIDATION WITH GLUCURONIDATION IN ISOLATED HEPATOCYTES

Abstracts ◽  
1978 ◽  
pp. 461
Author(s):  
P. Moldéus ◽  
B. Andersson ◽  
A. Norling ◽  
M. Berggren
Keyword(s):  
Ethanol Oxidation ◽  
Isolated Hepatocytes

Regulation of ethanol metabolism in the rat

1987 ◽  
Vol 65 (5) ◽  
pp. 458-466 ◽  
Author(s):  
S. Cheema-Dhadli ◽  
F. A. Halperin ◽  
K. Sonnenberg ◽  
V. MacMillan ◽  
M. L. Halperin
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Maximum Velocity ◽  
Reducing Power ◽  
Ammonium Bicarbonate ◽  
Ethanol Metabolism ◽  
Urea Synthesis ◽  
Major Pathway ◽  
Nadh Generation

The purpose of these experiments was to examine the factors which regulate ethanol metabolism in vivo. Since the major pathway for ethanol removal requires flux through hepatic alcohol dehydrogenase, the activity of this enzyme was measured and found to be 2.9 μmol/(min∙g liver). Ethanol disappearance was linear for over 120 min in vivo and the blood ethanol fell 0.1 mM/min; this is equivalent to removing 20 μmol ethanol/min and would require that flux through alcohol dehydrogenase be about 60% of its measured maximum velocity. To test whether ethanol metabolism was limited by the rate of removal of one of the end products (NADH) of alcohol dehydrogenase, fluoropyruvate was infused to reoxidize hepatic NADH and to prevent NADH generation via flux through pyruvate dehydrogenase. There was no change in the rate of ethanol clearance when fluoropyruvate was metabolized. Furthermore, enhancing endogenous hepatic NADH oxidation by increasing the rate of urea synthesis (converting ammonium bicarbonate to urea) did not augment the steady-state rate of ethanol oxidation. Hence, transport of cytoplasmic reducing power from NADH into the mitochondria was not rate limiting for ethanol oxidation. In contrast, ethanol oxidation at the earliest time periods could be augmented by increasing hepatic urea synthesis.

scholarly journals Pseudomonas aeruginosa Ethanol Oxidation by AdhA in Low-Oxygen Environments

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Alex W. Crocker ◽  
Colleen E. Harty ◽  
John H. Hammond ◽  
Sven D. Willger ◽  
Pedro Salazar ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Physiological Role ◽  
Wild Type ◽  
Low Oxygen ◽  
Content Type ◽  
Anoxic Conditions ◽  
Oxygen Conditions ◽  
Acetate Accumulation

ABSTRACT Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA. The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosa. IMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

scholarly journals Inhibition of Horse Liver Alcohol Dehydrogenase by Methyltin Compounds

2005 ◽  
Vol 3 (3-4) ◽  
pp. 191-199 ◽  
Author(s):  
Pavel V. Bychkov ◽  
Tatyana N. Shekhovtsova ◽  
Elena R. Milaeva
Keyword(s):  
Alcohol Dehydrogenase ◽  
Ethanol Oxidation ◽  
Calculated Data ◽  
Enzyme Protein ◽  
Horse Liver Alcohol Dehydrogenase ◽  
Tin Compounds ◽  
Liver Alcohol Dehydrogenase ◽  
Horse Liver ◽  
Oxidative Agents ◽  
Slight Inhibition

The study of inorganic tin (SnCl2, SnCl4) and methyltin compounds (MeSnCl3, Me2SnCI2, Me3SnCl) effects on the enzymatic activity of alcohol dehydrogenase (ADH) in the reaction of ethanol oxidation has been carried out. The experimental results of the study show that inorganic tin and methyltin substances induce slight inhibition of the catalytic activity of horse liver alcohol dehydrogenase (HLADH), unable to be improved during pre-incubation with the enzyme. The conditions for carrying out the kinetic investigation of the mentioned phenomenon were optimized and as it turned out the mechanism of methyltin trichloride action, as the most effective methyltin inhibitor, is more complex than the proposed interaction of the metal atom with SH-groups of the enzyme protein. It was demonstrated that the tin compounds act in the same manner as methylmercury compounds and might serve as oxidative agents towards the co-enzyme NADH. Kinetic data on MeSnCl3were calculated. Data acquired on NAD-dependent ADH from horse liver and those regarding NAD-dependent LDH from sturgeon liver were compared.

scholarly journals The active (ADHa) and inactive (ADHi) forms of the PQQ-alcohol dehydrogenase from Gluconacetobacter diazotrophicus differ in their respective oligomeric structures and redox state of their corresponding prosthetic groups

2012 ◽  
Vol 328 (2) ◽  
pp. 106-113 ◽  
Author(s):  
Saúl Gómez-Manzo ◽  
Alejandra Abigail González-Valdez ◽  
Jesús Oria-Hernández ◽  
Horacio Reyes-Vivas ◽  
Roberto Arreguín-Espinosa ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Redox State ◽  
Gluconacetobacter Diazotrophicus ◽  
Prosthetic Groups
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