Der Einfluß der Wurzelanaerobiose auf Äthanol-, Laktat- und Glukosegehalte sowie auf die Aktivitäten von ADH und LDH in Weizenkeimpflanzen

The effect of anaerobiosis of wheat seedling roots during 6 consecutive days on contents of ethanol, lactate and glucose in roots and shoots and on the exudation of ethanol from roots to the medium was examined. Activities of alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) were determined. After 36 h of anaerobiosis the concentration of ethanol in roots increased temporarily about 6 times and after 6 days it decreased to the level of control plants. The exudation of ethanol from roots to the medium showed similar pattern. The content of lactate was unaffected by anaerobiosis. In contrast, the content of glucose in roots of seedlings increased already after 1 day of anaerobiosis about 2 times and this higher level of glucose was noticed during consecutive 5 days. Anaerobiosis of roots caused an increase in the activity of ADH in both roots and shoots but the increase was not related to the content of ethanol in tissues, or exudated to the medium. The activity of LDH was unaffected by this factor. The results are discussed in relation to the limitation of energy supply of plants grown under root anaerobiosis.     

beta-Amylase from Mustard (Sinapis alba L.) Cotyledons : Immunochemical Evidence for Synthesis de Novo during Photoregulated Seedling Development

In barley (Hordeum vulgare L.), alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) are induced by anaerobiosis in both aleurone layers and roots. Under aerobic conditions, developing seeds of cv Himalaya accumulate ADH activity, which survives seed drying and rehydration. This activity consists almost entirely of the ADH1 homodimer. Activity of LDH also increases during seed development, but the level of activity in dry or rehydrated seeds is very low, indicating that this enzyme may not be involved in anaerobic glycolysis during the initial stages of germination. In contrast to ADH, the LDH isozymes present in developing seeds are similar to those found in uninduced and induced roots. Developmental expression of ADH and LDH was monitored from 0 to 24 days postgermination. Neither activity was induced to any extent in the germinating seeds; however, both enzymes were highly induced by anoxia in root tissue during development. Based on gel electrophoresis, this increase in activity results from the differential expression of different Adh and Ldh genes in root tissue. The changes in ADH and LDH activity levels were matched by changes in the amount of these particular proteins, indicating that the increase in activity results from de novo synthesis of these two proteins. The level of inducible LDH activity in an ADH1⁻ mutant was not found to differ from cv Himalaya. We suggest that although the ADH⁻ plants are more susceptible to flooding, they are not capable of responding to the lack of ADH1 activity by increasing the amount of LDH activity in root tissue.     

Gene regulation during anaerobiosis in soya roots

The proteins of soybean roots undergoing anaerobiosis can be grouped into three classes. Class 1 proteins are induced severalfold and at least 28 of these were identified by in vivo labeling. These proteins include the enzymes alcohol dehydrogenase (ADH), fructose aldolase, pyruvate decarboxylase, phosphoglucomutase, and lactate dehydrogenase. Class 2 proteins include such enzymes as glucose phosphate isomerase, sucrase, and malate dehydrogenase; their specific activity remains constant in aerobiosis or anaerobiosis. The third class of proteins includes those enzymes such as peroxidase whose activity decreases more than 90% after just 1 day in anaerobiosis. Immunoblotting coupled with two-dimensional chromatography of in vitro translated plant extracts demonstrated that ADH level during anaerobiosis is controlled by its mRNA concentration. Little or no mRNA for ADH was detected in aerobically grown roots. This suggests that the increased level of ADH activity is due to de novo synthesis of the mRNA rather than activation of a sequestered mRNA or superactivation of the protein.     

The variations in isoenzyme patterns of alcohol dehydrogenase and their substrate specificity in germinating pea seeds

In pea alcohol dehydrogenase (PADH) four isoenzymes were detected with the same mobility in one-and two-day germinating seeds; in three-and four-day seedlings the isoenzyme fastest moving towards the anode was lacking. These isoenzymes did not differ in substrate specificity to ethanol, propanol, butanol, and allyl alcohol, but only three of them reacted with isobutanol, and two with cyclohexanol. On germination of seeds in actinomycin D at a concentration of 30 μg ml^-1 two isoenzymes disappeared and the activity of the other two was considerably lower.     

Losses of Alcohol and Alcohol Dehydrogenase Activity in Germinating Seeds

Losses of alcohol, which had accumulated under anaerobic conditions,occurred during the germination of several species of seedswhich could not be attributed to the volatility of the alcohol.It is suggested that utilization of the alcohol by the seedsmay occur. From the seeds, an active alcohol dehydrogenase,which is mainly confined to the cotyledons in pea seeds, canbe extracted. The activity of the enzyme decreases as the cotyledonsgrow older during germination. The properties of the enzymehave been investigated.     

Inhibition of hepatic gluconeogenesis by ethanol

1. Gluconeogenesis from 10mm-lactate in the perfused liver of starved rats is inhibited by ethanol. The degree of inhibition reached a maximum of 66% at 10mm-ethanol under the test conditions and decreased at higher ethanol concentrations. The concentration-dependence of the inhibition is paralleled by the concentration-dependence of the activity of alcohol dehydrogenase. The enzyme is also inhibited by ethanol concentrations above 10mm. 2. Gluconeogenesis from pyruvate is not inhibited by ethanol. 3. The degree of the inhibition of gluconeogenesis from lactate by ethanol depends on the concentration of lactate and other oxidizable substances, e.g. oleate, in the perfusion medium. 4. Ethanol also inhibits, to different degrees, gluconeogenesis from glycerol, dihydroxyacetone, proline, serine, alanine, fructose and galactose. 5. The inhibition of gluconeogenesis from lactate by ethanol is reversed by acetaldehyde. 6. Pyrazole, a specific inhibitor of alcohol dehydrogenase, also reverses the inhibition of gluconeogenesis by ethanol. 7. Gluconeogenesis in kidney cortex, where the activity of alcohol dehydrogenase is very low, is not inhibited by ethanol. 8. Kidney cortex, testis, ovary, uterus and certain tissues of the alimentary tract were the only rat tissues, apart from the liver, that showed measurable alcohol dehydrogenase activity. 9. The concentrations of pyruvate in the liver were decreased to about one-fifth by ethanol. 10. The concentration of lactate in the perfused liver was about 3mm below that of the perfusion medium 30min. after the addition of 10mm-lactate. 11. The great majority of the findings support the view that the inhibition of gluconeogensis by ethanol is caused by the alcohol dehydrogenase reaction, which decreases the [free NAD(+)]/[free NADH] ratio. The decrease lowers the concentration of pyruvate and this is the immediate cause of the inhibition of gluconeogenesis from lactate, alanine and serine: the fall in the concentration of pyruvate lowers the rate of the pyruvate carboxylase reaction, one of the rate-limiting reactions of gluconeogenesis. The cause of the inhibition of gluconeogenesis from other substrates is discussed.     

Identification and overexpression of a bifunctional aldehyde/alcohol dehydrogenase responsible for ethanol production in Thermoanaerobacter mathranii

Thermoanaerobacter mathranii contains four genes, adhA, adhB, bdhA and adhE, predicted to code for alcohol dehydrogenases involved in ethanol metabolism. These alcohol dehydrogenases were characterized as NADP(H)-dependent primary alcohol dehydrogenase (AdhA), secondary alcohol dehydrogenase (AdhB), butanol dehydrogenase (BdhA) and NAD(H)-dependent bifunctional aldehyde/alcohol dehydrogenase (AdhE), respectively. Here we observed that AdhE is an important enzyme responsible for ethanol production in T. mathranii based on the constructed adh knockout strains. An adhE knockout strain fails to produce ethanol as a fermentation product, while other adh knockout strains showed no significant difference from the wild type. Further analysis revealed that the ΔadhE strain was defective in aldehyde dehydrogenase activity, but still maintained alcohol dehydrogenase activity. This showed that AdhE is the major aldehyde dehydrogenase in the cell and functions predominantly in the acetyl-CoA reduction to acetaldehyde in the ethanol formation pathway. Finally, AdhE was conditionally expressed from a xylose-induced promoter in a recombinant strain (BG1E1) with a concomitant deletion of a lactate dehydrogenase. Overexpressions of AdhE in strain BG1E1 with xylose as a substrate facilitate the production of ethanol at an increased yield.     

New mutants resistant to glucose repression affected in the regulation of the NADH reoxidation

Spontaneous mutants resistant to vanadate, arsenate or thiophosphate were isolated from a haploid strain of Saccharomyces cerevisiae. These three anions have an inhibitory effect on some mitochondrial functions and at the level of glyceraldehyde 3-phosphate dehydrogenase, a glycolysis enzyme. All the selected mutants had the same phenotype: they were deficient in alcohol dehydrogenase I, the terminal enzyme of the glycolysis, and possessed a high content of cytochrome c oxidase, the terminal enzyme of the respiratory chain. Moreover, cytochrome c oxidase biosynthesis had become insensitive to the catabolite repression, while the biosynthesis of the other enzymes sensitive to this phenomenon were always inhibited by glucose. Metabolic effects of this pleiotropic mutation manifested themselves in the following ways. 1. Growth rate and final cell mass were enhanced, compared to the wild type, when cells were grown on glucose or on glycerol, but not on lactate or ethanol. 2. Growth under anaerobiosis was nil and mutants did not ferment. 3. Mitochondrial respiration of the mutant strains was identical to the wild type with succinate or 2-oxo-glutarate as substrate, and weak with ethanol. But with added NADH, respiration rate of the mutants was higher than that of the wild type and partially insensitive to antimycin, even when cells were grown in repression conditions. It is postulated that in mutants strains, NADH produced at the level of glyceraldehyde 3-phosphate dehydrogenase, failing to be reoxidized via alcohol dehydrogenase, could be reoxidized with a high turnover owing to the enhancement of the amount of cytochrome c oxidase. Since NADH reoxidation is partially insensitive to antimycin, a secondary pathway going from external NADH dehydrogenase to cytochrome c oxidase is suggested.     

Sull'Attivazione Funzionale dei Semi Germinanti

Abstract

On the functional activation of germinanting seeds. Note V. Alcohol dehydrogenase activity. — The activity of alcohol dehydrogenase is determined by spettrophotometric method on acetone precipitates of dry and germinating seeds of leguminous and graminaceous plants.

The activity of alcohol dehydrogenase is present, even with remarkable different values, in all seeds and in their various parts, singularly examined.

During germination in aerobic conditions, after a first, not strong, increase, the alcohol dehydrogenase activity sensibly decreases.     

Pyruvate metabolism in rice coleoptiles under anaerobiosis

Relative importance of ethanolic, lactate and alanine fermentation pathways was estimated in coleoptiles of rice seedlings (Oryza sativa L.) subjected to anoxic stress. The in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and alanine aminotransferase (AlaAT, EC 2.6.1.2) in the coleoptiles increased in anoxia, whereas no significant increase was measured in lactate dehydrogenase (LDH, EC 1.1.1.27) activity. At 48 h, the ADH, PDC and AlaAT activities in anoxic coleoptiles were 62-, 15- and 7.6-fold greater, respectively, than those in the presence of oxygen. Ethanol and alanine in the coleoptiles accumulated rapidly under anoxia, increasing by 48 h, 57- and 5.6-fold compared with those in the presence of oxygen, respectively. However, lactate concentration did not increase and no initial burst of lactate production was detected. The relative ratio of carbon flux from pyruvate to ethanol, lactate and alanine in anoxic coleoptiles was estimated to be 92, 1 and 7% of the total carbon flux, respectively. These results suggest that the potential carbon flux from pyruvate to ethanol may be much greater than the potential flux from pyruvate to lactate and alanine in rice coleoptiles during anoxia.     

Redox potential dependence of photophosphorylation and electron transfer in continuous illumination of Rhodopseudomonas sphaeroides chromatophores

The rate of ethanol elimination in fed and fasted rats can be predicted based on the liver content of alcohol dehydrogenase (EC 1.1.1.1), the steady-state rate equation, and the concentrations of substrates and products in liver during ethanol metabolism. The specific activity, kinetic constants, and multiplicity of enzyme forms are similar in fed and fasted rats, although the liver content of alcohol dehydrogenase falls 40% with fasting. The two major forms of the enzyme were separated and found to have very similar kinetic properties. The rat alcohol dehydrogenase is subject to substrate inhibition by ethanol at concentrations above 10 mM and follows a Theorell-Chance mechanism. The steady-state rate equation for this mechanism predicts that the in vivo activity of the enzyme is limited by NADH product inhibition at low ethanol concentrations and by both NADH inhibition and substrate inhibition at high ethanol concentrations. When the steady-state rate equation and the measured concentrations of substrates and products in freeze-clamped liver of fed and fasted rats metabolizing alcohol are employed to calculate alcohol oxidation rates, the values agree very well with the actual rates of ethanol elimination determined in vivo.     

Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t _d=2.7–6.4 h) and on ethanol (t _d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t _d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.     

Evaluation of the importance of lactate for the activation of ethanolic fermentation in lettuce roots in anoxia

According to the Davies–Roberts hypothesis, plants primarily respond to oxygen limitation by a burst of lactate production and the resulting pH drop in the cytoplasm activates ethanolic fermentation. To evaluate this system in lettuce ( Lactuca sativa L.), seedlings were subjected to anoxia and in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) and concentrations of ethanol, acetaldehyde and lactate were determined in roots of the seedlings. The in vitro activities of ADH and PDC in the roots increase in anoxia, whereas no significant increase was measured in LDH activity. At 6 h, the ADH and PDC activities in the roots kept in anoxia were 2.8- and 2.9-fold greater than those in air, respectively. Ethanol and acetaldehyde in the roots accumulated rapidly in anoxia and increased 8- and 4-fold compared with those in air by 6 h, respectively. However, lactate concentration did not increase and an initial burst of lactate production was not found. Thus, ethanol and acetaldehyde production occurred without an increase in lactate synthesis. Treatments with antimycin A and salicylhydroxamic acid, which are respiratory inhibitors, to the lettuce seedlings in the presence of oxygen increased the concentrations of ethanol and acetaldehyde but not of lactate. These results suggest that ethanolic fermentation may be activated without preceding activation of lactate fermentation and may be not regulated by oxygen concentration directly.     

Purification and Properties of Rape Alcohol Dehydrogenase

Germinating seeds with the highest specific activity (24 hour germination) were used for isolation of alcohol dehydrogenase, ADH, from rape (Brassica napus L. cv. T?ebi?ská). The rape ADH was purified by fractionation with ammonium sulphate, desalting on Sephadex G 25, chromatography on DEAE cellulose and gel filtration on Sephadex G 150. Using this isolation procedure, enzyme with a specific activity 85.6 times higher than that of the crude extract was obtained. The molecular weight of the enzyme obtained is 66.000. The enzyme is a metallo-enzyme containing sulfhydryl groups as evidence by the inhibitory effect of chelating compounds and thiol reagents. The optimum pH for the oxidation of ethanol is 8.5 and for reduction of acetaldehyde 7.0. The enzyme exhibits a relatively wide substrate specificity towards alcohols. Dimethyl-sulphoxide (DMSO), some amides and oximes and some intermediates of the carbohydrate metabolism act as ADH inhibitors, ATP as analogue of NAD also exhibits an inhibitory effect. The inhibitory effect of heterocyclic substances (pyrazol, imidazol, pyridine) is similar to the effect on liver alcohol dehydrogenase.     

Effect of Thiamine on Ethanol and Pyruvate Production in Helminthosporium maydis

Growth of the fungus Helminthosporium maydis race T in a basal glucose-l-asparagine liquid medium, pH 5, is inhibited by thiamine-HCl. Analysis of the media for organic acids reveals that the extracellular pyruvate concentration decreases as the thiamine-HCl concentration of the medium increases. Extracellular ethanol, in contrast to pyruvate, increases in concentration as the thiamine-HCl concentration of the medium increases under both aerobic and anaerobic conditions.The changes in ethanol and pyruvate levels in the presence of thiamine-HCl occur via a thiamine-mediated increase in the activity of pyruvate decarboxylase but not alcohol dehydrogenase. This increase in pyruvate decarboxylase activity appears to be due to an increase in the quantity of enzyme present rather than an activation of pre-existing enzyme. Whereas thiamine-pyrophosphate stimulates pyruvate decarboxylase activity in vitro, thiamine-HCl has no effect. Neither thiamine derivative affects alcohol dehydrogenase activity. The increase in pyruvate decarboxylase activity which accompanies an increase in the thiamine-HCl concentration of the medium is correlated with a decrease in the level of intracellular pyruvate.     

Investigations of the anaerobic metabolism of the polychaete wormNereis diversicolor M.

Summary The anaerobic metabolism ofNereis diversicolor M. was studied during various periods of experimental anaerobiosis.The degradation of glycogen is shown to be the main source of anaerobic energy production. During first hours of anaerobiosis, aspartate, in addition to glycogen, is metabolized in considerable quantities.Five acids were found to accumulate as end-products: alanine, D-lactate, succinate, acetate and propionate (Table 2).Alanine is accumulated only during the first hours of anaerobiosis. The increase in alanine is correlated with a decrease in aspartate.D-Lactate is the main end-product during the first 24 h of anaerobiosis, and continues to be produced even during prolonged anaerobiosis. In accordance with lactate production,Nereis diversicolor possesses a high glycolytic capacity (Table 4).The major end-products of long term fermentation are propionate and acetate. In contrast to other end-products, these acids are excreted in substantial amounts.Abbreviations GAPDH glyceraldehydephosphate dehydrogenase, EC 1.2.1.12 - LDH lactate dehydrogenase, EC 1.1.1.27 - GOT aspartate aminotransferase, EC 2.6.1.1 - GPT alanine aminotransferase, EC 2.6.1.2 - MDH malate dehydrogenase, EC 1.1.1.37 Supported by Deutsche Forschungsgemeinschaft (Gr 456/5 and Gr 456/6)     

The Appearance of Lactic Acid in Phaseolus Seeds Germinating under Wet Conditions

Under normal conditions of germination, there is never muchlactate in bean cotyledons, but when beans are germinated underwet conditions lactate accumulates for about 30 h and then slowlydisappears, as radicle emergence begins. Removal of the testaefrom soaked seeds prevents subsequent lactate accumulation.Lactate also appears in beans germinated under nitrogen. Lactate dehydrogenase activity is enhanced in wet or anaerobicconditions. Incubation with pyruvate increased lactate dehydrogenaseby 50 per cent. There was also a temporary accumulation of alcohol in beansgrown under wet conditions. Under wet conditions the production of respiratory carbon dioxidewas particularly slow during the period of lactate accumulation.     

Enzymatic Conversion of Volatile Metabolites in Dry Seeds during Storage

Dry seeds can transform volatile metabolites via enzymes pre-existingin them. The interconversion between acetaldehyde and ethanoloccurred when they were applied gaseously to seeds of rice,lettuce, pea, cocklebur during storage at different relativehumidities (RH) at either 23°C or –3.5°C. Interconversionsbetween the compounds decreased with decreasing RH from 75%to 12% regardless of seed species and storage temperature, butit was still detectable even at 12% RH at –3.5°C.However, the conversion from acetaldehyde to ethanol did notoccur when seeds were killed by heating prior to storage. 2-Methoxyethanol,a competitive inhibitor of alcohol dehydrogenase (ADH) (EC 1.1.1.1 [EC] ),suppressed the transformation from ethanol to acetaldehyde inlettuce seeds. Therefore, ADH pre-existing in dry seeds maybe involved in the interconversion between ethanol and acetaldehydein dry seeds. Propanal, also a substrate of ADH, could be transformedto propanol during storage. However, methanol, which is nota substrate of this enzyme, was hardly converted to formaldehyde.Ethylacetate, applied to seeds during storage, was hydrolyzedby both lettuce and rice seeds, and the amount of hydrolysisincreased with increasing RH. Similarly, other carboxylic esters,such as methylacetate, ethylformate and ethylpropionate, couldbe hydrolyzed by rice, pea, and lettuce seeds. Little hydrolysisof ethylacetate occurred in the seeds killed by heating beforestorage, suggesting that ethylacetate may be hydrolyzed by carboxylic-esterhydrolase (EC 3.1.1.1 [EC] ). On the other hand, the production ofethylacetate through esterification was demonstrated by additionof gaseous acetate and ethanol in lettuce seeds, which occurredmore greatly at 12% RH than at 75% RH. These findings were discussedin relation to the mechanism of seed aging. (Received September 3, 1994; Accepted November 14, 1994)     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

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

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.