Identification of Pig Brain Aldehyde Reductases with the High-Km Aldehyde Reductase, the Low-Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase

Four NADPH-dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high-Km aldehyde reductase, glucuronate reductase, and tissue-specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low-Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin-9-ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of gamma-hydroxybutyrate.     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.     

Effect of acetaldehyde and cyanamide on the metabolism of formaldehyde by hepatocytes, mitochondria, and soluble supernatant from rat liver

Formaldehyde can be metabolized primarily by two different pathways, one involving oxidation by the low-Km mitochondrial aldehyde dehydrogenase, the other involving a specific, glutathione-dependent, formaldehyde dehydrogenase. To estimate the roles played by each enzyme in formaldehyde metabolism by rat hepatocytes, experiments with acetaldehyde and cyanamide, a potent inhibitor of the low-Km aldehyde dehydrogenase were carried out. The glutathione-dependent oxidation of formaldehyde by 100,000g rat liver supernatant fractions was not affected by either acetaldehyde or by cyanamide. By contrast, the uptake of formaldehyde by intact mitochondria was inhibited 75 to 90% by cyanamide. Acetaldehyde inhibited the uptake of formaldehyde by mitochondria in a competitive fashion. Formaldehyde was a weak inhibitor of the oxidation of acetaldehyde by mitochondria, suggesting that, relative to formaldehyde, acetaldehyde was a preferred substrate. In isolated hepatocytes, cyanamide, which inhibited the oxidation of acetaldehyde by 75 to 90%, produced only 30 to 50% inhibition of formaldehyde uptake by cells as well as of the production of 14CO2 and of formate from [14C]formaldehyde. The extent of inhibition by cyanamide was the same as that produced by acetaldehyde (30-40%). In the presence of cyanamide, acetaldehyde was no longer inhibitory, suggesting that acetaldehyde and cyanamide may act at the same site(s) and inhibit the same formaldehyde-oxidizing enzyme system. These results suggest that, in rat hepatocytes, formaldehyde is oxidized by cyanamide- and acetaldehyde-sensitive (low-Km aldehyde dehydrogenase) and insensitive (formaldehyde dehydrogenase) reactions, and that both enzymes appear to contribute about equally toward the overall metabolism of formaldehyde.     

Purification and properties of low-Km aldehyde reductase from ox brain

A low-Km aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), which may be identical with aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21), has been purified from ox brain to homogeneity. It was shown to be a monomer with Mr values of 31 000 and 35 100 being obtained by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, respectively. The enzyme catalyses the NADPH-dependent reduction of a number of aromatic and sugar aldehydes. The activity of the enzyme with 133 microM NADH was about one-third of that with 120 microM NADPH. Activity with both these coenzymes was optimum at pH 6.2 and was inhibited by increasing the ionic strength with KCl, NaCl or NaNO3. In contrast, the activity was stimulated by sodium phosphate. The activity with NADH as the coenzyme was more sensitive to stimulation by phosphate and to inhibition by increasing ionic strength than that determined with NADPH.     

Inhibition of hepatic aldehyde dehydrogenases in the rat by calcium carbimide (calcium cyanamide)

Oral administration of 7.0 mg/kg calcium carbimide (calcium cyanamide, CC) to the rat produced differential inhibition of hepatic aldehyde dehydrogenase (ALDH) isozymes, as indicated by the time-course profiles of enzyme activity. The low-Km mitochondrial ALDH was most susceptible to inhibition following CC administration, with complete inhibition occurring at 0.5 h and return to control activity at 96 h. The low-Km cytosolic and high-Km mitochondrial, cytosolic, and microsomal ALDH isozymes were inhibited to a lesser degree and (or) for a shorter duration compared with the mitochondrial low-Km enzyme. The time course of carbimide, the hydrolytic product of CC, was determined in plasma following oral administration of 7.0 mg/kg CC to the rat. The maximum plasma carbimide concentration (102 ng/mL) occurred at 1 h and the apparent elimination half-life in plasma was 1.5 h. Carbimide was not measurable in the liver during the 6.5 h time interval when carbimide was present in the plasma. There were negative, linear correlations between plasma carbimide concentration and hepatic low-Km mitochondrial, low-Km cytosolic, and high-Km microsomal ALDH activities. In vitro studies demonstrated that carbimide, at concentrations obtained in plasma following oral CC administration, produced only 19% inhibition of low-Km mitochondrial ALDH and no inhibition of low-Km cytosolic and high-Km microsomal ALDH isozymes. These data demonstrate that carbimide, itself, is not primarily responsible for hepatic ALDH inhibition in vivo following oral CC administration. It would appear that carbimide must undergo metabolic conversion in vivo to inhibit hepatic ALDH enzymes, which is supported by the observation of no measurable carbimide in the liver when ALDH was maximally inhibited following oral CC administration.     

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the Ki for crotonaldehyde was about 5 to 20 microM. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low-Km mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.     

Purification and Characterization of Four NADPH-Dependent Aldehyde Reductases from Pig Brain

By a procedure involving ammonium sulfate precipitation, gel filtration, and affinity chromatography, four aldehyde reductases (ALRs) were purified to enzymatic homogeneity from pig brain. These enzymes, designated ALR1, ALR2, ALR3, and succinic semialdehyde reductase were chemically and physically identical with, respectively, the high-Km aldehyde reductase, the low-Km aldehyde reductase, carbonyl reductase, and succinic semialdehyde reductase of other tissues and species. The purification procedure allows the purification of these enzymes from the same tissue homogenate in amounts sufficient for characterization and other enzymatic studies. This methodology should be applicable to the simultaneous and rapid purification of aldehyde reductases from other tissues.     

Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase

During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.     

Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications

Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (k(cat)=142 min(-1) and k(cat)/K(m)=1.8x10(7) M(-1) min(-1)). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione.     

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme.

Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.     

Aldehyde dehydrogenase activity as the rate-limiting factor for acetaldehyde metabolism in rat liver

The velocity of acetaldehyde metabolism in rat liver may be governed either by the rate of regeneration of NAD from NADH through the electron transport system or by the activity of aldehyde dehydrogenase (ALDH). Measurements of oxygen consumption revealed that the electron transport system was capable of reoxidizing ALDH-generated NADH much faster than it was produced and hence was not rate-limiting for aldehyde metabolism. To confirm that ALDH activity was the rate-limiting factor, low-Km ALDH in slices or intact mitochondria was partially inhibited by treatment with cyanamide and the rate of acetaldehyde metabolism measured. Any inhibition of low-Km ALDH resulted in a decreased rate of acetaldehyde metabolism, indicating that no excess of low-Km ALDH existed. Approximately 40% of the metabolism of 200 microM acetaldehyde in slices was not catalyzed by low-Km ALDH. Fifteen of this 40% was catalyzed by high-Km ALDH. A possible contribution by aldehyde oxidase was ruled out through the use of a competitive inhibitor, quinacrine. Acetaldehyde binding to cytosolic proteins may account for the remainder. By measuring acetaldehyde accumulation during ethanol metabolism, it was also established that low-Km ALDH activity was rate-limiting for acetaldehyde oxidation during concomitant ethanol oxidation.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

Aldose and aldehyde reductases in human tissues

Immunochemical characterizations of aldose reductase and aldehyde reductases I and II, partially purified by DEAE-cellulose (DE-52) column chromatography from human tissues, were carried out by immunotitration, using antisera raised against the homogenous preparations of human and bovine lens aldose reductase and human placenta aldehyde reductase I and aldehyde reductase II. Anti-aldose reductase antiserum cross-reacted with aldehyde reductase I, anti-aldehyde reductase I antiserum cross-reacted with aldose reductase and anti-aldehyde reductase II antiserum precipitated aldehyde reductase II, but did not cross-react with aldose reductase or aldehyde reductase I from all the tissues examined. DE-52 elution profiles, substrate specificity and immunochemical characterization indicate that aldose reductase is present in human aorta, brain, erythrocyte and muscle; aldehyde reductase I is present in human kidney, liver and placenta; and aldehyde reductase II is present in human brain, erythrocyte, kidney, liver, lung and placenta. Monospecific anti-α and anti-β antisera were purified from placenta anti-aldehyde reductase I antiserum, using immunoaffinity techniques. Anti-α antiserum precipitated both aldehyde reductase I and aldose reductase, whereas anti-β antibodies cross-reacted with only aldehyde reductase I. Based on these studies, a three gene loci model is proposed to explain the genetic interrelationships among these enzymes. Aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α and β subunits and aldehyde reductase II is a monomer of δ subunits.     

Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte

Aldose reductase (EC 1.1.1.21) and aldehyde reductase II (L-hexonate dehydrogenase, EC 1.1.1.2) have been purified to homogeneity from human erythrocytes by using ion-exchange chromatography, chromatofocusing, affinity chromatography, and Sephadex gel filtration. Both enzymes are monomeric, Mr 32,500, by the criteria of the Sephadex gel filtration and polyacrylamide slab gel electrophoresis under denaturing conditions. The isoelectric pH's for aldose reductase and aldehyde reductase II were determined to be 5.47 and 5.06, respectively. Substrate specificity studies showed that aldose reductase, besides catalyzing the reduction of various aldehydes such as propionaldehyde, pyridine-3-aldehyde and glyceraldehyde, utilizes aldo-sugars such as glucose and galactose. Aldehyde reductase II, however, did not use aldo-sugars as substrate. Aldose reductase activity is expressed with either NADH or NADPH as cofactors, whereas aldehyde reductase II can utilize only NADPH. The pH optima for aldose reductase and aldehyde reductase II are 6.2 and 7.0, respectively. Both enzymes are susceptible to the inhibition by p-hydroxymercuribenzoate and N-ethylmaleimide. They are also inhibited to varying degrees by aldose reductase inhibitors such as sorbinil, alrestatin, quercetrin, tetramethylene glutaric acid, and sodium phenobarbital. The presence of 0.4 M lithium sulfate in the assay mixture is essential for the full expression of aldose reductase activity whereas it completely inhibits aldehyde reductase II. Amino acid compositions and immunological studies further show that erythrocyte aldose reductase is similar to human and bovine lens aldose reductase, and that aldehyde reductase II is similar to human liver and brain aldehyde reductase II.     

On-bead combinatorial techniques for the identification of selective aldose reductase inhibitors

Aldose reductase (AKR1B1; ALR2; E.C. 1.1.1.21) is an NADPH-dependent carbonyl reductase which has long been associated with complications resulting from the elevated blood glucose often found in diabetics. The development of effective inhibitors has been plagued by lack of specificity which has led to side effects in clinical trials. To address this problem, a library of bead-immobilized compounds was screened against fluorescently labeled aldose reductase in the presence of fluorescently labeled aldehyde reductase, a non-target enzyme, to identify compounds which were aldose reductase specific. Picked beads were decoded via novel bifunctional bead mass spec-based techniques and kinetic analysis of the ten inhibitors which were identified using this protocol yielded IC50 values in the micromolar range. Most importantly, all of these compounds showed a preference for aldose reductase with selectivities as high as approximately 7500-fold. The most potent of these exhibited uncompetitive inhibition versus the carbonyl-containing substrate D/L-glyceraldehyde with a Ki of 1.16 microM.     

Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications

Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (k_cat=142 min⁻¹ and k_cat/K_m=1.8×10⁷ M⁻¹ min⁻¹). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione.     

Age-dependent peculiarities modulation of activity of aldehyde scavenger enzymes in mitochondria of rat thigh muscle during stress

The purpose of this study was a comparative investigation of activity of aldehyde scavenger enzymes in mitochondrial fraction of a thigh muscle in intact and immobilized rats of different ages. It has been shown that 12-month-old (adult) rats have high basal levels of aldehyde dehydrogenase, aldehyde reductase and glutathione transferase activity in mitochondrial fraction of thigh muscle. Aldehyde dehydrogenase activity increases during immobilization stress in adult rats. This change promote to enhance the effectiveness of utilization of carbonyl products of free radical oxidation in mitochondria of skeletal muscle of 12-month-old rats during stress. Immobilization of old and pubertal rats is accompanied by metabolic preconditions leading to accumulation of endogenous aldehydes in mitochondria, and, as a result, to the injury of muscular fibers and intensification of sarcopenia manifestations.     

Stable preparation of aldose reductase isoenzymes from human placenta

An efficient, large-scale purification has been achieved for two aldose reductase isoenzymes from human placenta in stable form. The procedure included ammonium sulfate fractionation (45-75%), followed by chromatographies on Matrex Red A, DE-52 cellulose, and Matrex Orange A. The preparations were stable for at least 3 months at 3 degrees C. IC50 values toward sorbinil were similar to those reported for crude or partially purified enzymes, indicating that they retained native structures during the purification steps. The molecular weights of purified GAR1 and GAR2, named according to their order of elution with a salt gradient from a Matrex Red A column, were 36,600 and 40,300, respectively. Kinetic studies indicate that GAR1 belongs to an aldose reductase (a low-Km form) and GAR2 to an aldehyde reductase (a high-Km form). GAR2, an aldehyde reductase, was also active in the reduction of D-glucose, with an apparent Km comparable to that of GAR1 but with a Vmax only 14% that of GAR1.     

Microquantitative determination of intra-acinar distribution profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver

Microquantitative measurements of total and of low-Km aldehyde dehydrogenase (ALDH) activity with millimolar and micromolar concentrations of acetaldehyde and propionaldehyde were carried out on the livers of male and female rats. Lyophilized cryostat sections of liver parenchyma were microdissected along the entire sinusoidal length from the terminal afferent vessels to the terminal efferent venule. ALDH activity was measured in a microbiochemical assay using the oil-well technique with luminometric determination of NADH. On the basis of single measurements, mean values of total, low-Km and high-Km ALDH activity could be calculated and the specific distribution patterns graphically demonstrated. The two substrates acetaldehyde and propionaldehyde yielded similar values of ALDH activity, the intraacinar distribution profiles of which showed characteristic sex differences. In the liver of the male rat high-Km ALDH activity has two flat peaks in the periportal and the perivenous area, while low-Km ALDH activity is almost evenly distributed throughout the acinus. In the livers of female rats, both high-Km and low-Km ALDH activity shows a continuous gradient which decreases from the periportal to the perivenous zone (pp/pv = 1.4:1). It was therefore possible to demonstrate that the maxima of alcohol dehydrogenase activity and of low-Km ALDH activity are localized in opposite parts of the liver acinus of the female rat. This heterotopy should have consequences with respect to hepatotoxicity after alcohol ingestion.