Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Hydride transfer stereospecificity of rat liver aldehyde dehydrogenases

The stereospecificity of hydride transfer to NAD+ by several forms of rat liver aldehyde dehydrogenase was determined by a nuclear magnetic resonance method. The forms included several mitochondrial and microsomal isozymes from normal liver, as well as isozymes from xenobiotic-treated and tumor cells. The proton added to NAD+ comes exclusively from the aldehyde substrate and in all cases was A (pro-R)-stereospecific.     

Subcellular distribution and properties of aldehyde dehydrogenase in the rat liver

The subcellular distribution and certain properties of rat liver aldehyde dehydrogenase are investigated. The enzyme is shown to be localized in fractions of mitochondria and microsomes. Optimal conditions are chosen for detecting the aldehyde dehydrogenase activity in the mentioned fractions. The enzyme of mitochondrial fraction shows the activity at low (0,03-0.05 mM; isoenzyme I) and high (5 mM; isoenzyme II) concentrations of the substrate. The seeming Km and V of aldehyde dehydrogenase from fractions of mitochondria and microsomes of rat liver are calculated, the acetaldehyde and NAD+ reaction being used as a substrate.     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver

The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity of aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.     

The role of aldehyde dehydrogenases in the malonic dialdehyde metabolism in the rat liver

The enzymes catalyzing the NAD-dependent oxidation of malonic dialdehyde (MDA) were isolated from rat liver extracts. Upon 5'-AMP-Sepharose chromatography MDA dehydrogenase was separated into two isoforms, I and II. Isoform I was eluted from the affinity carrier with a 0.1 M phosphate buffer pH 8.0. This isoform had a broad substrate specificity towards aliphatic and aromatic aldehydes. Kinetic studies showed that short- and medium-chain aliphatic aldehydes (C2-C6) were characterized by the lowest Km values and the highest Vmax values. The Km' values for MDA and acetaldehyde were 2.8 microM and 0.69 microM, respectively. Isoform II was eluted with a 0.1 M phosphate buffer pH 8.0 containing 0.5 mM NAD, was the most active with medium- and long-chain aliphatic aldehydes (C6-C11) and had Km values for MDA and acetaldehyde equal to 37 microM and 52 microM, respectively. Isoform I was much more sensitive towards disulfiram inhibition than isoform II. Both isoforms had an identical molecular mass (93 kD) upon gel filtration. It is concluded that MDA dehydrogenase isoform I is identical to mitochondrial aldehyde dehydrogenase having a low Km for acetaldehyde, whereas isoform II may be localized in liver cytosol. The role of aldehyde dehydrogenases in the metabolism of aldehydes derived from lipid peroxidation is discussed.     

The subcellular distribution of fumarase isozymes in rat liver

Between 13 and 25% of the fumarase activity of rat liver was found to be cytosolic in origin the remainder being localised in the mitochondria. Electrophoretic analysis on cellulose acetate showed that mitochondria do not contain detectable levels of cytosolic isozyme or vice versa.     

The effect of ethanol ingestion on the aldehyde dehydrogenases of rat liver.

The effect of ethanol ingestion on aldehyde dehydrogenase activity in the subcellular fractions of livers from 14 pair-fed male Sprague-Dawley rats was tested. Enzymatic assays were performed at two different concentrations of propionaldehyde (0.068 and 13.6 mM) sufficient to saturate enzymes with high and low affinities for propionaldehyde, respectively. The effect of alcohol ingestion varied depending on the subcellular fraction tested and the propionaldehyde concentration used in the assay. There was a 60% increase in the activity of aldehyde dehydrogenase with high affinity for propionaldehyde in the mitochondrial membranes. Conversely there was a 50% decrease in the activity of aldehyde dehydrogenases with high affinity for propionaldehyde in the microsomal fraction. There was also a 58% decrease in the activity of enzymes from the mitochondrial matrix with low affinity for propionaldehyde. The results suggest that differences in the assay systems employed may account for the conflicting results obtained by previous investigators of the effect of ethanol feeding.     

The subcellular distribution of rat liver serine-pyruvate aminotransferase.

1. The subcellular distribution of L-serine-pyruvate aminotransferase activity in rat liver was investigated. About 80% was recovered from cell-free homogenates in a 'total-particles' fraction and the remainder in the cytosol. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients showed a distribution for serine-pyruvate aminotransferase activity closely matching that observed for mitochondrial marker enzymes. 3. A study of the solubilization of enzymes from combined subcellular particles by digitonin at various concentrations also indicated a common subcellular location for serine-pyruvate aminotransferase and established mitochondrial enzymes. 4. The increase in liver serine-pyruvate amino-transferase activity induced by glucagon injection was accounted for as an increased mitochondrial activity.     

Purification and properties of sheep-liver aldehyde dehydrogenases.

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with a specific activity of 380 nmol NADH min(-1) mg(-1). An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions. On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6-fold enhancement of nucleotide fluorescence. The protein fluorescence (lambdaexcit = 290 nm, lambdaemisson = 340 nm) is quenched on the binding of NAD+ and NADH. The enhancement of nucleotide fluorescence on the binding of NADH has been utilised to determine the dissociation constant for the enzyme . NADH complex (Kd = 1.2 +/- 0.2 muM). A Hill plot of the data gave a straight line with a slope of 1.0 +/- 0.3 indicating the absence of co-operative effects. Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecylsulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme. Molecular weights were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212 000 +/- 8 000 and 205 000 respectively. Each enzyme consisted of four subunits with molecular weight of 53 000 +/- 2 000. Properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases.     

Uptake and subcellular distribution of injected transferrin in rat liver

Radioactively labelled transferrin was injected into rats intravenously and its uptake and subcellular distribution in the liver was investigated. The amount of radioactivity in the liver remained constant from 10 min after injection. It was not influenced by asialoglycoproteins. The radioactive label was identified as asialotransferrin. After subcellular fractionation by differential and zonal sucrose density-gradient centrifugation the label was enriched in a low-density endocytic compartment showing fluorescence quenching of acridine orange and N-ethylmaleimide-sensitive ATPase activity. The data fit into a model of continuous transferrin-receptor-mediated recycling through the hepatocyte's endocytic compartment.     

Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases

Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step.     

The effect of thyroidectomy on the subcellular Mg distribution in rat liver

The subcellular distribution of Mg²⁺ in livers of normal and thyroparathyroidectomized rats has been studied. Significant increases in Mg²⁺ are found in the mitochondrial fractions of thyroparathyroidectomized rats accompanied by a decrease in the Ca²⁺ content. In the microsomal fractions no significant modifications of both ions were detected. Propylthiouracil administration reproduced the ionic alterations found after surgical thyroidectomy and a triiodothyronine replacement therapy to thyroidectomized rats resulted in a reversion of the Mg²⁺ content back to normal levels. The possible participation of the parathyroid in the above mentioned phenomena could be excluded in the present work.     

Comparison of phenobarbital-and carcinogen-induced aldehyde dehydrogenases in the rat

There is a genetically determined variation in the inducibility of a high-K_m cytoplasmic aldehyde dehydrogenase activity in the rat liver by treatment with phenobarbital. In the present experiments this activity increased after phenobarbital administration in the phenobarbital-responsive rats also in the intestinal postmitochondrial supernatant fraction. Phenobarbital-nonresponsive rats did not exhibit such an increase after drug treatment. Intraperitoneal administration of 2,3,7,8,-tetrachlorodibenzo-pdioxin, strongly enchanced the cytoplasmic enzyme activity in the liver of both responsive and nonresponsive rats. This effect was also seen in the serum but not in the intestinal or hte kidney. Intragastric administration of 3-methylcholanthrene, 3,4,-benzpyrene or chrysene induced the activity in liver and intestine but not in serum or kidney. The activity in liver was also induced by long-term feeding with 2-acetamido-fluorene. The activities induced by tetrachlorodibenzodioxin or the carcinogens had similar behaviour in isoelectric focusing in gel slabs and in gel chromatography, suggesting a possible common identity of these induced enzymes. The activity induced by these agents could be clearly differentiated both from the activity induced by phenobarbital and from the normal cytoplasmic activities.     

Intralobular distribution of rat liver aldehyde dehydrogenase and alcohol dehydrogenase

1. The activity of liver microsomal high Km-ALDH and mitochondrial low Km-ALDH, which may be primarily responsible for the oxidation of acetaldehyde after ethanol administration was found to be predominantly distributed in the centrilobular area. 2. The activities of other ALDH isozymes in mitochondrial and soluble fractions were evenly distributed in periportal and perivenous regions. 3. The activity of ADH which is involved in production of acetaldehyde was predominantly located in the periportal area. 4. From these results it seems unlikely that a concentration of acetaldehyde after ethanol ingestion is higher in perivenous hepatocytes than in periportal ones. Additional data would be needed to understand fully the mechanism by which ethanol induces predominantly centrilobular liver injury.     

Some comparisons of pig and sheep liver cytosolic aldehyde dehydrogenases

1. The pig enzyme was purified to homogeneity and was found to be a tetramer of apparently identical subunits. 2. The pig enzyme was found to contain 1 mol NADH/mol enzyme which is tightly bound, which is not directly involved in catalysis and which so far has not been removed from the enzyme so as to produce an active apoenzyme. 3. The pig enzyme seems to contain only one functioning active site/tetramer. 4. The pig and sheep enzymes are compared in respect of NADH binding, substrate specificity, immunological response and surface charge.     

The effect of disulfiram on the aldehyde dehydrogenases of sheep liver.

1. The effect of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. 2. Disulfiram causes an immediate inhibition of the enzyme reaction. The effect on the cytoplasmic enzyme is much greater than on the mitochondrial enzyme. 3. In both cases, the initial partial inhibition is followed by a gradual irreversible loss of activity. 4. The pH-rate profile of the inactivation of the mitochondrial enzyme by disulfiram and the pH-dependence of the maximum velocity of the enzyme-catalysed reaction are both consistent with the involvement of a thiol group. 5. Excess of 2-mercaptoethanol or GSH abolishes the effect of disulfiram. However, equimolar amounts of either of these reagents and disulfiram cause an effect greater than does disulfiram alone. It was shown that the mixed disulphide, Et2N-CS-SS-CH2-CH2OH, strongly inhibits aldehyde dehydrogenase. 6. The inhibitory effect of diethyldithiocarbamate in vitro is due mainly to contamination by disulfiram.