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.     

Rat liver aldehyde dehydrogenase. II. Isolation and characterization of four inducible isozymes

The purification and properties of 4 inducible cytosolic rat liver aldehyde dehydrogenase isozymes are described. Based on their behavior during purification and their properties, the activities can be grouped into 2 classes. The isozyme inducible in normal liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the tumor-specific isozyme found in hepatocellular carcinomas have apparent molecular weights of 110,000, prefer NADP+ as coenzyme, and preferentially oxidize benzaldehyde-like aromatic aldehydes, but not phenylacetaldehyde. They also have identical pH profiles and responses to effectors. These isozymes differ slightly in isoelectric point and thermal stability. The normal liver phenobarbital-inducible isozyme and the isozyme appearing during the promotion phase of hepatocarcinogenesis appear to be identical. Both have apparent molecular weights of 165,000, are NAD-specific and prefer aliphatic aldehydes. They can oxidize phenylacetaldehyde, but not benzaldehyde-like aromatic aldehydes. They also have identical pH and thermal stability profiles and responses to effectors. While the 4 inducible isozymes share identical subunit molecular weights (54,000) with the normal liver millimolar Km aldehyde dehydrogenases, they are distinctly different enzymatic species. The interrelationships of the various normal liver and inducible rat liver aldehyde dehydrogenases are discussed.     

Horse liver aldehyde dehydrogenase. Purification and characterization of two isozymes.

Two isozymes of horse liver aldehyde dehydrogenase (aldehyde, NAD oxidoreductase (EC 1.2.1.3)), F1 and F2, have been purified to homogeneity using salt fractionation followed by ion exchange and gel filtration chromatography. The specific activities of the two isozymes in a pH 9.0 system with propionaldehyde as substrate were approximately 0.35 and 1.0 mumol of NADH/min/mg of protein for the F1 and F2 isozymes, respectively. The multiporosity polyacrylamide gel electrophoresis molecular weights of the F1 and F2 isozymes were approximately 230,000 and 240,000 respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave subunit molecular weight estimates of 52,000 and 53,000 for the F1 and F2 isozymes, respectively. The amino acid compositions of the two isozymes were found to be similar; the ionizable amino acid contents being consistent with the electrophoretic and chromatographic behavior of the two isozymes. Both isozymes exhibited a broad aldehyde specificity, oxidizing a wide variety of aliphatic and aromatic aldehydes and utilized NAD as coenzyme, but at approximately 300-fold higher coenzyme concentration could use NADP. The F1 isozyme exhibited a very low Km for NAD (3 muM) and a higher Km for acetaldehyde (70 muM), while the F2 isozyme was found to have a higher Km for NAD (30 muM) and a low Km for acetaldehyde (0.2 muM). The two isozymes showed similar chloral hydrate and p-chloromercuribenzoate inhibition characteristics, but the F1 isozyme was found to be several orders of magnittude more sensitive to disulfiram, a physiological inhibitor of acetaldehyde oxidation. Based on its disulfiram inhibition characteristics, it has been suggested that the F1 isozyme may be the primary enzyme for oxidizing the acetyldehyde produced during ethanol oxidation in vivo.     

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.     

Glucose dehydrogenase (hexose 6-phosphate dehydrogenase) and the microsomal electron transport system. Evidence supporting their possible functional relationship.

The ability of a microsomal enzyme, glucose dehydrogenase (hexose 6-phosphate dehydrogenease) to supply NADPH to the microsomal electron transport system, was investigated. Microsomes could perform oxidative demethylation of aminopyrine using microsomal glucose dehydrogenase in situ as an NADPH generator. This demethylation reaction had apparent Km values of 2.61 X 10(-5) M for NADP+, 4.93 X 10(-5) m for glucose 6-phosphate, and 2.14 X 10(-4) m for 2-deoxyglucose 6-phosphate, a synthetic substrate for glucose dehydrogenase. Phenobarbital treatment enhanced this demethylation activity more markedly than glucose dehydrogenase activity itself. Latent activity of glucose dehydrogenase in intact microsomes could be detected by using inhibitors of microsomal electron transport, i.e. carbon monoxide and p-chloromercuribenzoate (PCMB), and under anaerobic conditions. These observations indicate that in microsomes the NADPH generated by glucose dehydrogenase is immediately oxidized by NADPH-cytochrome c reductase, and that glucose dehydrogenase may be functioning to supply NADPH.     

Dihydrodiol dehydrogenase activities of rabbit liver are associated with hydroxysteroid dehydrogenases and aldo-keto reductases.

1. Dihydrodiol dehydrogenase activities were investigated in rabbit liver. Using a five-step purification scheme, eight isoenzymes of dihydrodiol dehydrogenase with isoelectric points of 5.55-9.3 and promoter molecular masses of 34-35 kDa were purified to apparent homogeneity and designated CF-1 to CF-6, CM-1 and CM-2. 2. CF-1 and CF-2 had near-neutral isoelectric points of 7.4 and 6.8 and molecular masses of about 125 kDa in the native state. Both enzymes readily accepted NAD+ as well as NADP+ as coenzymes, had relatively low Km values of 0.33 mM and 0.47 mM for benzene dihydrodiol and resembled previously described carbonyl reductases in their substrate specificity towards ketones and quinones. 3. CF-5 and CF-6 had acidic isoelectric points of 5.9 and 5.55 and native molecular masses of approximately 60 kDa. They displayed a strong preference for NADP(H) as coenzyme and had high Km and Vmax with benzene dihydrodiol. Since these enzymes reduced p-nitrobenzaldehyde and glucuronic acid efficiently, they appeared to be closely related to aldehyde reductase. 4. CF-4 had a high 3 alpha-hydroxysteroid dehydrogenase activity for the diagnostic substrate androsterone, a moderate activity for other 3 alpha-hydroxysteroids as well as 17 alpha-hydroxysteroids, and relatively low activities for 3 beta-hydroxysteroids and 17 beta-hydroxysteroids. CF-5 and CM-1 had high 17 beta-hydroxysteroid dehydrogenase activity for the diagnostic substrate 5 alpha-dihydrotestosterone, and low to moderate activities for other 17 beta-hydroxysteroids as well as 3 alpha-hydroxysteroids. 5. The isoenzyme CM-2 had an isoelectric point of 9.3 and was a very active quinone reductase with phenanthrene-9,10-quinone as substrate. It was potently inhibited by phenobarbital. 6. We conclude that the dihydrodiol dehydrogenase activities of rabbit liver are associated with aldehyde and carbonyl reductase and with 3 alpha-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases.     

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.     

Purification and properties of an NADPH-linked aldehyde reductase from rat kidney

Rat kidney was shown to contain two NADPH-linked aldehyde reductases (alcohol:NADP+) oxidoreductase, EC 1.1.1.2) with different substrate affinities. The high-Km aldehyde reductase, which was purified to apparent homogeneity, had a molecular weight of 32 000 as determined by Sephadex G-100 gel filtration, and of 37 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme reduced various aliphatic aldehydes of different carbon-chain lengths besides many chemicals containing aldehyde groups. The Km values for n-hexadecanal and n-octadecanal were 8 microM and 4 microM, respectively. Bovine serum albumin (1.8 mM) stimulated the reduction of n-hexadecanal and n-octadecanal, and increased the Vmax values by about 15-fold without changing the Km values. The kidney enzyme was not distinguishable from the brain and liver high-Km aldehyde reductases in mobility on polyacrylamide gel electrophoresis, immunological properties, peptide maps or substrate specificity.     

Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus.

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.     

Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland.

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.     

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.     

Intraparticulate localization and some properties of a clofibrate-induced peroxisomal aldehyde dehydrogenase from rat liver

A study was made of the effect of chronic administration of the hypolipidemic drug clofibrate on the activity and intracellular localization of rat liver aldehyde dehydrogenase. The enzyme was assayed using several aliphatic and aromatic aldehydes. Clofibrate treatment caused a 1.5 to 2.3-fold increase in the liver specific aldehyde dehydrogenase activity. The induced enzyme has a high Km for acetaldehyde and was found to be located in peroxisomes and microsomes. Clofibrate did not alter the enzyme activity in the cytoplasmic fraction. The total peroxisomal aldehyde dehydrogenase activity increased 3 to 4-fold under the action of clofibrate. Disruption of the purified peroxisomes by the hypotonic treatment or in the alkaline conditions resulted in the release of catalase from the broken organelles, while aldehyde dehydrogenase as well as nucleoid-bound urate oxidase and the peroxisomal membrane marker NADH:cytochrome c reductase remained in the peroxisomal 'ghosts'. At the same time, treatment by Triton X-100 led to solubilization of the membrane-bound NADH:cytochrome c reductase and aldehyde dehydrogenase from intact peroxisomes and their 'ghosts'. These results indicate that aldehyde dehydrogenase is located in the peroxisomal membrane. The peroxisomal aldehyde dehydrogenase is active with different aliphatic and aromatic aldehydes, except for formaldehyde and glyceraldehyde. The enzyme Km values lie in the millimolar range for acetaldehyde, propionaldehyde, benzaldehyde and phenylacetaldehyde and in the micromolar range for nonanal. Both NAD and NADP serve as coenzymes for the enzyme. Aldehyde dehydrogenase was inhibited by disulfiram, N-ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic)acid. According to its basic kinetic properties peroxisomal aldehyde dehydrogenase seems to be similar to a clofibrate-induced microsomal enzyme. The functional role of both enzymes in the liver cells is discussed.     

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.     

Wheat Alcohol Dehydrogenase Isozymes: PURIFICATION, CHARACTERIZATION, AND GENE EXPRESSION

Evidence in support of the hypothesis of gene expression and subunit association suggested earlier for Triticum alcohol dehydrogenase has been obtained through purification and partial characterization of the enzyme from tetraploid wheat. Three isozymes of alcohol dehydrogenase were separated and purified to apparent homogeneity using streptomycin sulfate precipitation, gel filtration chromatography, and anion exchange chromatography. The isozymes are dimers with the same molecular weight (116,000 ± 2,000), but significantly different isoelectric pH values. The Michaelis constants for NAD⁺ and ethanol are 0.1 millimolar and 12 millimolar, respectively. The substrate specificity of the three alcohol dehydrogenase isozymes was investigated.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

Purification of aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation from rabbit intestinal microsomes

This work presents the purification and further characterization of the aldehyde dehydrogenase reconstitutively active in fatty alcohol oxidation, from rabbit intestinal microsomes. Microsomal aldehyde dehydrogenase was solubilized with cholate and purified by using chromatography on 6-amino-n-hexyl-Sepharose and 5'-AMP-Sepharose. The purified enzyme migrated as a single polypeptide band with molecular weight of 60,000 on SDS-polyacrylamide gel. By gel filtration in the presence of detergent, its apparent molecular weight was estimated to be 370,000. In the detergent-free solution, in contrast, it had a much higher molecular weight, indicating its association in forming large aggregates. The pH optimum was 9.0 when pyrophosphate buffer was used. The enzyme was active toward various aliphatic aldehydes with more than three carbons. The Km value for substrate seemed to decrease with increase in the chain length. The microsomal aldehyde dehydrogenase was not affected by disulfiram and MgCl2, which were, in contrast, highly inhibitory towards the activity of the cytosolic aldehyde dehydrogenase separated from intestinal mucosa.     

Subcellular distribution and kinetic parameters of HS mouse liver aldehyde dehydrogenase

1. Aldehyde dehydrogenase subcellular distribution studies were performed in a heterogeneous stock (HS) of male and female mice (Mus musculus) with propionaldehyde (5 mM and 50 microM) and formaldehyde (1 mM) and NAD+ or NADP+. 2. The relative percents of distribution were: cytosolic 55-68%, mitochondrial 12-20%, microsomal 9-18% and lysosomal 3-15% for both propionaldehyde concentrations and NAD+. 3. Kinetic experiments using propionaldehyde and acetaldehyde with NAD+ revealed two separate enzymes, Enzyme I (low Km) and Enzyme II (high Km) in the cytosolic and mitochondrial fractions. 4. The kinetic data also indicated a spectrum of cytosolic low Km values that exhibited a bimodal distribution with one congruent to 40 microM and one congruent to 5 microM. 5. It was concluded that there was no significant difference in aldehyde-metabolizing capability between male and female HS mice, compared on a per gram of liver basis. The cytosolic low Km enzyme plays a major role in aldehyde oxidation at moderate to low aldehyde concentrations.     

Purification and properties of polyol dehydrogenase from Cephalosporium chrysogenus.

A polyol dehydrogenase of broad specificity was purified 178-fold from extracts of the filamentous fungus Cephalosporium chrysogenum. The enzyme was found to act as an oxido-reductase in two substrate-coenzyme systems: D-sorbitol (or xylitol)-nicotinamide-adenine dinucleotide (NAD) and D-mannitol-nicotinamide adenine dinucleotide phosphate (NADP). The dehydrogenase was composed of five isozymes, which, as a mixture, exhibited these properties: Km to D-sorbitol and D-mannitol, 7.15 to 10(-2) M; PH optimum, 9 to 10; molecular weight, 300,000 subunit weight, 29,000; PI, 5.8 to 7.5. The NADP-linked activity was labile to treatment with heat or ethylenediaminetetraacetic acid. Mixed substrate assays support the hypothesis that both NAD-, and NADP-linked activities are associated with isozymes of a single dehydrogenase.     

Some observations on the NADP+-linked oxidation of methylglyoxal catalysed by 2-Oxoaldehyde dehydrogenase.

In the oxidation of methylglyoxal by 2-oxoaldehyde dehydrogenase, the apparent Km value for NADP+ was about 2.5 times lower than the corresponding Km for NAD+; the apparent Km values for methylglyoxal and for the amine activator L-2-aminopropan-1-ol, with NADP+ as cofactor, were also different from those obtained with NAD+. In the presence of NADP+, the enzyme was not activated by P1, in contrast with the activation of the enzyme when NAD+ was used. The significance of the results is discussed.     

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.