Molecular basis for thermoprotection in Bemisia: structural differences between whitefly ketose reductase and other medium-chain dehydrogenases/reductases

The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) accumulates sorbitol as a thermoprotectant in response to elevated temperature. Sorbitol synthesis in this insect is catalyzed by an unconventional ketose reductase (KR) that uses NADPH to reduce fructose. A cDNA encoding the NADPH-KR from adult B. argentifolii was cloned and sequenced to determine the primary structure of this enzyme. The cDNA encoded a protein of 352 amino acids with a calculated molecular mass of 38.2 kDa. The deduced amino acid sequence of the cDNA shared 60% identity with sheep NAD(+)-dependent sorbitol dehydrogenase (SDH). Residues in SDH involved in substrate binding were conserved in the whitefly NADPH-KR. An important structural difference between the whitefly NADPH-KR and NAD(+)-SDHs occurred in the nucleotide-binding site. The Asp residue that coordinates the adenosyl ribose hydroxyls in NAD(+)-dependent dehydrogenases (including NAD(+)-SDH), was replaced by an Ala in the whitefly NADPH-KR. The whitefly NADPH-KR also contained two neutral to Arg substitutions within four residues of the Asp to Ala substitution. Molecular modeling indicated that addition of the Arg residues and loss of the Asp decreased the electric potential of the adenosine ribose-binding pocket, creating an environment favorable for NADPH-binding. Because of the ability to use NADPH, the whitefly NADPH-KR synthesizes sorbitol under physiological conditions, unlike NAD(+)-SDHs, which function in sorbitol catabolism.     

Preparation and characterization of the NAD vinylogue, 3-pyridylacryloamide adenine dinucleotide

The vinylogue of NAD, 3-pyridylacryloamide adenine dinucleotide, was prepared from NAD and 3-pyridylacryloamide through the snake venom NADase-catalyzed transglycosidation reaction. The analog, purified by ion-exchange chromatography, was obtained in a 55% yield. The cyanide adduct and reduced form of the analog exhibited absorbance maxima at 358 nm and 378 nm, respectively, with extinction coefficients in each case being 2.3-times higher than those reported for the corresponding NAD derivatives. 3-Pyridylacryloamide adenine dinucleotide served as a coenzyme with bovine liver glutamic dehydrogenase and to a lesser extent with malate and lactate dehydrogenases. The analog was not reduced in reactions catalyzed by yeast and horse liver alcohol dehydrogenases, sheep liver sorbitol dehydrogenase, and rabbit muscle glycerophosphate dehydrogenase. Substitution of the pyridylacryloamide analogs for NAD and NADH in the assay of substrates for glutamic dehydrogenase was demonstrated.     

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/K(sorbitol) for E154C decreased below an apparent pK of 9.1+/-0.3, reflecting a shift in pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7+/-0.2 (E154C, 1.7+/-0.1) and 1.9+/-0.3 (E154C, 2.4+/-0.2) on kcat/K(sorbitol) respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687+/-12 s(-1) in the pre-steady state, which features a turnover of 0.9+/-0.1 enzyme equivalents as NADH was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.     

Purification and characterization of the multiple forms of 3 alpha-hydroxysteroid dehydrogenase in rat liver cytosol

The presence of multiple forms of 3 alpha-hydroxysteroid dehydrogenase in the cytosol of male rat livers was demonstrated. The enzyme activity was separated into two fractions (F3 and F4) by DEAE-cellulose chromatography, and further fractionation of F3 into four (I-IV) and F4 into three (I-III) fractions was achieved by subsequent CM-Sephadex chromatography. Six forms (F3-II-IV and F4-I-III) were further purified by chromatofocusing and Red-Sepharose 4B chromatography. Two (F4-II and III) of the isolated enzymes were homogeneous, based on polyacrylamide gel electrophoresis. No shift of pI values was observed, when isoelectric focusing was performed with the F4 enzyme species in the presence of NAD(P)+ or NAD(P)H. All six enzyme species migrated closely with each other on dodecyl sulfate-polyacrylamide gel electrophoresis, from which the molecular masses were estimated to be 32 500 Da. Gel filtration gave similar values for the F4 enzyme species, indicating that each enzyme is a monomeric peptide. All enzyme species were able to catalyse the dehydrogenation of 3 alpha-hydroxysteroids (C19 to C26), and not C27 compound having a 1,5-dimethylhexyl side chain. The catalytic properties with steroids were very similar for five of the enzyme species, but F3-IV especially preferred androsterone. When male rat livers were used for isolation, the enzyme activity ratio of F3 to F4 for androsterone was about 1 to 8, whereas the ratio was about 1 to 20 for female rat livers. Considering the biosynthetic pathway of bile acids, the enzymes isolated here might play a specific role in the conversion of a 3 beta-hydroxy group to a 3 alpha-hydroxy group via a 3-oxo group of an intermediate in the synthesis of bile acids.     

The enzymic reduction of nicotinamide–adenine dinucleotide by 2-mercaptoethanol

1. The reduction of NAD(+), by an enzyme preparation from rat liver, in the presence of 2-mercaptoethanol is reported. 2. It is suggested that the NAD(+)-linked alcohol dehydrogenase in the extract transfers hydrogen from 2-mercaptoethanol to NAD(+). 3. Both yeast and horse-liver alcohol dehydrogenases were observed to reduce NAD(+) in the presence of 2-mercaptoethanol. 4. Some interactions of 2-mercaptoethanol, cysteine or hydroxylamine with the alcohol dehydrogenases from rat liver, horse liver and yeast are discussed.     

Lipoamide dehydrogenase from baker's yeast. Improved purification and some molecular, kinetic, and immunochemical properties

The flavoprotein lipoamide dehydrogenase was purified, by an improved method, from commercial baker's yeast about 700-fold to apparent homogeneity with 50-80% yield. The enzyme had a specific activity of 730-900 U/mg (about twice the value of preparations described previously). The holoenzyme, but not the apoenzyme, possessed very high stability against proteolysis, heat, and urea treatment and could be reassociated, with fair yield, with the other components of yeast pyruvate dehydrogenase complex to give the active multienzyme complex. The apoenzyme was reactivated when incubated with FAD but not FMN. As other lipoamide dehydrogenases, the yeast enzyme was found to possess diaphorase activity catalysing the oxidation of NADH with various artificial electron acceptors. Km values were 0.48 mM for dihydrolipoamide and 0.15 mM for NAD. NADH was a competitive inhibitor with respect to NAD (Ki 31 microM). The native enzyme (Mr 117000) was composed of two apparently identical subunits (Mr 56000), each containing 0.96 FAD residues and one cystine bridge. The amino acid composition differed from bacterial and mammalian lipoamide dehydrogenases with respect to the content of Asx, Glx, Gly, Val, and Cys. The lipoamide dehydrogenases of baker's and brewer's yeast were immunologically identical but no cross-reaction with mammalian lipoamide dehydrogenases was found.     

Acute and chronic ethanol treatment in vivo increases malate-aspartate shuttle capacity in perfused rat liver

The effects of acute and chronic treatment with ethanol on transport of reducing equivalents into mitochondria via the malate-aspartate shuttle were studied in perfused rat liver. The shuttle capacity was estimated from the decrease in rates of glucose production from the reduced substrate sorbitol caused by an increase in the NADH/NAD+ ratio in the cytosol due to metabolism of ethanol. The greater the capacity of the malate-aspartate shuttle, the smaller the inhibition of glucose synthesis by ethanol. Glucose synthesis was decreased about 2-fold less in livers from fasted rats treated acutely 2.5 h earlier with ethanol than in untreated controls. Chronic treatment with ethanol for 3-5 weeks prevented completely the decrease in glucose synthesis from sorbitol due to ethanol oxidation. Rates of ethanol uptake were elevated significantly from 69 +/- 7 mumols/g/h in livers from control rats up to 92 +/- 7 mumols/g/h in livers from SIAM rats. Similarly, rates of ethanol uptake were stimulated by chronic ethanol treatment from 71 +/- 6 to 222 +/- 15 mumols/g/h; this increase was largely sensitive to aminooxyacetate. Taken together, these data indicate that flux of reducing equivalents over the malate-aspartate shuttle is increased by both acute and chronic treatment with ethanol and that movement of reducing equivalents from the cytosol into the mitochondria via the malate-aspartate shuttle is an important rate determinant in hepatic ethanol oxidation.     

Cloning and expression of a fungal L-arabinitol 4-dehydrogenase gene

L-Arabinitol 4-dehydrogenase (EC ) was purified from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). It is an enzyme in the L-arabinose catabolic pathway of fungi catalyzing the reaction from L-arabinitol to L-xylulose. The amino acid sequence of peptide fragments was determined and used to identify the corresponding gene. We named the gene lad1. It is not constitutively expressed. In a Northern analysis we found it only after growth on L-arabinose. The gene was cloned and overexpressed in Saccharomyces cerevisiae, and the enzyme activity was confirmed in a cell extract. The enzyme consists of 377 amino acids and has a calculated molecular mass of 39,822 Da. It belongs to the family of zinc-binding dehydrogenases and has some amino acid sequence similarity to sorbitol dehydrogenases. It shows activity toward L-arabinitol, adonitol (ribitol), and xylitol with K(m) values of about 40 mM toward L-arabinitol and adonitol and about 180 mM toward xylitol. No activity was observed with D-sorbitol, D-arabinitol, and D-mannitol. NAD is the required cofactor with a K(m) of 180 microM. No activity was observed with NADP.     

Molecular aspects of functional differences between alcohol and sorbitol dehydrogenases

The amino acid sequence of sheep liver sorbitol dehydrogenase has been fitted to the high-resolution model of the homologous horse liver alcohol dehydrogenase by computer graphics. This has allowed construction of a model of sorbitol dehydrogenase that provides explanations why sorbitol is not a substrate for alcohol dehydrogenase, why ethanol is not a substrate for sorbitol dehydrogenase, and what determines its specificity for polyols. An important feature of the model is that one of the ligands to the active site zinc atom is a glutamic acid residue instead of a cysteine residue, which is the corresponding ligand in the homologous alcohol dehydrogenases. This is one component of the structural change that can be related to the different substrate specificities, showing how altered enzymic activity might be brought about by structural changes of the kind that it is now possible to introduce by site-directed mutagenesis and recombinant DNA techniques.     

Uridine diphosphate glucuronic acid production and utilization in various tissues actively synthesizing glycosaminoglycans

1. UDP-glucose dehydrogenase has been partially purified from sheep nasal septum cartilage, neonatal rat skin and bovine corneal epithelium. 2. The pH profile, K(m) values for NAD(+) and UDP-glucose, activation energy and molecular weight have been determined for the enzyme from several of the tissues. 3. The sugar nucleotide concentrations in each of the tissues have been related to the spectrum of glycosaminoglycans produced by each tissue. 4. The presence of an allosteric UDP-xylose-binding site distinct from the active site(s) in sheep nasal septum UDP-glucose dehydrogenase has been demonstrated. 5. An active UDP-glucuronic acid nucleotidase has been demonstrated in sheep nasal cartilage. 6. Tissue-space experiments have shown the cell water content of sheep nasal septum cartilage to be 14% of the wet weight. 7. Glucuronic acid 1-phosphate does not occur in measurable amounts in sheep nasal septum cartilage and no UDP-glucuronic acid pyrophosphorylase activity could be detected in this tissue. 8. The inhibition by UDP-xylose with respect to both substrates, UDP-glucose and NAD(+), has been examined, and shown to be allosteric.     

Nitrogen metabolism in the isolated perfused rat liver. Nitrogen balance, redox state and rates of proteolysis

Nitrogen balances were measured in isolated perfused rat livers in the presence and absence of nitrogen donors. In all instances the balance apparently was incomplete. The expression [alanine][alpha-oxoglutarate]/[pyruvate][glutamate] remained fairly constant under the metabolic conditions studied, indicating that it may be at near-equilibrium. The source of the extra nitrogen seems to be derived from increased hepatic proteolysis. The addition of a nitrogen donor to the perfusate arrested proteolysis, as did the addition of pyruvate. The free mitochondrial [NAD(+)]/[NADH] ratio, calculated from the glutamate dehydrogenase and beta-hydroxybutyrate dehydrogenase reactants, showed similar values and exhibited parallel changes under most metabolic situations studied. These results suggest that, under the reported experimental conditions, both dehydrogenases share a common mitochondrial NAD pool. Glutamate dehydrogenase plays an important role in hepatic nitrogen metabolism in vivo.     

Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies

Five different immobilized NAD+ derivatives were employed to compare the behavior of four amino acid dehydrogenases chromatographed using kinetic-based enzyme capture strategies (KBECS): S6-, N6-, N1-, 8'-azo-, and pyrophosphate-linked immobilized NAD+. The amino acid dehydrogenases were NAD+-dependent phenylalanine (EC 1.4.1.20), alanine (EC 1.4.1.1), and leucine (EC 1.4.1.9) dehydrogenases from various microbial species and NAD(P)+-dependent glutamate dehydrogenase from bovine liver (GDH; EC 1.4.1.3). KBECS for bovine heart L-lactate dehydrogenase (EC 1.1.1.27) and yeast alcohol dehydrogenase (EC 1.1.1.1) were also applied to assist in a preliminary assessment of the immobilized cofactor derivatives. Results confirm that the majority of the enzymes studied retained affinity for NAD+ immobilized through an N6 linkage, as opposed to an N1 linkage, replacement of the nitrogen with sulfur to produce an S6 linkage, or attachment of the cofactor through the C8 position or the pyrophosphate group of the cofactor. The one exception to this was the dual-cofactor-specific GDH from bovine liver, which showed no affinity for N6-linked NAD+ but was biospecifically adsorbed to S6-linked NAD+ derivatives in the presence of its soluble KBEC ligand. The molecular basis for this is discussed together with the implications for future development and application of KBECS.     

The stimulation of tricarboxylic acid-cycle flux by alpha-adrenergic agonists in perfused rat liver.

Output of 14CO2 from 1-14C-labelled glutamate, 2-oxoglutarate or octanoate and from 4-methyl-2-oxo[2-14C]pentanoate was increased by more than 100% after infusion of phenylephrine into perfused livers of fed rats. Infusion of ethanol or sorbitol raised 3-hydroxybutyrate/acetoacetate ratios and decreased the output of 14CO2. Increases in 14CO2 output induced by phenylephrine were observed in the presence or absence of ethanol or sorbitol and were accompanied by elevated 3-hydroxybutyrate/acetoacetate ratios under all conditions examined. Phenylephrine had no effect on total tissue ATP/ADP ratios in livers from fed or starved rats. The data suggest that phenylephrine-induced increases in tricarboxylic acid-cycle flux do not arise from lowered matrix NADH/NAD+ or ATP/ADP ratios.     

Purification and structural characterization of placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase. The primary structure reveals the enzyme to belong to the short-chain alcohol dehydrogenase family

Human placental NAD(+)-linked 15-hydroxyprostaglandin dehydrogenase was purified to homogeneity according to a five-step method, with chromatography on DEAE-Sepharose, Blue Sepharose, and Mono-Q FPLC as principal steps. Final yield was 23% and purification about 13,000-fold, with a specific activity of 24,000 milliunits/mg. The subunit molecular weight is about 29,000 as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and the native protein molecular weight is about 54,000 as estimated by Sephadex G-100 chromatography, establishing the enzyme to be a dimer of similar-sized protein chains. The subunit N-terminal residue is methionine, and the alpha-amino group is free. The complete primary structure was determined by peptide analysis, based essentially on four different proteolytic treatments (Lys-specific protease, Glu-specific protease, Asp-specific protease, and CNBr). The protein chain is composed of 266 residues, with C-terminal glutamine. A microheterogeneity was detected at position 217, with both Cys and Tyr, in about equal amounts, from a preparation starting with a single placenta. No other subunit heterogeneities were detected. The protein is clearly but distantly related to insect alcohol dehydrogenases, characterized bacterial dehydrogenases of sugar metabolism, and bacterial and eukaryotic steroid dehydrogenases. Together, these results establish that placental 15-hydroxyprostaglandin dehydrogenase is a member of the short-chain nonmetalloenzyme alcohol dehydrogenase protein family. The protein has four cysteine residues (five with the positional microheterogeneity), but there is no evidence for functional importance of any of these residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sorbitol dehydrogenase is a zinc enzyme.

Evidence is given that tetrameric sorbitol dehydrogenase from sheep liver contains one zinc atom per subunit, most probably located at the active site, and no other specifically bound zinc or iron atom. In alcohol dehydrogenases that are structurally related to sorbitol dehydrogenase, more than one zinc atom per subunit can complicate investigations of zinc atom function. Therefore, sorbitol dehydrogenase will be particularly valuable for defining the precise roles of zinc in alcohol and polyol dehydrogenases, and for establishing correlations of structure and function with other important zinc-containing proteins.     

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD(+)-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD(+)-dependent retinaldehyde activity was associated with the E1 isozyme (coded for by aldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD(+)-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD(+)-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1 gene) and broad substrate specificity E1 (a member of EC 1. 2.1.3 aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

Purification and properties of sorbitol dehydrogenase from mouse liver

1. The sorbitol dehydrogenase (L-iditol: NAD oxidoreductase, EC 1.1.1.14) from mouse liver has been purified to homogeneity. 2. The enzyme has a mol. wt of 140,000 and is composed of four identical subunits of mol. wt 35,000. 3. the purified enzyme catalyses both sorbitol oxidation and fructose reduction. 4. It is specific for NAD+ (NADH) and does not function with NADP+ (NADPH). 5. The Michaelis constants for sorbitol, fructose, NAD+ and NADPH are 1.54 and 154 mM, 58.8 and 15 microM, respectively. 6. The enzyme is SH-group reagent sensitive and is strongly inhibited by 1,10-phenanthroline.     

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.