Purification and characterization of a rat brain aldehyde dehydrogenase able to metabolize gamma-aminobutyraldehyde to gamma-aminobutyric acid.

An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE-cellulose and affinity chromatography on 5'-AMP-Sepharose, phosphocellulose and Blue Agarose, followed by gel filtration. Such an enzyme was first purified from mammalian brain tissues, and was identified as an isoenzyme of aldehyde dehydrogenase. It has an Mr of 210,000 determined by polyacrylamide-gradient-gel electrophoresis, and appeared to be composed of subunits of Mr 50,000. The close similarity of substrate specificity toward acetaldehyde, propionaldehyde and glycolaldehyde between the enzyme and other aldehyde dehydrogenases previously reported was observed. But substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E1 and E2), and was lower than those of ABAL dehydrogenases from human liver (E3), Escherichia coli and Pseudomonas species. The Mr and relative amino acid composition of the enzyme are also similar to those of E1 and E2. The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway (alternative pathway) for GABA synthesis from putrescine.     

Aldehyde dehydrogenase (EC 1.2.1.3): comparison of subcellular localization of the third isozyme that dehydrogenates gamma-aminobutyraldehyde in rat, guinea pig and human liver.

1. Subcellular fractionation of rat, guinea pig and human livers showed that aldehyde dehydrogenase metabolizing gamma-aminobutyraldehyde was exclusively localized in the cytoplasmic fraction in all three mammalian species. 2. Total gamma-aminobutyraldehyde activity of aldehyde dehydrogenase was found to be ca 0.41, 0.3 and 0.24 mumol NADH min-1 g-1 tissue, respectively in rat, guinea pig and human liver, with more than 95% of activity in the cytoplasm. 3. Partially purified cytoplasmic isozyme from rat liver showed similar chromatographic behavior and kinetic properties to the E3 isozyme isolated from human liver. 4. The rat isozyme was insensitive to disulfiram (40 microM) and to magnesium (160 microM) and had Km values of 5 microM (pH 7.4) for gamma-aminobutyraldehyde, 7.5 microM (pH 9.0) for propionaldehyde and 4 microM (pH 7.4) for NAD.     

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

Purification and characterization of bovine brain gamma-aminobutyraldehyde dehydrogenase.

gamma-Aminobutyraldehyde dehydrogenase was purified to homogeneity from bovine brain. The molecular weight of the native enzyme and subunit were 230,000 and 58,000, respectively. The Km value for gamma-aminobutyraldehyde and NAD+ were 154 microM and 53 microM, respectively. The optimum pH and temperature were 8.0 and 37 degrees C, respectively. N-terminal sequence of the enzyme is as follows: NH2-S-A-A-T-Q-A-V-P-T-P-N-Q-Q-COOH. The enzyme migrates on isoelectric focusing with pI = 6.5. Enhancement of the enzyme activity by polyamine, Mn2+, Mg2+ and inhibition by gamma-aminobutyric acid and Zn2+ will enhance the limited information on regulation of the gamma-ABALDH activity and GABA metabolism to some extent.     

A pathway for putrescine catabolism in Escherichia coli

Escherichia coli mutants able to grow in putrescine have been isolated from gamma-aminobutyrate mutants. These mutants show putrescine-alpha-ketoglutarate transaminase and gamma-aminobutyraldehyde dehydrogenase activities. Both enzymes have been characterized, the first of them showing an apparent Km for putrescine of 22.5 microM and the second an apparent Km of 37 microM for NAD and 18 microM for delta-1-pyrroline; the optimum pH values were 7.2 and 5.4, respectively, for the two enzymes.     

Identification of Escherichia coli K12 YdcW protein as a gamma-aminobutyraldehyde dehydrogenase

Gamma-aminobutyraldehyde dehydrogenase (ABALDH) from wild-type E. coli K12 was purified to apparent homogeneity and identified as YdcW by MS-analysis. YdcW exists as a tetramer of 202+/-29 kDa in the native state, a molecular mass of one subunit was determined as 51+/-3 kDa. Km parameters of YdcW for gamma-aminobutyraldehyde, NAD+ and NADP+ were 41+/-7, 54+/-10 and 484+/-72 microM, respectively. YdcW is the unique ABALDH in E. coli K12. A coupling action of E. coli YgjG putrescine transaminase and YdcW dehydrogenase in vitro resulted in conversion of putrescine into gamma-aminobutyric acid.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes.

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.     

Preparation and kinetic properties of 5-ethylphenazine-lactate-dehydrogenase-NAD+ conjugate, a semisynthetic lactate oxidase showing a hide-and-seek effect.

5-Ethylphenazine-lactate-dehydrogenase-NAD+ conjugate (EP(+)-LDH-NAD+) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min-1 and Km for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min-1 and Km for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD+ moieties of the conjugate are in the free state (i.e. not bound in the coenzyme-binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s-1 and 2.1 s-1 for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 microM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety.     

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.     

Human prostatic aldehyde dehydrogenase of healthy controls and diseased prostates.

Aldehyde dehydrogenase (ALDH, EC 1.2.1.3) of the human prostate was the subject of investigation in this study. The possible physiological role of aldehyde dehydrogenase in the human prostate might be to detoxify aldehydes arising from the oxidation of the polyamines via monoamine or diamine oxidases. The specific activity of the enzyme with 1 mM propionaldehyde as substrate and 0.5 mM NAD at pH 7.4 in the control normal prostates and prostates afflicted with the disease, benign prostatic hyperplasia (BPH), was 26.06 +/- 2.96 and 5.17 +/- 0.48 nmol/g prostate per min, respectively. When 100 microM gamma-aminobutyraldehyde was used as a substrate, the specific activity in the normal controls and prostates with benign prostatic hyperplasia was 19.80 +/- 1.33 and 2.95 +/- 2.46 nmol/g prostate per min, respectively. Upon isoelectric focusing of the extracts of the control prostates when the gels were developed for aldehyde dehydrogenase activity, there were three aldehyde dehydrogenase activity bands visible, pI 4.9 (mitochondrial), 5.4 (cytosolic) and about 6.0-6.5, on the IEF gels developed with gamma-aminobutyraldehyde as a substrate. With the extracts of prostates with benign prostatic hyperplasia the pI 4.9 band was significantly reduced, the pI 5.4 band enhanced and the approx. pI 6.0 band was not detectable on the IEF gels with propionaldehyde as a substrate. There was no detectable aldehyde dehydrogenase activity in the extract of the prostate with cancer on IEF gels nor in the activity assays with propionaldehyde or gamma-aminobutyraldehyde as substrates.     

Human aldehyde dehydrogenase. Activity with aldehyde metabolites of monoamines, diamines, and polyamines

Two isozymes (E1 and E2) of human aldehyde dehydrogenase (EC 1.2.1.3) were purified to homogeneity 13 years ago and a third isozyme (E3) with a low Km for gamma-aminobutyraldehyde only recently. Comparison with a variety of substrates demonstrates that substrate specificity of all three isozymes is broad and similar. With straight chain aliphatic aldehydes (C1-C6) the Km values of the E3 isozyme are identical with those of the E1 isozyme. All isozymes dehydrogenate naturally occurring aldehydes, 5-imidazoleacetaldehyde (histamine metabolite) and acrolein (product of beta-elimination of oxidized polyamines) with similar catalytic efficiency. Differences between the isozymes are in the Km values for aminoaldehydes. Although all isozymes can dehydrogenate gamma-aminobutyraldehyde, the Km value of the E3 isozyme is much lower: the same appears to apply to aldehyde metabolites of cadaverine, agmatine, spermidine, and spermine for which Km values range between 2-18 microM and kcat values between 0.8-1.9 mumol/min/mg. Thus, the E3 isozyme has properties which make it suitable for the metabolism of aminoaldehydes. The physiological role of E1 and E2 isozymes could be in dehydrogenation of aldehyde metabolites of monoamines such as 3,4-dihydroxyphenylacetaldehyde or 5-hydroxyindoleacetaldehyde; the catalytic efficiency with these substrates is better with E1 and E2 isozymes than with E3 isozyme. Isoelectric focusing of liver homogenates followed by development with various physiological substrates together with substrate specificity data suggest that aldehyde dehydrogenase (EC 1.2.1.3) is the only enzyme in the human liver capable of catalyzing dehydrogenation of aldehydes arising via monoamine, diamine, and plasma amine oxidases. Although the enzyme is generally considered to function in detoxication, our data suggest an additional function in metabolism of biogenic amines.     

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of gamma-aminobutyraldehyde dehydrogenase from Escherichia coli

gamma-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an Mr of 195,000 +/- 10,000 in its dimeric form with an Mr of 95,000 +/- 1,000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. Km values are 31.3 +/- 6.8 microM and 53.8 +/- 7.4 microM for delta-1-pyrroline and NAD+, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

Xanthine:NAD+ oxidoreductase from embryo liver of hen Gallus gallus

1. Xanthine:NAD+ oxidoreductase from chick embryo liver is unconvertible to the O2-dependent form, as is the enzyme from the adult hen. The Km for NAD+ (approximately 3 microM) of the embryonic enzyme is equal to, and the Km for xanthine (approximately 5 microM) is 2.5-fold lower, when compared with respective Km values of the "adult" hen enzyme. The inhibition of embryonic enzyme by NADH begins at 10 microM NADH and attains 13% at 35 microM NADH (respective data for the "adult" enzyme: 50 microM and 20% at 80 microM NADH). 2. The course of hypoxanthine----xanthine----uric acid hydroxylation catalyzed by the embryonic and "adult" enzymes is similar, however the rate of the first reaction is 2-fold lower for the embryonic enzyme. Under conditions of the limited nutritional system in the developing chick embryo, the low rate of hypoxanthine hydroxylation may promote reutilization of hypoxanthine for nucleotide synthesis.     

Isolation, characterization, and biological activity of ferredoxin-NAD+ reductase from the methane oxidizer Methylosinus trichosporium OB3b.

A ferredoxin-NAD+ oxidoreductase (EC 1.18.1.3) has been isolated from extracts of the obligate methanotroph Methylosinus trichosporium OB3b. This enzyme was shown to couple electron flow from formate dehydrogenase (NAD+ requiring) to ferredoxin. Ferredoxin-NAD+ reductase was purified to homogeneity by conventional chromatography techniques and was shown to be a flavoprotein with a molecular weight of 36,000 +/- 1,000. This ferredoxin reductase was specific for NADH (Km, 125 microM) and coupled electron flow to the native ferredoxin and to ferredoxins from spinach, Clostridium pasteurianum, and Rhodospirillum rubrum (ferredoxin II). M. trichosporium ferredoxin saturated the ferredoxin-NAD+ reductase at a concentration 2 orders of magnitude lower (3 nM) than did spinach ferredoxin (0.4 microM). Ferredoxin-NAD+ reductase also had transhydrogenase activity which transferred electrons and protons from NADH to thionicotinamide adenine dinucleotide phosphate (Km, 9 microM) and from NADPH to 3-acetylpyridine adenine dinucleotide (Km, 16 microM). Reconstitution of a soluble electron transport pathway that coupled formate oxidation to ferredoxin reduction required formate dehydrogenase, NAD+, and ferredoxin-NAD+ reductase.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

Coenzyme specificity of mammalian liver D-glycerate dehydrogenase

D-Glycerate dehydrogenase (glyoxylate reductase) was partially purified from rat liver by anion- and cation-exchange chromatography. When assayed in the direction of D-glycerate or glycolate formation, the enzyme was inhibited by high (greater than or equal to 0.5 mM), unphysiological concentrations of hydroxypyruvate or glyoxylate much more potently in the presence of NADPH than in the presence of NADH. However, the dehydrogenase displayed a much greater affinity for NADPH (Km less than 1 microM) than for NADH (Km = 48-153 microM). Furthermore, NADP was over 1000-fold more potent than NAD in inhibiting the enzyme competitively with respect to NADH. NADP also inhibited the reaction competitively with respect to NADPH whereas NAD, at concentrations of up to 10 mM had no inhibitory effect. When measured by the formation of hydroxypyruvate from D-glycerate, the enzyme also displayed a much greater affinity for NADP than for NAD. These properties indicate that liver D-glycerate dehydrogenase functions physiologically as an NADPH-specific reductase. In agreement with this conclusion, the addition of hydroxypyruvate or glyoxylate to suspensions of rat hepatocytes stimulated the pentose-phosphate pathway. The coenzyme specificity of D-glycerate dehydrogenase is discussed in relation to the biochemical findings made in D-glyceric aciduria and in primary hyperoxaluria type II (L-glyceric aciduria).     

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.     

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.