Mitochondrial aldehyde dehydrogenase from higher plants

Aldehyde dehydrogenase has been purified to homogeneity from mitochondria of potato tubers and pea epicotyls. Although the enzyme had a high affinity for glycolaldehyde it also had a high affinity for a number of other aliphatic and arylaldehydes. It is proposed that the codification glycolaldehyde dehydrogenase (EC 1.2.1.22) should be abandoned in favour of mitochondrial aldehyde dehydrogenase (EC 1.2.1.3). The purified enzyme showed esterase activity and had properties similar to those reported for the mammalian mitochondrial aldehyde dehydrogenase. Although the natural substrate(s) for the enzyme is not known, the kinetic properties of the enzyme are consistent with it playing a role in the oxidation of acetaldehyde, glycolaldehyde and indoleacetaldehyde.     

Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis

Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form. Purification of the enzyme was achieved using triazine dye affinity chromatography. C. perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield). Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically. Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM. Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase. Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C. perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C. bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase. Statistical comparison validated the use of C. perfringens 3 alpha- and C. bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.     

Partial purification of d-glucose-6-phosphate dehydrogenase from bakers' yeast by affinity partitioning using polymer-bound triazine dyes

d-Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate:NADP⁺ 1-oxidoreductase EC 1.1.1.49) has been purified from bakers' yeast by liquid-liquid extraction using phase-restricted triazine dyes (Procion Yellow HE-3G, Procion Olive MX-3G, Procion Navy MX-RB and Cibacron Blue F3G-A). This method was combined with fractional precipitation with poly(ethylene) glycol) and batchwise treatment with DEAE-cellulose. This rapid procedure gave an enzyme preparation with a specific activity of 0.92 kat per kg protein within 5 h. The affinity extraction step can easily be scaled up and the good recovery of ligand-poly(ethylene glycol) should make the process useful for larger amounts of enzyme. The technical possibilities are discussed.     

Partial purification of d-glucose-6-phosphate dehydrogenase from bakers' yeast by affinity partitioning using polymer-bound triazine dyes

d-Glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate:NADP⁺ 1-oxidoreductase EC 1.1.1.49) has been purified from bakers' yeast by liquid-liquid extraction using phase-restricted triazine dyes (Procion Yellow HE-3G, Procion Olive MX-3G, Procion Navy MX-RB and Cibacron Blue F3G-A). This method was combined with fractional precipitation with poly(ethylene) glycol) and batchwise treatment with DEAE-cellulose. This rapid procedure gave an enzyme preparation with a specific activity of 0.92 kat per kg protein within 5 h. The affinity extraction step can easily be scaled up and the good recovery of ligand-poly(ethylene glycol) should make the process useful for larger amounts of enzyme. The technical possibilities are discussed.     

Purification and characterization of human liver sorbitol dehydrogenase

Sorbitol dehydrogenase from human liver was purified to homogeneity by affinity chromatography on immobilized triazine dyes, conventional cation-exchange chromatography, and high-performance liquid chromatography. The major form is a tetrameric, NAD-specific enzyme containing one zinc atom per subunit. Human liver sorbitol dehydrogenase oxidizes neither ethanol nor other primary alcohols. It catalyzes the oxidation of a secondary alcohol group of polyol substrates such as sorbitol, xylitol, or L-threitol. However, the substrate specificity of human liver sorbitol dehydrogenase is broader than that of the liver enzymes of other sources. The present report describes the stereospecific oxidation of (2R,3R)-2,3-butanediol, indicating a more general function of sorbitol dehydrogenase in the metabolism of secondary alcohols. Thus, the enzyme complements the substrate specificities covered by the three classes of human liver alcohol dehydrogenase.     

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.     

Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase from porcine liver. Isolation of a dehydrogenase-cyclohydrolase fragment from the multifunctional enzyme

Tryptic digestion of a multifunctional enzyme from porcine liver containing methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5), methenyltetrahydrofolate cyclohydrolase (5,10-methenyltetrahydrofolate 5-hydrolase, EC 3.5.4.9) and formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase, EC 6.3.4.3) activities destroys the synthetase. A fragment containing both dehydrogenase and cyclohydrolase activities has been isolated by affinity chromatography on an NADP+-Sepharose affinity column. The purified fragment is homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis where its molecular weight was determined as 33 000 +/- 1200 compared with 100 000 for the undigested protein. The cyclohydrolase activity retains sensitivity to inhibition by NADP+, MgATP and ATP.     

Human brain "high Km" aldehyde dehydrogenase: purification, characterization, and identification as NAD+ -dependent succinic semialdehyde dehydrogenase

NAD-dependent succinic semialdehyde dehydrogenase (EC 1.2.1.24) has been purified to homogeneity from human brain via ion-exchange chromatography and affinity chromatography employing Blue Sepharose and 5'-AMP Sepharose. Succinic semialdehyde dehydrogenase was never previously purified to homogeneity from any species; this preparation therefore allows the determination of its molecular weight, subunit molecular weight, subunit composition, isoelectric points, and substrate specificity for the first time. The enzyme is a tetramer of Mr230,000 to 245,000 and consists of weight-nonidentical subunits (Mr 61,000 and 63,000). On isoelectric focusing the enzyme separates into five bands with the following isoelectric points: 6.3, 6.6, 6.8, 6.95, and 7.15. Its substrates include glutaric semialdehyde, nitrobenzaldehyde, and short chain aliphatic aldehydes in addition to succinic semialdehyde which is the best substrate. The Km values for succinic semialdehyde, acetaldehyde, and propionaldehyde are 1,875, and 580 microM, respectively. The enzyme is inactive with 3,4-dihydroxyphenylacetaldehyde and indole-3-acetaldehyde as substrates. Its subcellular localization is in the mitochondrial fraction. Succinic semialdehyde dehydrogenase is sensitive to inhibition by disulfiram (a drug used therapeutically to produce alcohol aversion) resembling, in this respect, aldehyde dehydrogenase (EC 1.2.1.3). It does not, however, interact with the antibody developed in the rabbit vs aldehyde dehydrogenase, suggesting that the two enzymes are structurally distinct.     

Malate dehydrogenase in phototrophic purple bacteria: purification, molecular weight, and quaternary structure.

The citric acid cycle enzyme malate dehydrogenase was purified to homogeneity from the nonsulfur purple bacteria Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodomicrobium vannielii, and Rhodocyclus purpureus. Malate dehydrogenase was purified from each species by either a single- or a two-step protocol: triazine dye affinity chromatography was the key step in purification of malate dehydrogenase in all cases. Purification of malate dehydrogenase resulted in a 130- to 240-fold increase in malate dehydrogenase specific activity, depending on the species, with recoveries ranging from 30 to 70%. Homogeneity of malate dehydrogenase preparations from the four organisms was determined by sodium dodecyl sulfate and nondenaturing polyacrylamide gel electrophoresis; a single protein band was observed in purified preparations by both techniques. The molecular weight of native malate dehydrogenases was determined by four independent methods and estimated to be in the range of 130,000 to 140,000 for the enzyme from R. capsulatus, R. rubrum, and R. vannielii and 57,000 for that from R. purpureus. It is concluded that malate dehydrogenase from R. capsulatus, R. rubrum, and R. vannielii is a tetramer composed of four identical subunits, while the enzyme from R. purpureus is a dimer composed of two identical subunits.     

Malate dehydrogenase from Chlorobium vibrioforme, Chlorobium tepidum, and Heliobacterium gestii: purification, characterization, and investigation of dinucleotide binding by dehydrogenases by use of empirical methods of protein sequence analysis.

Malate dehydrogenase (MDH; EC 1.1.1.37) from strain NCIB 8327 of the green sulfur bacterium Chlorobium vibrioforme was purified to homogeneity by triazine dye affinity chromatography followed by gel filtration. Purification of MDH gave an approximately 1,000-fold increase in specific activity and recoveries of typically 15 to 20%. The criteria of purity were single bands on sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide electrophoresis (PAGE) and the detection of a single N terminus in an Edman degradation analysis. MDH activity was detected in purified preparations by activity staining of gels in the direction of malate oxidation. PAGE and gel filtration (Sephadex G-100) analyses showed the native enzyme to be a dimer composed of identical subunits both at room temperature and at 4 degrees C. The molecular weight of the native enzyme as estimated by gel filtration was 77,000 and by gradient PAGE was 74,000. The subunit molecular weight as estimated by SDS-gradient PAGE was 37,500. N-terminal sequences of MDHs from C. vibrioforme, Chlorobium tepidum, and Heliobacterium gestii are presented. There are obvious key sequence similarities in MDHs from the phototrophic green bacteria. The sequences presented probably possess a stretch of amino acids involved in dinucleotide binding which is similar to that of Chloroflexus aurantiacus MDH and other classes of dehydrogenase enzymes but unique among MDHs.     

Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli.

D-Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and D-glucitol-6-phosphate dehydrogenase (EC 1.1.1.140) were purified to apparent homogeneity in good yields from Escherichia coli. The amino acid compositions, N-terminal amino acid sequences, sensitivities to chemical reagents, and catalytic properties of the two enzymes were determined. Both enzymes showed absolute specificities for their substrates. The subunit molecular weights of mannitol-1-phosphate and glucitol-6-phosphate dehydrogenases were 40,000 and 26,000, respectively; the apparent molecular weights of the native proteins, determined by gel filtration, were 40,000 and 117,000, respectively. It is therefore concluded that whereas mannitol-1-phosphate dehydrogenase is a monomer, glucitol-6-phosphate dehydrogenase is probably a tetramer. These two proteins differed in several fundamental respects.     

Improved purification and properties of glucose dehydrogenase from Bacillus subtilis

Glucose dehydrogenase (EC 1.1.1.47) from Bacillus subtilis was purified about 5240-fold, using an aqueous two-phase system and triazine-dye affinity chromatography. The specific activity of the purified preparation was about 460 units/mg of protein with a final recovery of enzyme activity of about 75%. The affinity column could be regenerated and reused again several times. The purified enzyme appeared to be homogeneous when analyzed both on SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the activity stain. ATP acts apparently as a competitive inhibitor for this enzyme with respect to NAD and protects the enzyme from dissociation into partially inactive dimers. In the absence of either glycerol or ATP, the enzyme dissociates into partially inactive dimers.     

Further characterization of L-beta-hydroxyacid dehydrogenase from Drosophila

L-beta-Hydroxyacid dehydrogenase (L-beta-hydroxyacid-NAD-oxidoreductase, EC 1.1.1.45) of Drosophila is composed of two, identical subunits with a molecular weight of approx. 33 300. The enzyme was purified 938-fold from Drosophila melanogaster. An isoelectric point of 8.6 was determined for L-beta-hydroxyacid dehydrogenase. An amino acid analysis was conducted of the purified enzyme. A single subunit was obtained by SDS-gel electrophoresis of the purified enzyme. Translation of larval and adult mRNA in a mRNA-dependent reticulocyte lysate, followed by immune precipitation using anti-L-beta-hydroxyacid dehydrogenase IgG revealed a single L-beta-hydroxyacid dehydrogenase subunit of 33 300. Larval and adult proteins were the same size. The enzyme does not appear to be subjected to substantial post-translational modifications.     

Purification of IMP dehydrogenase from rat hepatoma 3924A

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

Purification and properties of the enzyme 6-phosphogluconate dehydrogenase from Dicentrarchus labrax L. liver

The enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP+ oxidoreductase, decarboxylating EC 1.1.1.44) from bass liver has been purified to over 95% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight was estimated by gel filtration chromatography to about 100,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed to be a dimeric protein. The effect of pH and kinetic properties were studied.     

Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus

A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

Molecular and biochemical characterization of rat gamma-trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis

The penultimate step in carnitine biosynthesis is mediated by gamma-trimethylaminobutyraldehyde dehydrogenase (EC 1.2.1.47), a cytosolic NAD(+)-dependent aldehyde dehydrogenase that converts gamma-trimethylaminobutyraldehyde into gamma-butyrobetaine. This enzyme was purified from rat liver, and two internal peptide fragments were sequenced by Edman degradation. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA containing an open reading frame of 1485 base pairs encoding a polypeptide of 494 amino acids with a calculated molecular mass of 55 kDa. Expression of the coding sequence in Escherichia coli confirmed that the cDNA encodes gamma-trimethylaminobutyraldehyde dehydrogenase. The previously identified human aldehyde dehydrogenase 9 (EC 1.2.1.19) has 92% identity with rat trimethylaminobutyraldehyde dehydrogenase and has been reported to convert substrates that resemble gamma-trimethylaminobutyraldehyde. When aldehyde dehydrogenase 9 was expressed in E. coli, it exhibited high trimethylaminobutyraldehyde dehydrogenase activity. Furthermore, comparison of the enzymatic characteristics of the heterologously expressed human and rat dehydrogenases with those of purified rat liver trimethylaminobutyraldehyde dehydrogenase revealed that the three enzymes have highly similar substrate specificities. In addition, the highest V(max)/K(m) values were obtained with gamma-trimethylaminobutyraldehyde as substrate. This indicates that human aldehyde dehydrogenase 9 is the gamma-trimethylaminobutyraldehyde dehydrogenase, which functions in carnitine biosynthesis.     

Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

NAD⁺-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD⁺ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD⁺ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.