Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.     

Comparison of mandelate dehydrogenases from various strains of Acinetobacter calcoaceticus: similarity of natural and 'evolved' forms

In previous work it had been shown that Acinetobacter calcoaceticus wild-type strain NCIB 8250 had only an L-mandelate deydrogenase but it could give rise to mutants that contained an evolved D-mandelate dehydrogenase; conversely, wild-type strain EBF 65/65 had only a D-mandelate dehydrogenase but gave rise to mutants that possessed an evolved L-mandelate dehydrogenase. Several other wild-type strains of A. calcoaceticus have now been shown to grow on both enantiomers of mandelate. In every case the L-mandelate dehydrogenases were found to be much more heat-stable and insensitive to inhibition by p-chloromercuribenzoate than were the D-mandelate dehydrogenases when measured in bacterial extracts. All the D-mandelate dehydrogenases in the wild-type strains were inactivated to about the same extent by an antiserum that had been raised in a rabbit against an evolved D-mandelate dehydrogenase. An evolved D-mandelate deydrogenase (from a mutant strain derived from strain NCIB 8250) and an original D-mandelate dehydrogenase (from a mutant strain derived from strain EBF 65/65) were purified to homogeneity by the same procedure and were indistinguishable as judged by immunological cross-reactivity of the native and the sodium-dodecyl-sulphate-denatured enzymes, solubility in cholate, net charge at pH 7.5, pI value, salting-out properties, Mr value, apparent K(m) value for D-mandelate, heat-stability and sensitivity to p-chloromercuribenzoate. The most likely explanation for the appearance of evolved mandelate dehydrogenases in strains of A. calcoaceticus is that cryptic genes become expressed.     

D-lactate metabolism in starved Octopus ocellatus

The concentrations of D- and L-lactate, methylglyoxal and pyruvate were measured in tissues of normal and starved Octopus ocellatus. D-Lactate was always more abundant than L-lactate in the tissues. D-Lactate, pyruvate and methylglyoxal were present in 320, 94 and 43 times higher concentrations in tentacle of O. ocellatus of control group than those in normal rat skeletal muscle. The D-lactate concentration in the tentacle of O. ocellatus was 17-fold higher than that in Octopus vulgars. The activities of enzymes involved with D-lactate metabolism such as pyruvate kinase, octopine dehydrogenase, glyoxalase I and II and lactate dehydrogenase were measured in those tissues. The activities of glyoxalase I and II, and D-lactate dehydrogenase were increased in mantle and tentacle of starved octopus, while the levels of D-lactate and related metabolites were lowered in these tissues. The experimental results presented in this report and up to the present indicate that D-lactate is actively used for energy production in the tentacle and mantle of the starved animals. In octopus, especially starved octopus D-lactate was actively produced from methylglyoxal, which is formed via aminoacetone from threonine and glycine.     

Comparison of the D-lactate stereospecific dehydrogenase of Limulus polyphemus with active-site regions of L-lactate dehydrogenases

Lactate dehydrogenase (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) from the horseshoe crab, Limulus polyphemus, a dimeric enzyme stereospecific for D-lactate, has been purified by affinity chromatography. Maleyl tryptic peptides containing arginine residues isolated from the Limulus enzyme have been characterized and sequenced. The small peptides obtained from similarly treated L-lactate-specific enzyme homologs define major portions of the substrate and coenzyme binding regions and are virtually identical among L-lactate-specific enzymes. Although the six small peptides and free arginine isolated from the Limulus enzyme indicate that the small number of arginine tryptic peptides are located in a few discrete consecutive clusters similarly to the L-lactate dehydrogenases, the peptides nevertheless show no obvious sequence homology to the corresponding peptides from L-lactate dehydrogenases. These results indicate that this lactate dehydrogenase of altered substrate specificity either evolved with major rearrangements of the active site if it evolved from an L-lactate dehydrogenase, or that D-lactate dehydrogenases have evolved from a different protein. The results contradict proposed models which suggest that minor changes in the spatial orientation of pyruvate resulting from minimal rearrangement of the active site could accommodate the change in substrate specificity.     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide     

Anaerobic metabolism of the common cockle, Cardium edule. II. Partial purification and properties of lactate dehydrogenase and octopine dehydrogenase. A comparative study.

1. Octopine dehydrogenase and lactate dehydrogenase were purified 190-fold and 10-fold respectively from the adductor muscle of the marine bivalve Cardium edule by gel filtration on Sephadex G-100 and chromatography on DEAE-Sephadex A-50. 2. Lactate dehydrogenase was capable to convert D- and L-lactate, had a molecular weight of about 70 000 and 280 000 daltons, exhibits no distinct pH optimum and was not inhibited by lactate. The enzyme showed apparent Km values of 0.16 mM for pyruvate and 16 mM and 48 mM for D- and L-lactate respectively. 3. In comparison to the purified enzymes from other species, octopine dehydrogenase from Cardium edule showed similar biochemical properties : pH optima of 6.8 and 8.7 respectively, Km values of 0.9 mM (for pyruvate) and 2.0 mM (for arginine), a molecular weight of 37 000 daltons and inhibition by octopine. Electrophoretic studies on standard polyacrylamide gels showed five isoenzymes. 4. The biochemical properties of both dehydrogenases are compared to the conditions in vivo of these animals and the biological role of the octopine dehydrogenase is discussed.     

A reagentless amperometric electrode based on carbon paste, chemically modified with D-lactate dehydrogenase, NAD(+), and mediator containing polymer for D-lactic acid analysis. I. Construction, composition, and characterization

A reagentless carbon paste electrode was designed for D-lactic acid analysis in a flow injection system for the monitoring of the production of D-lactate in a batch fermentation. D-Lactate dehydrogenase, nicotinamide adenine dinucleotide (NAD(+)), a synthetic redox polymer containing covalently attached toluidine blue O as mediator, graphite powder, and paraffin oil were used for the construction of the modified carbon paste electrode. D-Lactate selectivity was indicated by insignificant responses from a variety of possible interfernces including L-lactate. The electrodes gave a linear response in the range between 0.05 and 5 mM D-lactate, with a detecting limit of 30 muM, allowing a sample throughput of 20 h(-1). Preliminary investigations were made by covering the electrode surface with electropolymerized membranes. Satisfactory stability was observed, indicated by a reproducibility of 3.3% relative standard deviation (RSD, n = 31), with a non-membrane-covered electrode for the analysis of D-lactate in fermentation broth. A long-term stability (230 broth samples) was proven, suggesting the electrodes to have a good potential for use in on-line monitoring of fermentation processes. (c) 1995 John Wiley & Sons, Inc.     

Sodium ion/L-lactate co-transport in rabbit small-intestinal brush-border-membrane vesicles.

Uptake of L-lactate into rabbit jejunal brush-border-membrane vesicles prepared by a Ca2+-precipitation procedure was studied by a rapid filtration technique with L-[14C]-lactate as tracer. Transport of L-lactate into an intravesicular (osmotically reactive) space could be established. An inwardly directed NaCl gradient (outside 21 mM/inside 0mM) stimulated the uptake of L-lactate at 15 s 2-4-fold compared with that observed with an equal KCl gradient. A transient accumulation of L-lactate inside the vesicles (overshoot) was observed in the presence of an NaCl gradient. Gradients of LiCl, RbCl, CsCl or choline chloride were not able to replace NaCl in the stimulation of L-lactate uptake. L-Lactate uptake was saturable only in the presence of Na+. D-Lactate, DL-thiolactate (2-DL-mercaptopropionate), pyruvate and propionate inhibited the Na+-stimulated L-lactate uptake; D-lactate, thiolactate and pyruvate provoked trans-stimulation of L-lactate uptake. Artificially imposed diffusion potentials (inside negative) did not exert any effect on the Na+-dependent L-lactate uptake. The results are consistent with the existence of an electroneutral Na+/L-lactate co-transport system in the brush border of rabbit small intestine.     

Lactate dehydrogenases in cyanobacteria

NAD-linked lactate dehydrogenases specific for the D- and L-lactate have been demonstrated in a number of strains of unicellular cyanobacteria. The D-lactate dehydrogenase of one strain (Synechococcus 6716) was partially purified and its properties were studied. The enzyme has a molecular weight of ca. 115000-120000, is highly specific, autooxidizable, and susceptible to inhibition by iodoacetamide, oxamate and ATP. The possible physiological functions of the enzyme in the metabolism of the organism were investigated. D-lactate carbon was incorporated in cell material during photosynthetic growth with CO2, but lactate was not used as sole source for carbon for photosynthetic or chemosynthetic development. D-lactate and pyruvate were oxidized aerobically in the dark by resting cell suspensions with the assimilation mainly of the C2 and the C3 carbon atoms. In the oxidation of lactate, acetate was excreted into the medium. No fermentation of glucose was found, but a small amount of D-lactate was detected as a product of endogenous dark metabolism of the cell. All enzymes required for the production of lactate from glucose and from glycogen were found in exponentially growing cells, but the activity of some key enzymes was low or undetectable in old cultures.     

Glyceraldehyde metabolism in human erythrocytes in comparison with that of glucose and dihydroxyacetone

Metabolism of D-glyceraldehyde in human erythrocytes in comparison with that of glucose and dihydroxyacetone was studied. Both trioses were metabolized to produce L-lactate at rates comparable to that of L-lactate formation from glucose. Almost complete inactivation of glyceraldehyde-3-phosphate dehydrogenase by treatment of cells with iodoacetate resulted in a 95% decrease in L-lactate formation from the ketotriose as well as from glucose, whereas L-lactate formation from the aldotriose was only partially reduced (60%). D-Lactate was produced faster from either the aldotriose or the ketotriose than from glucose, but the ability of the two trioses to produce D-lactate was far lower than that to produce L-lactate. Almost complete inhibition of aldehyde dehydrogenase by disulfiram and of both aldose reductase and aldehyde reductase II by sorbinil, had no effect on L-lactate formation from D-glyceraldehyde. The present study suggests that D-glyceraldehyde is metabolized via two or more pathways including the glycolytic pathway after its phosphorylation by triokinase, and that neither oxidation to D-glyceric acid nor reduction to glycerol is a prerequisite for D-glyceraldehyde metabolism.     

Two forms of NAD-dependent D-mandelate dehydrogenase in Enterococcus faecalis IAM 10071

Two forms of NAD-dependent D-mandelate dehydrogenase (D-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different K(m) and V(max) values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.     

Purification and in vitro complementation of mutant histidinol dehydrogenases.

The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the hisO1242 (constitutive overproducer) attenuator mutation and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl2, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Neither mutant protein showed negative complementation with wild-type enzyme. The Vmax for hybrid histidinol dehydrogenase was 11% of that for native enzyme, with only minor changes in Km values for substrate or coenzyme. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound 54Mn2+ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound 54Mn2+ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 2-mercaptoethanol was prevented by low levels of MnCl2. It thus appears that mutant histidinol dehydrogenase molecules bind metal ion poorly. The complementation procedure may allow for formation of a functional Mn2+-binding site, perhaps at the subunit interface.     

D-lactate dehydrogenase is a member of the D-isomer-specific 2-hydroxyacid dehydrogenase family. Cloning, sequencing, and expression in Escherichia coli of the D-lactate dehydrogenase gene of Lactobacillus plantarum.

The gene encoding D-lactate dehydrogenase (D-lactate: NAD+ oxidoreductase, EC 1.1.1.28) of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter. Comparison of the sequence of D-lactate dehydrogenase with L-lactate dehydrogenases, including the L. plantarum L-lactate dehydrogenase, showed no significant homology. In contrast, the D-lactate dehydrogenase is homologous to E. coli D-3-phosphoglycerate dehydrogenase and Lactobacillus casei D-2-hydroxyisocaproate dehydrogenase. This indicates that D-lactate dehydrogenase is a member of a new family of 2-hydroxyacid dehydrogenases recently proposed, being distinct from L-lactate dehydrogenase and L-malate dehydrogenase, and strongly suggests that the new family consists of D-isomer-stereospecific enzymes. In the reductive reaction, the enzyme showed a broad substrate specificity, although pyruvate was the most favorable of all 2-ketocarboxylic acids tested. In particular, hydroxypyruvate is effectively reduced by the enzyme, the reaction rate, and Km value being comparable to those in the case of pyruvate, indicating that the enzyme has not only D-lactate dehydrogenase activity but also D-glycerate dehydrogenase activity. The conserved residues in this family appear to be the residues involved in the substrate binding and the catalytic reaction, and thus to be targets for site-directed mutagenesis.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Algal glyceraldehyde-3-phosphate dehydrogenase. Pyridine-nucleotide requirements of two enzymes purified from Scenedesmus obliquus.

Two enzymes with glyceraldehyde-3-phosphate dehydrogenase activity have been purified from heterotrophically grown Scenedesmus obliquus by ion-exchange chromatography and gel filtration. The D-enzyme has a molecular weight of 550000 and a VNADH: VNADPH ratio of 16 whereas the T-enzyme has a molecular weight of 140000 and a VNADH:VNADPH ratio of 0.15. The two enzymes, however, are very similar with regard to their Michaelis constants for the reduced pyridine nucleotides, pH optimum, subunit size and ultraviolet absorption.     

Schistosoma mansoni: Purification and characterization of malate dehydrogenases

Isoelectric focusing of a homogenate of Schistosoma mansoni, followed by malate dehydrogenase-specific staining, showed the presence of two major and five minor malate dehydrogenase isoenzymes (EC 1.1.1.37), with isoelectric points ranging from 7.3 to 9.5. The malate dehydrogenase isoenzymes were purified by gel filtration, followed by ion-exchange chromatography on DEAE- and CM-cellulose. The isoenzymes could be differentiated by their susceptibility to substrate inhibition. No differences in the Michaelis-Menten constants for substrate were found. One of the isoenzymes is inhibited by 5′-AMP. Further purification of this particular isoenzyme was achieved by affinity chromatography on 5′-AMP-Sepharose 4B. Analysis after subcellular fractionation indicated a mitochondrial origin for this isoenzyme. The mitochondrial isoenzyme (at a recovery of 80%) was purified 218-fold compared to the crude soluble extract, and contained about 40% of the total malate dehydrogenase activity. The enzyme has a molecular weight of 65,500 and showed absolute specificity for l-malic acid, NAD, and NADH. The final preparation has a specific activity of 451 U/mg protein. Physicochemical studies, including binding constants, substrate inhibition, thermostability, and pH optima, demonstrated differences between the mitochondrial and cytoplasmic enzymes. A role for malate dehydrogenase in Schistosoma mansoni metabolism is discussed.     

Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.     

Null and electrophoretic mobility mutants in the structural gene for L-lactate dehydrogenase of Saccharomyces cerevisiae

A mutant lacking L-lactate dehydrogenase (EC 1.1.2.3) of Saccharomyces cerevisiae was isolated by its inability to grow on minimal medium with L-lactate as a carbon source. A simple activity gel assay for visualization of this enzyme and the two D-lactate dehydrogenases in this organism (EC 1.1.2.4 and 1.1.1.28) was developed. This enabled us to screen spontaneous and ethylmethanesulfonate-induced back mutants for electrophoretic mobility. Two mutants with a mobility faster than that of the wild type were isolated, and proved to be allelic to the L-lactate dehydrogenase negative mutant.     

Transport of L-Lactate, D-Lactate, and glycolate by the LldP and GlcA membrane carriers of Escherichia coli.

To examine the substrate specificity of the membrane transport carriers LldP (L-lactate permease) and GlcA (glycolate permease) of Escherichia coli, a mutant strain lacking their structural genes and blocked in the metabolism of the tested substrates was constructed and transformed with a plasmid bearing either the lldP or the glcA gene. Each transformant acquired the ability to accumulate L-lactate, D-lactate, and glycolate against a high concentration gradient. Substrate accumulation was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force. Competition of (14)C-L-lactate transport by nonradioactive L-lactate, D-lactate, and glycolate in LldP synthesizing cells and competition of (14)C-glycolate transport by the same three substrates in GlcA synthesizing cells showed that both carriers effectively transported all three substrates with a K(i) value ranging from 10 to 20 microM. D-Lactate does not appear to have a permease of its own. Utilization of the compound depends mainly on LldP.     

Comparison of the properties of the alcohol dehydrogenases from wild-type and mutant Chinese hamster somatic cells

Alcohol dehydrogenases (alcohol: NAD oxidoreductase, E.C. 1.1.1.1.) from allyl alcohol-resistant and wild-type Chinese hamster cells were purified using gel filtration, ion-exchange, and affinity-column chromatography. Both enzymes exhibited the same isozyme band patterns on electrophoresis and isoelectric focusing. Physicochemical properties of the two enzymes such as pH and temperature optima, Km values, and temperature stability were found to be the same within the experimental errors. The genetic significance of these findings is discussed.