Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 1. Purification and molecular characterization

The complete purification of malate dehydrogenase (EC 1.1.1.37) from extremely halophilic bacteria of the Dead Sea is described. The purification procedure includes (a) precipitation by ammonium sulfate, (b) fractionation on Sepharose 4B using a decreasing concentration gradient of ammonium sulfate, (c) gel permeation chromatography on Sephadex G-100, (d) chromatography on hydroxylapatite, and (e) affinity chromatography on 8-(6-aminohexyl)amino-NAD+-Sepharose at 4.26 M NaCl. The absorption and fluorescence spectra of the native and denatured enzyme were measured, and the extinction coefficient at 280 nm in 4.26 M NaCl was found to be 0.803 cm2mg-1. The amino acid composition analysis showed an excess of 10.4 mol % of acidic amino acids. The apparent specific "volume" phi' of the active enzyme at 4.26 M NaCl was found to be 0.680 +/- 0.015 mL/g. The molecular weight of the native enzyme was found to be 84 000 +/- 4000 determined in 4.26 M NaCl from equilibrium sedimentation data. The molecular weight of the subunits is 39 000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the native enzyme is composed of two subunits.     

Interaction between citrate synthase and malate dehydrogenase. Substrate channeling of oxaloacetate

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase.     

Molecular weight of cytoplasmic malate dehydrogenase, mitochondrial malate dehydrogenase and lactate dehydrogenase of a freshwater catfish

The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) were studied in the liver and skeletal muscle of the freshwater catfish, Clarias batrachus. There were two electrophoretically distinguishable bands (AA and BB) of cMDH and mMDH which suggests that they are apparently encoded at two gene loci (A and B) in both the tissues. However, the presence of a single band (LDH-1) of LDH in liver and double bands (LDH-1 and LDH-2) in skeletal muscle in which LDH-2 was predominant reflects the differential expression of LDH genes in different metabolic tissues to meet the requirement of energy production. The AA isoform (74 kd) of liver cMDH was smaller than those of the AA form (110 kd) of skeletal muscle. In contrast, the BB isoform of liver (42 kd) and skeletal muscle (54 kd) were more or less similar in size. Unlike the case of cMDH, the molecular weight of AA isoform (115 kd) of liver mMDH was higher than those of the AA form (87 kd) of skeletal muscle. Whereas the molecular weight of BB isoform (58 kd) of liver was in proximity to the weight of BB form (44 kd) of skeletal muscle mMDH. The size of AA isoform (74 kd) of liver cMDH was smaller, while the AA isoform (110 kd) of skeletal muscle was larger as compared to AA form of mMDH in the liver (115 kd) and skeletal muscle (87 kd). But the size of BB isoform of both the isozymes was almost equal in these metabolic tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of L-acetylcarnitine on different cerebral mitochondrial populations from cerebral cortex

The maximum rate (Vmax) of some mitochondrial enzymatic activities related to the energy transduction (citrate synthase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, cytochrome oxidase) and amino acid metabolism (glutamate dehydrogenase, glutamate-pyruvate-transaminase, glutamate-oxaloacetate-transaminase) was evaluated in non-synaptic (free) and intra-synaptic mitochondria from rat brain cerebral cortex. Three types of mitochondria were isolated from rats subjected to i.p. treatment with L-acetylcarnitine at two different doses (30 and 60 mg.kg-1, 28 days, 5 days/week). In control (vehicle-treated) animals, enzyme activities are differently expressed in non-synaptic mitochondria respect to intra-synaptic "light" and "heavy" ones. In fact, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glutamate-pyruvate-transaminase and glutamate-oxaloacetate-transaminase are lower, while citrate synthase, cytochrome oxidase and glutamate dehydrogenase are higher in intra-synaptic mitochondria than in non-synaptic ones. This confirms that in various types of brain mitochondria a different metabolic machinery exists, due to their location in vivo. Treatment with L-acetylcarnitine decreased citrate synthase and glutamate dehydrogenase activities, while increased cytochrome oxidase and alpha-ketoglutarate dehydrogenase activities only in intra-synaptic mitochondria. Therefore in vivo administration of L-acetylcarnitine mainly affects some specific enzyme activities, suggesting a specific molecular trigger mode of action and only of the intra-synaptic mitochondria, suggesting a specific subcellular trigger site of action.     

Purification, characterization, and sequence analysis of 2-aminomuconic 6-semialdehyde dehydrogenase from Pseudomonas pseudoalcaligenes JS45

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.     

Isoenzyme analysis of Arthrobotrys, a nematode-trapping fungus

Extraction and isoenzyme analysis of four isolates of Arthrobotrys including A. musiformis, A. robusta and A. conoides were conducted. Among the 14 enzymes studied by starch gel electrophoresis, using morpholine-citrate as gel/electrode buffer, the following nine enzymes showed interpretable banding patterns: alpha-esterase, fumarase, hexokinase, isocitrate dehydrogenase, leucine aminopeptidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase and phosphoglucoisomerase. All isolates studied displayed typical isoenzyme phenotypes for each species. Two isolates of A. conoides differed in their alpha-isoesterase banding patterns, but no differences were observed for the other enzymes. The assay was satisfactory for enzyme extraction and resolution of Arthrobotrys and could be used in future taxonomic and genetic studies of this organism.     

Malate dehydrogenase from the mesophile Chlorobium vibrioforme and from the mild thermophile Chlorobium tepidum: molecular cloning, construction of a hybrid, and expression in Escherichia coli

The genes (mdh) encoding malate dehydrogenase (MDH) from the mesophile Chlorobium vibrioforme and the moderate thermophile C. tepidum were cloned and sequenced, and the complete amino acid sequences were deduced. When the region upstream of mdh was analyzed, a sequence with high homology to an operon encoding ribosomal proteins from Escherichia coli was found. Each mdh gene consists of a 930-bp open reading frame and encodes 310 amino acid residues, corresponding to a subunit weight of 33,200 Da for the dimeric enzyme. The amino acid sequence identity of the two MDHs is 86%. Homology searches using the primary structures of the two MDHs revealed significant sequence similarity to lactate dehydrogenases. A hybrid mdh was constructed from the 3' part of mdh from C. tepidum and the 5' part of mdh from C. vibrioforme. The thermostabilities of the hybrid enzyme and of MDH from C. vibrioforme and C. tepidum were compared.     

Amino acid sequence of human muscle glyceraldehyde-3-phosphate dehydrogenase. Isolation and amino acid sequences of tryptic peptides

1. The amino acid compostion, N- and C-terminal amino acid sequences, and the subunit molecular weight of glyceraldehyde phosphate dehydrogenase from human muscle, were determined. The obtained results and the maps of tryptic peptides suggest that the enzyme is composed of four identical or very similar polypeptide chains. 2. From the tryptic digest of performic acid-oxidized enzyme, 32 peptides were isolated. The amino acid sequence analysis showed a high degree of homology with the corresponding tryptic peptides of the dehydrogenase from pig muscle, with 9 replacements and probably two additional amino acids in the examined sequences of the human muscle enzyme.     

Malate dehydrogenase from the green gliding bacterium Chloroflexus aurantiacus is phylogenetically related to lactic dehydrogenases

The gene encoding malate dehydrogenase (MDH) from Chloroflexus aurantiacus was cloned, sequenced, and analyzed. The mdh gene corresponded to a polypeptide of 309 amino acids with a molecular mass of 32,717 Da. The primary structure and the coenzyme-binding domain showed a high degree of similarity to lactate dehydrogenase (LDH), whereas the conserved amino acids that participate in substrate binding were those typical of MDHs. Using PCR techniques, the mdh gene was cloned in the expression vector pET 11a, and large amounts of active C. aurantiacus MDH were produced in Escherichia coli after induction with isopropyl beta-D-thiogalactoside. The expressed enzyme thus obtained was purified and retained full activity at 55 degrees C. High levels of expression of mdh were also observed when the gene and its flanking sequences were cloned into pUC18/19, indicating that the putative sigma 70 promoter sequences found upstream of the C. aurantiacus mdh functioned in E. coli. When these sequences were deleted, the expression in E. coli was reduced dramatically.     

Isolation and characterization of rat olfactory marker protein

The olfactory marker protein was isolated and characterized from rat olfactory bulbs. Its properties and those of the olfactory marker protein isolated from the mouse are described. The rat protein was less acidic (pI = 5.0) than the mouse protein (pI = 4.7). However, the amino acid compositions were very similar: in both proteins arginine plus lysine accounted for 13 mol% and glutamate plus aspartate for 30 mol% of the total residues. Molecular weights of both proteins estimated by sodium dodecyl sulfate gel electrophoresis were indistinguishable and estimated to be 16,500. The molecular weight of the native rat olfactory marker protein estimated by gel filtration techniques was 30,000, which is identical to the molecular weight of the native mouse and garfish olfactory marker proteins. This suggested a dimeric structure. The purified rat and mouse proteins behaved like species of 35,000 molecular weight on gel filtration.     

Coenzyme binding at different ionization states of cytoplasmic and mitochondrial malate dehydrogenase

pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pKa in the range 6.8-8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotinamide-ribosyl-P2-ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD+ give evidence for the participation of a group in the mitochondrial enzyme with pKa 6.8, deprotonation of which is necessary for detectable association of NAD+. In contrast the fixation of NAD+ to the cytoplasmic enzyme is independent of pH.     

Biochemical and genetic characterization of 2-carboxybenzaldehyde dehydrogenase, an enzyme involved in phenanthrene degradation by Nocardioides sp. strain KP7

2-Carboxybenzaldehyde dehydrogenase from the phenanthrene-degrading bacterium Nocardioides sp. strain KP7 was purified and characterized. The purified enzyme had a molecular mass of 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 205 kDa by gel filtration chromatography. Thus, the homotetramer of the 53-kDa subunit constituted an active enzyme. The apparent Km and kcat values of this enzyme for 2-carboxybenzaldehyde were 100 microM and 39 s(-1), respectively, and those for NAD+ were 83 microM and 32 s(-1), respectively. The structural gene for this enzyme was cloned and sequenced. The length of the gene was 1,455 bp. The nucleotide sequence of the 10,279 bp of DNA around the gene for 2-carboxybenzaldehyde dehydrogenase was also determined, and seven open reading frames were found in this DNA region. These were the genes for 1-hydroxy-2-naphthoate dioxygenase (phdI) and trans-2'-carboxybenzalpyruvate aldolase (phdJ), orf1, the gene for 2-carboxybenzaldehyde dehydrogenase (phdK), orf2/orf3, and orf4. The amino acid sequence of the orf1 product was similar to that of the aromatic hydrocarbon transporter gene (pcaK) in Pseudomonas putida PRS2000. The amino acid sequence of the orf4 product revealed a similarity to cytochrome P-450 proteins. The region between phdK and orf4 encoded orf2 and orf3 on different strands. The amino acid sequences of the orf2 and orf3 products exhibited no significant similarity to the reported sequences in protein databases.     

Subnanomolar detection limit for sodium dodecyl sulfate-capillary gel electrophoresis using a fluorogenic, noncovalent dye

Picomolar limits of detection are obtained using the noncovalent, fluorogenic dye, Sypro Red. The size separation of four commonly used sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) molecular weight markers with 8% linear polyacrylamide (PAA) as the sieving matrix is used to construct a calibration curve for molecular weight determinations. SDS-CGE purity and molecular weight determination of purified chorismate mutase-prephenate dehydrogenase (CMPD) from Escherichia coli is shown to be comparable in accuracy with slab gel SDS-polyacrylamide gel electrophoresis (SDS-PAGE). A migration time precision study indicates excellent reproducibility. Sypro red labeling of SDS-bovine serum albumin (SDS-BSA) complexes at nanomolar protein concentrations suggests assay detection limits surpassing those of silver staining. This detectability exceeds that achieved in previous SDS-CGE laser-induced fluorescence studies. This approach is expected to be easily adapted for use with commercial polymer formulations and automated instrumentation.     

Electrostatic channeling of oxaloacetate in a fusion protein of porcine citrate synthase and porcine mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase and citrate synthase are sequential enzymes in the Krebs tricarboxylic acid cycle. We have shown [Lindbladh, C., Rault, M., Hagglund, C., Small, W. C., Mosbach, K., Bülow, L., Evans, C., and Srere, P.A (1994) Biochemistry 33, 11692-11698] that a fusion protein of yeast mitochondrial citrate synthase and yeast mitochondrial malate dehydrogenase channels oxaloacetate between the active sites. A Brownian dynamics simulation model of porcine mitochondrial enzymes of citrate synthase and malate dehydrogenase was used [Elcock, A. H., and McCammon, A. M. (1996) Biochemistry 35, 12652-12658], showing that a positive electrostatic surface potential between the active sites of the fusion protein could account for the channeling of oxaloacetate we observed with the yeast fusion protein. Since the data were established with a yeast fusion protein and the model was with porcine fusion protein, we have now prepared and studied the porcine fusion protein. The channeling of the oxaloacetate intermediate was the same for the porcine fusion protein as it was for the yeast fusion protein. This channeling behavior is eliminated at high ionic strength. A fusion protein of porcine citrate synthase and porcine cytosolic malate dehydrogenase does not exhibit any channeling of oxaloacetate. A model of the fusion protein with the cytosolic malate dehydrogenase shows no clear positive electrostatic potential surface between the two active sites, thus distinguishing it from the fusion protein with the mitochondrial malate dehydrogenase. These results establish the electrostatic nature of channeling in mitochondrial fusion proteins.     

Purification, crystallization and properties of triosephosphate isomerase from human skeletal muscle

1. Triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketoisomerase, EC 5.3.1.1) from human skeletal muscle was purified to homogeneity and crystallized. The crystalline enzyme preparation was resolved on polyacrylamide-gel electrophoresis into three isoenzymes. 2. The molecular weight of the enzyme estimated by gel filtration method was found to be 57,400 +/- 3000. Molecular weight determination under dissociation conditions indicated a dimeric subunit structure of the enzyme. 3. The apparent Km for D-glyceraldehyde-3-phosphate as substrate is 0.34 mM, and for dihydroxyacetone phosphate, 0.61 mM. Vmax of the reaction is, respectively, 7200 and 660 units/mg protein at 25 degrees C and pH 7.5. 4. Molecular and kinetic properties of triosephosphate isomerase from human skeletal muscle are very similar to those of rabbit muscle enzyme.     

Isoenzyme spectra of lactate dehydrogenase in tissues of pigs at different stages of embryonic development

Dynamics of the activity and correlation of isoenzyme spectra of lactate dehydrogenase in extracts of the liver, hip muscle and blood serum of the 30-60- and 108-day pig embryos was studied by polyacrilamide gel diskelectrophoresis. A high specificity of isoenzymes sets in all the tissues under study is established.     

Isolation and characterization of a unique glycoprotein from lavage of chicken lungs and lamellar organelles

A major glycoprotein with an estimated molecular weight by sodium dodecylsulfate-acrylamide gel electrophoresis of 36,000 daltons has been found in lavage material of normal chicken lung. Amino acid and carbohydrate analyses of this glycoprotein indicated the presence of hydroxyproline, a high percentage of glycine, sialic acid, mannose, galactose, fucose, and glucosamine. Threonine is the NH2-terminal residue of the glycoprotein. A similar glycoprotein of the same molecular weight, NH2-terminus, amino acid, and carbohydrate composition has been found in lamellar organelles isolated from normal chicken lung. The data indicate that this unique collagen-like glycoprotein, which is similar to a major alveolar glycoprotein found in mammals, including patients with alveolar proteinosis, is also a major protein of avian airways. Because avian airways contain no Clara, ciliated, or goblet cells but do contain type II cells as the only secretory airway cells with lamellar organelles, it is likely that in birds this glycoprotein may be a product of type II cells.     

Isolation of a relatively pure outer membrane fraction of Moraxella nonliquefaciens and a comparison of its characteristics with the cytoplasmic membrane-containing material

Cell envelope fractions of Moraxella nonliquefaciens were isolated by a slight modification of Osborn's method. Two main membrane fractions were characterized chemically and morphologically. The density of the fraction containing cytoplasmic membrane material was 1.17 to 1.18 g cm-3 compared with 1.24 to 1.27 g cm-3 for the outer membrane fraction. Lipopolysaccharide was detected almost exclusively in the outer membrane fraction and sodium dodecyl sulphate polyacrylamide gel electrophoresis of this fraction revealed one dominant protein band with an apparent molecular weight of 45 000. Cross-contamination of the fractions was estimated to be about 10%, as calculated on the basis of the lipopolysaccharide fatty acid 3-hydroxydodecanoic acid and on the relative activities of D-lactate dehydrogenase and succinate dehydrogenase.