Malate Dehydrogenase (malate + dehydrogenase)

Distribution by Scientific Domains

Selected Abstracts

Population genetics of Escherichia coli in a natural population of native Australian rats

Gulietta M. Pupo
Escherichia coli, a normal inhabitant of the intestinal tract of mammals and birds, is a diverse species. Most studies on E. coli populations involve organisms from humans or human-associated animals. In this study, we undertook a survey of E. coli from native Australian mammals, predominantly Rattus tunneyi, living in a relatively pristine environment in the Bundjalung National Park. The genetic diversity was assessed and compared by multilocus enzyme electrophoresis (MLEE), sequence analysis of the mdh (malate dehydrogenase) gene and biotyping using seven sugars. Ninety-nine electrophoretic types were identified from the 242 isolates analysed by MLEE and 15 sequences from the mdh genes sequenced from 21 representative strains. The Bundjalung isolates extend the diversity represented by the E. coli reference (ECOR) set, with new MLEE alleles found in six out of 10 loci. Many of the Bundjalung isolates fell into a discrete group in MLEE. Other Bundjalung strains fell into the recognized E. coli ECOR set groups, but tended to be at the base of both the MLEE and mdh gene trees, implying that these strains are derived independently from ancestral forms of the ECOR groups and that ECOR strains represent only a subset of E. coli adapted to humans and human-associated animals. Linkage disequilibrium analysis showed that the Bundjalung population has an ,epidemic' population structure. The Bundjalung isolates were able to utilize more sugars than the ECOR strains, suggesting that diet plays a prominent role in adaptation of E. coli. [source]

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L)

FEBS JOURNAL, Issue 24 2007
Andre Müller
cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5,- and 3,-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH,5His was purified to homogeneity using metal,chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in Km and decreases in kcat values for pyruvate and l -arginine, but had little effect on the Km and kcat values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid,base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329. [source]

,- d -Mannopyranosyl-(1,2)-,- d -glucopyranosyl-(1,2)-glycerate in the thermophilic bacterium Petrotoga miotherma , structure, cellular content and function

FEBS JOURNAL, Issue 12 2007
Carla D. Jorge
The intracellular accumulation of low molecular mass organic compounds in response to stressful conditions was investigated in the thermophilic bacterium Petrotoga miotherma, a member of the order Thermotogales. This led to the discovery of a new solute, whose structure was established as ,- d -mannopyranosyl-(1,2)-,- d -glucopyranosyl-(1,2)-glycerate (MGG) by MMR spectroscopy and MS. Under optimum growth conditions (3% NaCl; 55 °C), MGG was the major solute [up to 0.6 µmol·(mg protein),1]; ,-glutamate and proline were also present but in minor amounts [below 0.08 µmol·(mg protein),1]. The level of MGG increased notably with the salinity of the growth medium up to the optimum NaCl concentration. At higher NaCl concentrations, however, the level of MGG decreased, whereas the levels of proline and ,-glutamate increased about five-fold and 10-fold, respectively. MGG plays a role during low-level osmotic adaptation of Petrotoga miotherma, whereas ,-glutamate and, to a lesser extent, proline are used for osmoprotection under salt stress. MGG is not part of the cell strategy for coping with heat or oxidative stress. Nevertheless, MGG was an efficient protector of pig heart malate dehydrogenase against heat inactivation and freeze-drying, although mannosylglycerate was better. This is the first report on the occurrence of MGG in living systems. [source]

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu

FEBS JOURNAL, Issue 18 2004
Damodara Rao
The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 °C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation. [source]

A proteomic study of Escherichia coli O157:H7 NCTC 12900 cultivated in biofilm or in planktonic growth mode

Frédéric Trémoulet
Abstract Escherichia coli 0157:H7 biofilms were studied by a new method of cultivation in order to identify some of the proteins involved in the biofilm phenotype. A proteomic analysis of sessile or planktonic bacteria of the same age was carried out by two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and database searching. Comparison of two-dimensional gels showed clear differences between protein patterns of sessile and planktonic cells. Fourteen proteins increased in biofilms, whereas three decreased. From these 17 proteins, 10 were identified by MALDI-TOF-MS and could be classified into four categories according to their function: (1) general metabolism proteins (malate dehydrogenase, thiamine-phosphate pyrophosphorylase), (2) sugar and amino acid transporters (d -ribose-binding periplasmic protein, d -galactose-binding protein, YBEJ), (3) regulator proteins (DNA starvation protein and H-NS) and (4) three proteins with unknown function. The results of this study showed that E. coli O157:H7 modified the expression of several proteins involved in biofilm growth mode. [source]

Elevated activity of the oxidative and non-oxidative pentose phosphate pathway in (pre)neoplastic lesions in rat liver

Wilma M. Frederiks
Summary (Pre)neoplastic lesions in livers of rats induced by diethylnitrosamine are characterized by elevated activity of the first irreversible enzyme of the oxidative branch of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase (G6PD), for production of NADPH. In the present study, the activity of G6PD, and the other NADPH-producing enzymes, phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (ICD) and malate dehydrogenase (MD) was investigated in (pre)neoplastic lesions by metabolic mapping. Transketolase (TKT), the reversible rate-limiting enzyme of the non-oxidative branch of the PPP, mainly responsible for ribose production, was studied as well. Activity of G6PD in (pre)neoplastic lesions was highest, whereas activity of PGD and ICD was only 10% and of MD 5% of G6PD activity, respectively. Glucose-6-phosphate dehydrogenase activity in (pre)neoplastic lesions was increased 25 times compared with extralesional parenchyma, which was also the highest activity increase of the four NADPH-producing dehydrogenases. Transketolase activity was 0.1% of G6PD activity in lesions and was increased 2.5-fold as compared with normal parenchyma. Transketolase activity was localized by electron microscopy exclusively at membranes of granular endoplasmic reticulum in rat hepatoma cells where G6PD activity is localized as well. It is concluded that NADPH in (pre)neoplastic lesions is mainly produced by G6PD, whereas elevated TKT activity in (pre)neoplastic lesions is responsible for ribose formation with concomitant energy supply by glycolysis. The similar localization of G6PD and TKT activity suggests the channelling of substrates at this site to optimize the efficiency of NADPH and ribose synthesis. [source]

Organic acids: old metabolites, new themes

Israel Goldberg
Abstract Fumaric, L -malic and citric acids are intermediates of the oxidative tricarboxylic acid (TCA) cycle which in eukaryotes is localized in mitochondria. These organic acids are synthesized and accumulated in the medium to very high concentrations by filamentous fungi such as Aspergillus spp. and Rhizopus sp. This article reviews basic research on the unusual acid production capability and the associated metabolic pathways operating under defined stress conditions in these specific fungi. In particular, we describe and discuss the importance of the cytosolic reductive TCA pathway, which includes the cytosolic activities of pyruvate carboxylase, malate dehydrogenase and fumarase, for production of fumaric and L -malic acids. This article also describes the differences between fumaric acid, L -malic acid and citric acid production by different organisms (filamentous fungi, yeast, and higher eukaryotes), and the possible application of novel technologies (genetic engineering and bioinformatics) to fungal systems which may offer new industrial potential of filamentous fungi for the production of valuable metabolites. Copyright © 2006 Society of Chemical Industry [source]

Isozyme Analysis and Soluble Mycelial Protein Pattern in Iranian Isolates of Several formae speciales of Fusarium oxysporum

M. Mohammadi
Abstract A total of 13 representative isolates of Fusarium oxysporum f. sp. melonis (FOM) from Iran, USA and France, eight isolates of seven formae speciales from Iran and one isolate of F. oxysporum f. sp. niveum from the USA were compared based on isozyme analysis and soluble mycelial protein pattern. Isozyme analyses of alkaline phosphatase (ALP), catalase (CAT), esterase (EST), malate dehydrogenase (MDH), superoxide dismutase (SOD) and xanthine dehydrogenase (XDH) revealed polymorphism among the F. oxysporum isolates in which 22 electrophoretic phenotypes (EP) were determined. At least 10 putative loci for these six enzymes were detected and they were all polymorphic. Maximum genetic diversity was observed in CAT, EST and XDH loci. Using UPGMA, the 22 isolates were separated into three main groups with one of the groups divided into two subgroups. Group I included isolates belonging to five formae speciales from Iran, whereas group II that included FOM isolates from both Iran and the USA was divided into two subgroups each containing the vast majority of the respective isolates from either country. Group III constituted FOM isolates from France and one pathogenic isolate on pepper from Iran. FOM isolates representing five different geographical regions from Iran belonged to two different races of 1 and 1,2Y and one vegetative compatibility group (VCG)0134 and thus were genetically homologous. Isozyme polymorphism in these isolates was highly correlated with VCG and geographical origins and to a lesser extent with races. Variations in soluble protein profile in FOM isolates were correlated with genetic distances determined in isozyme analysis. This study suggests that isozyme analysis could be a useful tool for identifying genetic diversity not only in FOM but also several formae speciales of F. oxysporum. [source]

Identification of spore allergens from the indoor mould Aspergillus versicolor

ALLERGY, Issue 4 2008
D. Benndorf
Background:, Indoor mould growth and dampness are associated with respiratory health effects and allergies and several studies demonstrated that mainly Aspergillus versicolor and Penicillium expansum are responsible for indoor mould exposure. In contrast, commercialized test systems to diagnose allergic reactions to this mould species are not available. In this study, allergenic proteins from spores of the indoor relevant species A. versicolor and P. expansum should get detected and identified. Methods:, We used two-dimensional (2D)-gel electrophoresis of spore proteins and immunoblotting with sera from patients participating in an epidemiologic study about indoor exposure of moulds and their influence on the development of allergies (ESTERSPEGA). Sera were screened for IgE antibodies specific for proteins from A. versicolor, A. fumigatus and P. expansum in one-dimensional blots and in 2D immunoblots. From the 2D gels, the corresponding spots were picked and identified by mass spectrometry. Results:, More than 20 allergens from A. versicolor were identified; in particular, seven major allergens were selected, which were detected by more than 90% of the positive sera. The most abundant allergen was glyceraldehyde-3-phosphate dehydrogenase, followed by an unnamed protein, which displays a high homology to sobitol/xylose reductase. The other allergens were identified as catalase A, hypothetical protein AN6918.2, enolase, hypothetical protein AN0297.2 and a protein with homology to a fungal malate dehydrogenase. Conclusions:, The results indicate an important role of spore proteins from A. versicolor for sensitization against indoor moulds and identification of the major allergens might enable species-specific diagnosis of allergic reactions. [source]

The putative l -lactate dehydrogenase from Methanococcus jannaschii is an NADPH-dependent l -malate dehydrogenase

Dominique Madern
The enyme encoded by Methanococcus jannaschii open reading frame (ORF) 0490 was purified and characterized. It was shown to be an NADPH-dependent [lactate dehydrogenase (LDH)-like]l -malate dehydrogenase (MalDH) and not an l -lactate dehydrogenase, as had been suggested previously on the basis of amino acid sequence similarity. The results show the importance of biochemical data in the assignment of ORF function in genomic sequences and have implications for the phylogenetic distribution of members of the MalDH/LDH enzyme superfamilies within the prokaryotic kingdom. [source]

Redox enzymes in the plant plasma membrane and their possible roles

A. Bérczi
ABSTRACT Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low-molecular-mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K1), all of which are likely to participate in redox processes. A few enzymes have already been identified: Monodehydroascorbate reductase (EC is firmly bound to the cytosolic surface of the PM where it might be involved in keeping both cytosolic and, together with a b -type cytochrome, apoplastic ascorbate reduced. A malate dehydrogenase (EC is localized on the inner side of the PM. Several NAD(P)H-quinone oxidoreductases have been purified from the cytocolic surface of the PM, but their function is still unknown. Different forms of nitrate reductase (EC,3) are found attached to, as well as anchored in, the PM where they may act as a nitrate sensor and/or contribute to blue-light perception, although both functions are speculative. Ferric-chelate-reducing enzymes (EC are localized and partially characterized on the inner surface of the PM but they may participate only in the reduction of ferric-chelates in the cytosol. Very recently a ferric-chelate-reducing enzyme containing binding sites for FAD, NADPH and hemes has been identified and suggested to be a trans -PM protein. This enzyme is involved in the reduction of apoplastic iron prior to uptake of Fe2+ and is induced by iron deficiency. The presence of an NADPH oxidase, similar to the so-called respiratory burst oxidase in mammals, is still an open question. An auxin-stimulated and cyanide-insensitive NADH oxidase (possibly a protein disulphide reductase) has been characterized but its identity is still awaiting independent confirmation. Finally, the only trans -PM redox protein which has been partially purified from plant PM so far is a high-potential and ascorbate-reducible b -type cytochrome. In co-operation with vitamin K1 and an NAD(P)H-quinone oxidoreductase, it may participate in trans -PM electron transport. [source]

Prenatal alcohol exposure alters phosphorylation and glycosylation of proteins in rat offspring liver

Bourlaye Fofana
Abstract To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2-D PAGE with total, phospho- and glycoprotein staining and MALDI-MS/MS and database searching were conducted. The results, based on fold-change expression, revealed a down-regulation of total protein expression by prenatal alcohol exposure in 7-day-old and 3-month-old rats. There was an up-regulation of protein phosphorylation but a down-regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo-keto reductase, tumor rejection antigen gp96, fructose-1,6-bisphosphatase, glycerol-3-phosphate dehydrogenase, malate dehydrogenase, and ,-actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S-transferase, glucose regulated protein 58, ,-enolase, eukaryotic translation elongation factor 1 ,-2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3-hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications. [source]

A comparative proteomic approach to understand the adaptations of an H+ -ATPase-defective mutant of Corynebacterium glutamicum ATCC14067 to energy deficiencies

Liyuan Li
Abstract F172-8, an H+ -ATPase-defective mutant of the glutamic acid-producing bacterium Corynebacterium glutamicum ATCC 14067, exhibits enhanced rates of glucose consumption and respiration compared to the parental strain when cultured in a biotin-rich medium with glucose as the carbon source. We conducted a comparative proteomic analysis to clarify the mechanism by which the enhanced glucose metabolism in this mutant is established using a proteome reference map for strain ATCC 14067. A comparison of the proteomes of the two strains revealed the up-regulated expression of the several important enzymes such as pyruvate kinase (Pyk), malate:quinone oxidoreductase (Mqo), and malate dehydrogenase (Mdh) in the mutant. Because Pyk activates glycolysis in response to cellular energy shortages in this bacterium, its increased expression may contribute to the enhanced glucose metabolism of the mutant. A unique reoxidation system has been suggested for NADH in C. glutamicum consisting of coupled reactions between Mqo and Mdh, together with the respiratory chain; therefore, the enhanced expression of both enzymes might contribute to the reoxidation of NADH during increased respiration. The proteomic analysis allowed the identification of unique physiological changes associated with the H+ -ATPase defect in F172-8 and contributed to the understanding of the adaptations of C. glutamicum to energy deficiencies. [source]

Arabidopsis peroxisomal malate dehydrogenase functions in ,-oxidation but not in the glyoxylate cycle

Itsara Pracharoenwattana
Summary The aim was to determine the function of peroxisomal NAD+ -malate dehydrogenase (PMDH) in fatty acid ,-oxidation and the glyoxylate cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T-DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4-dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in ,-oxidation, even though microarray analysis shows that ,-oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal-2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in ,-oxidation in mutant seedlings, [14C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the glyoxylate cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid ,-oxidation and does not participate directly in the glyoxylate cycle. [source]

Liver gene expression in relation to hepatic steatosis and lipid secretion in two duck species

F. Hérault
Summary The susceptibility to development of hepatic steatosis is known to differ between Muscovy and Pekin ducks. Although some experiments were conducted to decipher these differences, few data have been produced to analyse the role of specific genes in this process. For this purpose, expression levels of genes involved in lipid (ATP citrate lyase, malic enzyme 1, fatty acid synthase, stearoyl-CoA desaturase 1, diacylglycerol O-acyl transferase 2, microsomal triglyceride transfer protein, apolipoprotein A1, apolipoprotein B, sterol regulatory element binding factor 1, hepatocyte nuclear factor 4, choline/ethanolamine phosphotransferase 1, carnitine palmitoyl transferase 1A, peroxisome proliferator-activated receptor alpha and sterol O-acyltransferase) and carbohydrate (activating transcription factor 4 or cAMP-response element binding protein, mitochondrial malate dehydrogenase 2 and carbohydrate responsive element binding protein) metabolism and in other functions were analysed in the liver of Pekin and Muscovy ducks fed ad libitum or overfed. A specific positive effect of feeding was observed on the expression of genes involved mainly in fatty acids and TG synthesis and glycolysis, and negative effect on genes involved in ,-oxidation. Interestingly, a strong species effect was also observed on stearoyl-CoA desaturase 1 and to a lesser extent on diacylglycerol O-acyl transferase 2 expression, leading to large differences in expression levels between Pekin and Muscovy overfed ducks, which could explain the difference in lipid metabolism and steatosis ability observed between the two duck species. These results should shed light on gene expression that might underlie susceptibility to hepatic steatosis in humans. [source]

Stress mitigating and immunomodulatory effect of dietary pyridoxine in Labeo rohita (Hamilton) fingerlings

Mohammad Shahbaz Akhtar
Abstract A 60-day experiment was carried out to delineate stress mitigating and immunomodulatory role of dietary pyridoxine (PN) in Labeo rohita fingerlings exposed to endosulfan. Two hundred and seventy fingerlings were randomly distributed into six treatments in triplicates. Five iso-caloric and iso-nitrogenous purified diets were prepared with graded levels of pyridoxine. Six treatment groups were T0 (10 mg PN+without endosulfan), T1 (0 mg PN+endosulfan), T2 (10 mg PN+endosulfan), T3 (50 mg PN+endosulfan), T4 (100 mg PN+endosulfan) and T5 (200 mg PN+endosulfan). The role of pyridoxine on stress mitigation and immunomodulation was assessed by biochemical and haemato-immunological parameters like aspartate aminotransaminase, alanine aminotransaminase, lactate dehydrogenase, malate dehydrogenase, superoxide dismutase and catalase were significantly (P<0.05) lower while acetylcholinesterase was significantly (P<0.05) higher in pyridoxine-fed groups. Erythrocytes count, haemoglobin content and total serum protein, albumin, globulin, nitroblue tetrazolium and lysozyme activity were significantly (P<0.05) higher while cortisol and blood glucose were decreased significantly (P<0.05) in pyridoxine-fed groups. Percentage survival after challenge with Aeromonas hydrophila was highest in T0 group. The results obtained in present study indicate that dietary pyridoxine supplementation at 100 mg PN kg,1 diet reduces the endosulfan-induced stress and triggers immune response in L. rohita fingerlings. [source]

Redox-active disulfides in a plant light switch: A Pbl problem,

Bernard Offmann
Abstract A problem for students on the mode of activation of light-regulated NADP-dependent malate dehydrogenase is presented here as a series of questions requiring the interpretation of experimental data. [source]

Structure of Escherichia coli malate dehydrogenase at 1.45,Ĺ resolution

Jelena Zaitseva
The structure of apo malate dehydrogenase from Escherichia coli has been determined to 1.45,Ĺ resolution. The crystals belonged to space group C2, with unit-cell parameters a = 146.0, b = 52.0, c = 168.9,Ĺ, , = 102.2°. The structure was determined with the molecular-replacement pipeline program BALBES and was refined to a final R factor of 18.6% (Rfree = 21.4%). The final model has two dimers in the asymmetric unit. In each dimer one monomer contains the active-site loop in the open conformation, whereas in the opposing monomer the active-site loop is disordered. [source]

Synergistic coordination of polyethylene glycol with ClpB/DnaKJE bichaperone for refolding of heat-denatured malate dehydrogenase

Rui Nian
Abstract The use of polyethylene glycol (PEG) as a refolding additive to a refolding cocktail comprising the molecular bichaperone ClpB and DnaKJE significantly enhances chaperone-mediated refolding of heat-denatured malate dehydrogenase (MDH). The critical factor to affect the refolding yield is the time point of introducing PEG to the refolding cocktail. The refolding efficiency reached approximately 90% only when PEG was added at the beginning of refolding reaction. The synergistic coordination of an inexpensive refolding additive PEG with the ClpB/DnaKJE bichaperone system may provide an economical route to further enhance the efficacy of ClpB/DnaKJE refolding cocktail approach, facilitating its implementation in large-scale refolding processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]

Design of a Molecular Chaperone-Assisted Protein Folding Bioreactor

Reto J. Kohler
Escherichia coli molecular chaperone GroEL and co-chaperone GroES are well known to assist the folding/refolding of a diverse range of substrate proteins. Despite this, there have been relatively few reports of the GroEL/GroES molecular chaperone system being used as a biotechnology tool for protein folding/refolding. In this paper, a solution-phase protein folding bioreactor is described that involves the complete GroEL/GroES system. The main features of this bioreactor are the use of a stirred-cell concentrator fitted with a 100 kDa molecular weight cutoff membrane and an attached buffer reservoir. This bioreactor system was used successfully for assisted-batch refolding of guanidinium chloride (Gu-HCl) unfolded mitochondrial malate dehydrogenase (mMDH). We believe that protein folding bioreactor systems of this type could have wide potential utility for the folding/refolding of unfolded protein substrates. [source]

Allergenicity and cross-reactivity of Senecio pollen: identification of novel allergens using the immunoproteomics approach

O. Luengo
Summary Background The genus Senecio is the largest genus of the family Asteraceae (Compositae). The allergenicity of Senecio has not been assessed previously. Objective The aim of this study was to investigate the allergens of Senecio jacobea pollen and to determine their immunological characteristics and clinical relevance. Methods Fifty patients with rhinoconjunctivitis and a positive skin prick test (SPT) to Senecio were recruited. The clinical relevance of this pollen was assessed by means of a nasal provocation test (NPT). Allergens were characterized by one-dimensional electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis and immunoblotting. Furthermore, characterization and identification of the allergens were performed by mass spectrometry (MS). In vitro inhibition tests were performed to evaluate cross-reactivity with other pollen. Results Three predominant allergens, both in the intensity of reaction and the frequency of recognition by human-allergic sera, were 59 (60%), 42 (50%) and 31 kDa (50%). The two-dimensional analysis allowed the identification of several allergens. One spot around 42 kDa was identified as a protein homologous to pectate lyase and three other spots were homologous to malate dehydrogenase by MS. S. jacobea proteins showed cross-reactivity with other proteins of the Asteraceae family and also with Parietaria judaica. This was demonstrated by immunoblotting and ELISA inhibition studies. Conclusion S. jacobea constitute a newly discovered allergenic source. It shows cross-reactivity with other members of the Asteraceae plant family as well as with P. judaica. [source]

Inhibition of calcineurin increases monocarboxylate transporters 1 and 4 protein and glycolytic enzyme activities in rat soleus muscle

Masataka Suwa
SUMMARY 1.,The present study was designed to examine the role of calcineurin in muscle metabolic components by the administration of the specific calcineurin inhibitor cyclosporine A (CsA) to rats. 2.,Male Wistar rats were divided into either a CsA-treated group (CT) or a vehicle-treated group (VT). Cyclosporine A was administered subcutaneously to rats at a rate of 25 mg/kg bodyweight per day for 10 successive days. Thereafter, changes in muscle enzyme activities and glucose transporter (GLUT)-4 and monocarboxylate transporter (MCT)-1 and MCT-4 proteins in the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles were examined. 3.,There was a significant increase in MCT-1 and MCT-4 proteins in the soleus muscle in the CT group, but not in the EDL muscle. The activities of hexokinase, pyruvate kinase and lactate dehydrogenase in the soleus muscle also increased significantly in the CT group, but a similar increase in enzyme activity was not seen in EDL muscle. The activities of citrate synthase or malate dehydrogenase and the GLUT-4 protein content were not altered by CsA treatment in either the soleus or EDL muscles. 4.,These results seem to imply that calcineurin negatively regulates the components of glucose/lactate metabolism, except for GLUT-4, especially in slow-twitch muscle. [source]