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Aldolase
Selected AbstractsA Mutant D -Fructose-6-Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone for the Synthesis of Polyhydroxylated CompoundsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2010Abstract A mutant of D -fructose-6-phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The kcat/KM value for DHA was 17-fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5-fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3-fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi-step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis. [source] A Thermostable Aldolase for the Synthesis of 3-Deoxy-2-ulosonic AcidsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2007Henry Abstract A stereochemically promiscuous 2-keto-3-deoxygluconate aldolase has been used as an efficient biocatalyst to catalyse the aldol reaction of pyruvate with C3 - and C4 -aldoses to afford syn - and anti -3-deoxy-2-ulosonic acids in poor to good de. A continuous flow bioreactor containing immobilised aldolase has been developed that enables gram quantities of C6 - and C7 -3-deoxyhept-2-ulosonic acids to be produced in an efficient manner. [source] Aldol Additions of Dihydroxyacetone Phosphate to N -Cbz-Amino Aldehydes Catalyzed by L -Fuculose-1-Phosphate Aldolase in Emulsion Systems: Inversion of Stereoselectivity as a Function of the Acceptor AldehydeCHEMISTRY - A EUROPEAN JOURNAL, Issue 5 2005Laia Espelt Dr. Abstract The potential of L -fuculose-1-phosphate aldolase (FucA) as a catalyst for the asymmetric aldol addition of dihydroxyacetone phosphate (DHAP) to N -protected amino aldehydes has been investigated. First, the reaction was studied in both emulsion systems and conventional dimethylformamide (DMF)/H2O (1:4 v/v) mixtures. At 100,mM DHAP, compared with the reactions in the DMF/H2O (1:4) mixture, the use of emulsion systems led to two- to three-fold improvements in the conversions of the FucA-catalyzed reactions. The N -protected aminopolyols thus obtained were converted to iminocyclitols by reductive amination with Pd/C. This reaction was highly diastereoselective with the exception of the reaction of the aldol adduct formed from (S)- N -Cbz-alaninal, which gave a 55:45 mixture of both epimers. From the stereochemical analysis of the resulting iminocyclitols, it was concluded that the stereoselectivity of the FucA-catalyzed reaction depended upon the structure of the N -Cbz-amino aldehyde acceptor. Whereas the enzymatic aldol reaction with both enantiomers of N -Cbz-alaninal exclusively gave the expected 3R,4R configuration, the stereochemistry at the C-4 position of the major aldol adducts produced in the reactions with N -Cbz-glycinal and N -Cbz-3-aminopropanal was inverted to the 3R,4S configuration. The study of the FucA-catalyzed addition of DHAP to phenylacetaldehyde and benzyloxyacetaldehyde revealed that the 4R product was kinetically favored, but rapidly disappeared in favor of the 4S diastereoisomer. Computational models were generated for the situations before and after CC bond formation in the active site of FucA. Moreover, the lowest-energy conformations of each pair of the resulting epimeric adducts were determined. The data show that the products with a 3R,4S configuration were thermodynamically more stable and, therefore, the major products formed, in agreement with the experimental results. [source] Light-induced gene expression of fructose 1,6-bisphosphate aldolase during heterotrophic growth in a cyanobacterium, Synechocystis sp.FEBS JOURNAL, Issue 1 2009PCC 680 Synechocystis sp. PCC 6803 exhibits light-activated heterotrophic growth (LAHG) under dark conditions with glucose as a carbon source. The light activation is remarkable at a late period of photoautotrophic preculture, such as the late-linear and stationary growth phases. To understand the physiological effects of light irradiation and glucose under LAHG conditions, their effects on the expression of soluble proteins were analyzed by means of 2D-PAGE. Various soluble proteins, which were minimal under photoautotrophic preculture conditions, were observed clearly under LAHG conditions, suggesting that proteins were synthesized actively under these conditions. Fructose 1,6-bisphosphate aldolase, one of the glycolytic enzymes, was found to be induced under LAHG conditions on 2D-PAGE. The activity of fructose 1,6-bisphosphate aldolase, which had decreased during photoautotrophic preculture, also increased under LAHG conditions, similar to the mRNA level of the encoding gene, fbaA. In addition, we found that a deletion mutant of sll1330, a putative gene containing a helix-turn-helix DNA-binding motif, could not grow under LAHG conditions, whereas it could grow photoautotrophically. The increases in the protein level of FbaA and fbaA gene expression observed in wild-type cells under LAHG conditions were greatly inhibited in the deletion mutant. These results suggest that the regulation of fbaA gene expression by way of sll1330 is one of the important processes in Synechocystis sp. PCC 6803 under light pulse LAHG conditions. [source] Metabolic fate of l -lactaldehyde derived from an alternative l -rhamnose pathwayFEBS JOURNAL, Issue 20 2008Seiya Watanabe Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alternative pathway of l -rhamnose metabolism, which is different from the known bacterial pathway. In a previous study (Watanabe S, Saimura M & Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l -rhamnose metabolism. J Biol Chem283, 20372,20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [l -rhamnose 1-dehydrogenase (LRA1), l -rhamnono-,-lactonase (LRA2), l -rhamnonate dehydratase (LRA3) and l -2-keto-3-deoxyrhamnonate aldolase (LRA4)]. In the known and alternative l -rhamnose pathways, l -lactaldehyde is commonly produced from l -2-keto-3-deoxyrhamnonate and l -rhamnulose 1-phosphate by each specific aldolase, respectively. To estimate the metabolic fate of l -lactaldehyde in fungi, we purified l -lactaldehyde dehydrogenase (LADH) from P. stipitis cells l -rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ionization-quadruple ion trap-time of flight mass spectrometry. In contrast, LADH of A. vinelandii (AvLADH) was clustered with the LRA1,4 gene on the genome. Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, l -lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH. In the phylogenetic tree of the aldehyde dehydrogenase superfamily, PsLADH is poorly related to the known bacterial LADHs, including that of Escherichia coli (EcLADH). However, despite its involvement in different l -rhamnose metabolism, AvLADH belongs to the same subfamily as EcLADH. This suggests that the substrate specificities for l -lactaldehyde between fungal and bacterial LADHs have been acquired independently. [source] Increased glucose metabolism and ATP level in brain tissue of Huntington's disease transgenic miceFEBS JOURNAL, Issue 19 2008Judit Oláh Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by multifarious dysfunctional alterations including mitochondrial impairment. In the present study, the formation of inclusions caused by the mutation of huntingtin protein and its relationship with changes in energy metabolism and with pathological alterations were investigated both in transgenic and 3-nitropropionic acid-treated mouse models for HD. The HD and normal mice were characterized clinically; the affected brain regions were identified by immunohistochemistry and used for biochemical analysis of the ATP-producing systems in the cytosolic and the mitochondrial compartments. In both HD models, the activities of some glycolytic enzymes were somewhat higher. By contrast, the activity of glyceraldehyde-3-phosphate dehydrogenase was much lower in the affected region of the brain compared to that of the control. Paradoxically, at the system level, glucose conversion into lactate was enhanced in cytosolic extracts from the HD brain tissue, and the level of ATP was higher in the tissue itself. The paradox could be resolved by taking all the observed changes in glycolytic enzymes into account, ensuing an experiment-based detailed mathematical model of the glycolytic pathway. The mathematical modelling using the experimentally determined kinetic parameters of the individual enzymes and the well-established rate equations predicted the measured flux and concentrations in the case of the control. The same mathematical model with the experimentally determined altered Vmax values of the enzymes did account for an increase of glycolytic flux in the HD sample, although the extent of the increase was not predicted quantitatively. This suggested a somewhat altered regulation of this major metabolic pathway in HD tissue. We then used the mathematical model to develop a hypothesis for a new regulatory interaction that might account for the observed changes; in HD, glyceraldehyde-3-phosphate dehydrogenase may be in closer proximity (perhaps because of the binding of glyceraldehyde-3-phosphate dehydrogenase to huntingtin) with aldolase and engage in channelling for glyceraldehyde-3-phosphate. By contrast to most of the speculation in the literature, our results suggest that the neuronal damage in HD tissue may be associated with increased energy metabolism at the tissue level leading to modified levels of various intermediary metabolites with pathological consequences. [source] Experimental validation of metabolic pathway modelingFEBS JOURNAL, Issue 13 2008An illustration with glycolytic segments from Entamoeba histolytica In the search for new drug targets in the human parasite Entamoeba histolytica, metabolic control analysis was applied to determine, experimentally, flux control distribution of amebal glycolysis. The first (hexokinase, hexose-6-phosphate isomerase, pyrophosphate-dependent phosphofructokinase (PPi -PFK), aldolase and triose-phosphate isomerase) and final (3-phosphoglycerate mutase, enolase and pyruvate phosphate dikinase) glycolytic segments were reconstituted in vitro with recombinant enzymes under near-physiological conditions of pH, temperature and enzyme proportion. Flux control was determined by titrating flux with each enzyme component. In parallel, both glycolytic segments were also modeled by using the rate equations and kinetic parameters previously determined. Because the flux control distribution predicted by modeling and that determined by reconstitution were not similar, kinetic interactions among all the reconstituted components were experimentally revised to unravel the causes of the discrepancy. For the final segment, it was found that 3-phosphoglycerate was a weakly competitive inhibitor of enolase, whereas PPi was a moderate inhibitor of 3-phosphoglycerate mutase and enolase. For the first segment, PPi was both a strong inhibitor of aldolase and a nonessential mixed-type activator of amebal hexokinase; in addition, lower Vmax values for hexose-6-phosphate isomerase, PPi -PFK and aldolase were induced by PPi or ATP inhibition. It should be noted that PPi and other metabolites were absent from the 3-phosphoglycerate mutase and enolase or aldolase and hexokinase kinetics experiments, but present in reconstitution experiments. Only by incorporating these modifications in the rate equations, modeling predicted values of flux control distribution, flux rate and metabolite concentrations similar to those experimentally determined. The experimentally validated segment models allowed ,in silico experimentation' to be carried out, which is not easy to achieve in in vivo or in vitro systems. The results predicted a nonsignificant effect on flux rate and flux control distribution by adding parallel routes (pyruvate kinase for the final segment and ATP-dependent PFK for the first segment), because of the much lower activity of these enzymes in the ameba. Furthermore, modeling predicted full flux-control by 3-phosphoglycerate mutase and hexokinase, in the presence of low physiological substrate and product concentrations. It is concluded that the combination of in vitro pathway reconstitution with modeling and enzyme kinetics experimentation permits a more comprehensive understanding of the pathway behavior and control properties. [source] Glycolysis in Ustilago maydisFEMS YEAST RESEARCH, Issue 8 2008Emma Saavedra Abstract The kinetic parameters of the 10 glycolytic enzymes and glycolytic fluxes were determined for the first time in Ustilago maydis. Enzyme activities in yeast grown in minimal medium and harvested in the stationary stage were twofold higher than those from yeast grown in rich medium. In contrast, in yeast harvested in the exponential stage, the enzyme activities were higher in cells grown in rich medium. Phosphofructokinase activity was the lowest in the four culture conditions analyzed, suggesting that this enzyme is a flux-controlling step in U. maydis glycolysis. The Vmax and Km values of hexokinase and pyruvate kinase were similar under all conditions. The results revealed that U. maydis aldolase belongs to the class II type of metalo-aldolases. 3-Phosphoglycerate mutase (PGAM) activity was 2,3-bisphosphoglycerate cofactor independent, which contrasted with the cofactor dependency predicted by the amino acid sequence alignment analysis. Pyruvate was secreted by U. maydis yeast in the presence and absence of external glucose. The glycolytic enzyme activities in the U. maydis mycelial form were similar to those found in yeast, except for one order of magnitude higher phosphofructokinase and PGAM activities, thus suggesting differences in the glycolysis regulatory mechanisms between the two cellular forms. [source] Six novel alleles identified in Italian hereditary fructose intolerance patients enlarge the mutation spectrum of the aldolase B gene,,HUMAN MUTATION, Issue 6 2004Gabriella Esposito Abstract Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired functioning of human liver aldolase (B isoform; ALDOB). To-date, 29 enzyme-impairing mutations have been identified in the aldolase B gene. Here we report six novel HFI single nucleotide changes identified by sequence analysis in the aldolase B gene. Three of these are missense mutations (g.6846T>C, g.10236G>T, g.10258T>C), one is a nonsense mutation (g.8187C>T) and two affect splicing sites (g.8180G>C and g.10196A>G). We have expressed in bacterial cells the recombinant proteins corresponding to the g.6846T>C (p.I74T), g.10236G>T (p.V222F), and g.10258T>C (p.L229P) natural mutants to study their effect on aldolase B function and structure. All the new variants were insoluble; molecular graphics data suggest this is due to impaired folding. © 2004 Wiley-Liss, Inc. [source] Response to alkaline stress by root canal bacteria in biofilmsINTERNATIONAL ENDODONTIC JOURNAL, Issue 5 2007L. E. Chávez de Paz Abstract Aim, To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. Methodology, Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. Results,Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. Conclusions, In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms. [source] A Mutant D -Fructose-6-Phosphate Aldolase (Ala129Ser) with Improved Affinity towards Dihydroxyacetone for the Synthesis of Polyhydroxylated CompoundsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2010Abstract A mutant of D -fructose-6-phosphate aldolase (FSA) of Escherichia coli, FSA A129S, with improved catalytic efficiency towards dihydroxyacetone (DHA), the donor substrate in aldol addition reactions, was explored for synthetic applications. The kcat/KM value for DHA was 17-fold higher with FSA A129S than that with FSA wild type (FSA wt). On the other hand, for hydroxyacetone as donor substrate FSA A129S was found to be 3.5-fold less efficient than FSA wt. Furthermore, FSA A129S also accepted glycolaldehyde (GA) as donor substrate with 3.3-fold lower affinity than FSA wt. This differential selectivity of both FSA wt and FSA A129S for GA makes them complementary biocatalysts allowing a control over donor and acceptor roles, which is particularly useful in carboligation multi-step cascade synthesis of polyhydroxylated complex compounds. Production of the mutant protein was also improved for its convenient use in synthesis. Several carbohydrates and nitrocyclitols were efficiently prepared, demonstrating the versatile potential of FSA A129S as biocatalyst in organic synthesis. [source] Activated ,,,-Unsaturated Aldehydes as Substrate of Dihydroxyacetone Phosphate (DHAP)-Dependent Aldolases in the Context of a Multienzyme SystemADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 17 2009Israel Sánchez-Moreno Abstract The utility for carbon-carbon bond formation of a multienzyme system composed of recombinant dihydroxyacetone kinase (DHAK) from Citrobacter freundii, the fructose bisphosphate aldolase from rabbit muscle (RAMA) and acetate kinase (AK) for adenosine triphosphate (ATP) regeneration has been studied. Several aldehydes with great structural diversity, including three ,,,-unsaturated aldehydes, have been analysed as acceptor substrates. It was found that ,,,-unsaturated aldehydes bearing an electron-withdrawing group in the , position to the double bond with a trans configuration are good acceptors for RAMA in this multienzyme system. The aldol reaction proceeds with excellent D - threo enantioselectivity and the aldol adduct is obtained in good overall yield. The L - threo and D - erythro enantiomers are also accessible from rhamnulose 1-phosphate aldolase (Rha-1PA) and fuculose 1-phosphate aldolase (Fuc-1PA) catalysed reactions, respectively. [source] Cover Picture: (Adv. Synth.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 16 2007Catal. The cover picture results from the seminal mechanistic work on DERA, deoxyribose-5-phosphate aldolase, a widely distributed catabolic enzyme, by Chi-Huey Wong and co-workers. [source] Cover Picture: (Adv. Synth.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 13 2007Catal. The cover picture results from the seminal mechanistic work on DERA, deoxyribose-5-phosphate aldolase, a widely distributed catabolic enzyme, by Chi-Huey Wong and co-workers. [source] Cover Picture: (Adv. Synth.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 8-9 2007Catal. The cover picture results from the seminal mechanistic work on DERA, deoxyribose-5-phosphate aldolase, a widely distributed catabolic enzyme, by Chi-Huey Wong and co-workers. [source] Cover Picture: (Adv. Synth.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 7 2007Catal. The cover picture results from the seminal mechanistic work on DERA, deoxyribose-5-phosphate aldolase, a widely distributed catabolic enzyme, by Chi-Huey Wong and co-workers. [source] Cover Picture: (Adv. Synth.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2007Catal. The cover picture results from the seminal mechanistic work on DERA, deoxyribose-5-phosphate aldolase, a widely distributed catabolic enzyme, by Chi-Huey Wong and co-workers. [source] A Thermostable Aldolase for the Synthesis of 3-Deoxy-2-ulosonic AcidsADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2007Henry Abstract A stereochemically promiscuous 2-keto-3-deoxygluconate aldolase has been used as an efficient biocatalyst to catalyse the aldol reaction of pyruvate with C3 - and C4 -aldoses to afford syn - and anti -3-deoxy-2-ulosonic acids in poor to good de. A continuous flow bioreactor containing immobilised aldolase has been developed that enables gram quantities of C6 - and C7 -3-deoxyhept-2-ulosonic acids to be produced in an efficient manner. [source] Cover Picture: (Adv. Synth.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 3 2007Catal. The cover picture results from the seminal mechanistic work on DERA, deoxyribose-5-phosphate aldolase, a widely distributed catabolic enzyme, by Chin-Huey Wong and co-workers. [source] Cover Picture: (Adv. Synth.ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 1-2 2007Catal. The cover picture results from the seminal mechanistic work on DERA, deoxyribose-5-phosphate aldolase, a widely distributed catabolic enzyme, by Chin-Huey Wong and co-workers. [source] Proteomic identification of nitrated brain proteins in early Alzheimer's disease inferior parietal lobuleJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Tanea T. Reed Abstract Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive decline in multiple cognitive domains. Its pathological hallmarks include senile plaques and neurofibrillary tangles. Mild cognitive impairment (MCI) is the earliest detectable stage of AD with limited symptomology and no dementia. The yearly conversion rate of patients from MCI to AD is 10,15%, although conversion back to normal is possible in a small percentage. Early diagnosis of AD is important in an attempt to intervene or slow the advancement of the disease. Early AD (EAD) is a stage following MCI and characterized by full-blown dementia; however, information involving EAD is limited. Oxidative stress is well-established in MCI and AD, including protein oxidation. Protein nitration also is an important oxidative modification observed in MCI and AD, and proteomic analysis from our laboratory identified nitrated proteins in both MCI and AD. Therefore, in the current study, a proteomics approach was used to identify nitrated brain proteins in the inferior parietal lobule from four subjects with EAD. Eight proteins were found to be significantly nitrated in EAD: peroxiredoxin 2, triose phosphate isomerase, glutamate dehydrogenase, neuropolypeptide h3, phosphoglycerate mutase1, H+, transporting ATPase, ,-enolase and fructose-1,6-bisphosphate aldolase. Many of these proteins are also nitrated in MCI and late-stage AD, making this study the first to our knowledge to link nitrated proteins in all stages of AD. These results are discussed in terms of potential involvement in the progression of this dementing disorder. [source] Genetic differentiation and natural hybridization between the Sardinian endemic Maniola nurag and the European Maniola jurtinaJOURNAL OF EVOLUTIONARY BIOLOGY, Issue 4 2007A. GRILL Abstract The Mediterranean island of Sardinia is known for its multitude of unique genetic lineages. We view one of them in a larger phylogeographic context. The endemic Sardinian Meadow Brown butterfly, Maniola nurag, is restricted to the mountainous areas of the island, whereas its widespread close relative, Maniola jurtina, also occurs on the coast. At intermediate altitudes the species' distributions overlap. There, a number of individuals exhibit phenotypic characteristics intermediate between the two species. We examined patterns of intra- and interpopulation variation in 10 M. nurag populations from Sardinia and 16 M. jurtina populations from Sardinia and continental Europe, as well as 17 intermediate individuals, sampled in 1999,2002, by means of allozyme markers, combining it with a morphometric analysis based on 18 wing-characters of 52 males. At the 15 loci studied (aldolase, aat-1, aat-2, g6pdh, gpd, idh-1, idh-2, mdh-1, mdh-2, mpi, me, leu-ala, pgi, pgm, and 6pgdh), 76 different alleles were detected, 63 of which were shared by M. nurag and M. jurtina. None of the loci was found to be alternatively fixed between the two species. In that respect, this study testifies to the difficulties that may arise when trying to identify hybrids from genotypic data. Levels of genetic variation in island populations (M. jurtina: HO = 0.137,0.189; M. nurag: HO = 0.141,0.270) were comparable to those of mainland M. jurtina (HO = 0.141,0.236). A Bayesian admixture analysis supported the hypothesis of mixed (hybrid) ancestry of individuals occurring at intermediate altitudes. Similarly, neighbour-joining and unweighted pair-group method with arithmetic averaging (UPGMA) analyses, as well as morphometrics hinted at the existence of a Maniola -hybrid zone in Sardinia at intermediate altitudes. We discuss the results in the light of the phylogeography of other Sardinian taxa with the aim to reach a general understanding of the biogeographic history of this island's endemic species. [source] Peak capacity of ion mobility mass spectrometry: the utility of varying drift gas polarizability for the separation of tryptic peptidesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2004Brandon T. Ruotolo Abstract Ion mobility mass spectrometry (IM-MS) peptide mass mapping experiments were performed using a variety of drift gases (He, N2, Ar and CH4). The drift gases studied cover a range of polarizabilities ((0.2,2.6) × 10,24 cm3) and the peak capacities obtained for tryptic peptides in each gas are compared. Although the different gases exhibit similar peak capacities (5430 (Ar) to 7580 (N2)) in some cases separation selectivity presumably based on peptide conformers (or conformer populations), is observed. For example the drift time profiles observed for some tryptic peptide ions from aldolase (rabbit muscle) show a dependence on drift gas. The transmission of high-mass ions (m/z > 2000) is also influenced by increased scattering cross-section of the more massive drift gases. Consequently the practical peak capacity for IM-MS separation cannot be assumed to be solely a function of resolution and the ability of a gas to distribute signals in two-dimensional space; rather, peak capacity estimates must account for the transmission losses experienced for peptide ions as the drift gas mass increases. Copyright © 2004 John Wiley & Sons, Ltd. [source] Changes in endoplasmic reticulum stress proteins and aldolase A in cells exposed to dopamineJOURNAL OF NEUROCHEMISTRY, Issue 1 2008April A. Dukes Abstract In Parkinson's disease, oxidative stress is implicated in protein misfolding and aggregation, which may activate the unfolded protein response by the endoplasmic reticulum (ER). Dopamine (DA) can initiate oxidative stress via H2O2 formation by DA metabolism and by oxidation into DA quinone. We have previously shown that DA quinone induces oxidative protein modification, mitochondrial dysfunction in vitro, and dopaminergic cell toxicity in vivo and in vitro. In this study, we used cysteine- and lysine-reactive fluorescent dyes with 2D difference in-gel electrophoresis, mass spectrometry, and peptide mass fingerprint analysis to identify proteins in PC12 cell mitochondrial-enriched fractions that were altered in abundance following DA exposure (150 ,M, 16 h). Quantitative changes in proteins labeled with fluorescent dyes indicated increases in a subset of proteins after DA exposure: calreticulin, ERp29, ERp99, Grp58, Grp78, Grp94 and Orp150 (149,260%), and decreased levels of aldolase A (39,42%). Changes in levels of several proteins detected by 2D difference in-gel electrophoresis were confirmed by western blot. Using this unbiased proteomics approach, our findings demonstrated that in PC12 cells, DA exposure leads to a cellular response indicative of ER stress prior to the onset of cell death, providing a potential link between DA and the unfolded protein response in the pathogenesis of Parkinson's disease. [source] Performance of wide-pore monolithic silica column in protein separationJOURNAL OF SEPARATION SCIENCE, JSS, Issue 15-16 2009Hironobu Morisaka Abstract A monolithic wide-pore silica column was newly prepared for protein separation. The wide distribution of the pore sizes of monolithic columns was evaluated by mercury porosimetry. This column, as well as the conventional monolithic column, shows high permeability in the chromatographic separation of low-molecular-sized substances. In higher-molecular-sized protein separation, the wide-pore monolithic silica column shows better performance than that of the conventional monolithic column. Under optimized conditions, five different proteins , ribonuclease A, albumin, aldolase, catalase, and ferritin , were baseline-separated within 3 min, which is faster than that using the particle-packed columns. In addition, the monolithic wide-pore silica column could also be prepared in fused silica capillary (600 mm long, 0.2 mm i.d.) for highly efficient protein separation. The peak capacity of the wide-pore monolithic silica capillary column is estimated to be approximately 300 in the case of protein separation, which is a characteristic performance. [source] Cytoskeleton-associated, carbohydrate-metabolizing enzymes in maize identified by yeast two-hybrid screeningPHYSIOLOGIA PLANTARUM, Issue 2 2005Daniela Holtgräwe We have used yeast two-hybrid screens and biochemical methods to identify glycolytic enzymes that interact with subcellular structures in hypoxic maize seedlings. As binding domain-bait fusion constructs, we have cloned actin, cytosolic aldolase, the three sucrose synthase (SUS) isoforms SUS1, SUS3, and SH1 as well as the SNF1-related protein kinase into yeast and identified cytosolic isoforms of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase, tubulin, and mitochondrial porin voltage-dependent anion channel protein (VDAC) as well as protein kinases and proteins involved in ubiquitinylation and proteasome-linked degradation as interacting activation domain-prey clones. The results were further confirmed using overlay blots (VDAC) as well as co-polymerization and co-precipitation assays (tubulin and actin). Some results were obtained that support the idea of metabolite and modification effects on the association, namely guanosine triphosphate (GTP)/MgCl2 was necessary for the binding of enolase to actin. GAPDH is inactivated upon association with tubulin but then serves to stabilize the microtubules. The findings support the idea of the dynamic formation of locally associated complexes of enzymes involved in sucrose breakdown and glycolysis in plant cells depending on their metabolic state. [source] Recuperative effect of Semecarpus anacardium linn. nut milk extract on carbohydrate metabolizing enzymes in experimental mammary carcinoma-bearing ratsPHYTOTHERAPY RESEARCH, Issue S1 2002Venugopal Sujatha Abstract Semecarpus anacardium Linn. of the family Anacardiaceae has many applications in the Ayurvedic and Siddha systems of medicine. We have tested the antitumour activity of Semecarpus anacardium nut extract against experimental mammary carcinoma in animals. As there is a direct relationship between the proliferation of tumour cells and the activities of the glycolytic and gluconeogenic enzymes, we studied changes in the activities of enzymes involved in this metabolic pathway in the liver and kidney. The enzymes investigated were glycolytic enzymes, namely hexokinase, phosphoglucoisomerase, aldolase and the gluconeogenic enzymes, namely glucose-6-phosphatase and fructose-1,6-biphosphatase in experimental rats. A significant rise in glycolytic enzyme activities and a simultaneous fall in gluconeogenic enzyme activities were found in mammary carcinoma bearing rats. Drug administration returned these enzyme activities to their respective control activities. Copyright © 2002 John Wiley & Sons, Ltd. [source] Many but not all Genes in Chlamydomonas reinhardtii are Regulated by the Circadian ClockPLANT BIOLOGY, Issue 6 2001S. Jacobshagen Abstract: Total RNA from autotrophic Chlamydomonas reinhardtii cultures grown in constant dim light and 17 °C constant temperature was subjected to Northern blot analyses. The mRNAs for cytochrome c, ,-tubulin, HSP70B (a chloroplastic heat shock protein of the 70 kD family), chloroplastic fructose-bisphosphate aldolase, and GAS3 (a "gamete-specific" protein of unknown function with high expression in gametes but low expression in vegetative cells) each exhibit a clear circadian rhythm in abundance. The rhythms differ significantly in phase and amplitude. The findings show that the genes for cytochrome c and ,-tubulin indeed are regulated by the circadian clock, as previously suggested. Experiments with cultures grown at 27 °C instead of 17 °C further revealed that the rhythms in mRNA abundance for HSP70B, chloroplastic aldolase, and GAS3 also occur with a similar period at the higher temperature. Thus, the rhythms conform to the criterion of temperature compensation for the period and therefore represent true circadian rhythms. In contrast, the combined amount of mRNA for ubiquitin 52 amino acid fusion protein and ubiquitin 78 to 81 amino acid fusion protein stays constant under both temperature conditions. Because the combined amount of mRNA for the ubiquitin fusion proteins was previously shown to cycle under diurnal conditions when cell division activity is high, our data suggest a regulation of these genes by the cell division cycle and not the circadian clock. In summary, our data, together with several other reports, suggest that the circadian clock regulates many but not all genes in Chlamydomonas reinhardtii. [source] Structure of human brain fructose 1,6-(bis)phosphate aldolase: Linking isozyme structure with functionPROTEIN SCIENCE, Issue 12 2004Tracy L. Arakaki Abstract Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceral-dehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 Ĺ resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes. [source] Proteomic changes in the crucian carp brain during exposure to anoxiaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2009Richard W. Smith Dr. Abstract During exposure to anoxia, the crucian carp brain is able to maintain normal overall protein synthesis rates. However, it is not known if there are alterations in the synthesis or expression of specific proteins. This investigation addresses this issue by comparing the normoxic and anoxic brain proteome. Nine proteins were found to be reduced by anoxia. Reductions in the glycolytic pathway proteins creatine kinase, fructose biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and lactate dehydrogenase reflect the reduced production and requirement for adenosine tri-phosphate during anoxia. In terms of neural protection, voltage-dependent anion channel, a protein associated with neuronal apoptosis, was reduced, along with gefiltin, a protein associated with the subsequent need for neuronal repair. Additionally the expression of proteins associated with neural degeneration and impaired cognitive function also declined; dihydropyrimidinase-like protein-3 and vesicle amine transport protein-1. One protein was found to be increased by anoxia; pre-proependymin, the precursor to ependymin. Ependymin fulfils multiple roles in neural plasticity, memory formation and learning, neuron growth and regeneration, and is able to reverse the possibility of apoptosis, thus further protecting the anoxic brain. [source] | |||||||||||||||||||||||||||||||