Acetate kinase from Veillonella alcalescens. Regulation by succinate and substrates.

The kinetic properties of acetate kinase from Veillonella alcalescens were investigated. In the presence of high concentrations of nucleotide both forward and reverse reactions were observed. In the presence of succinate the degree of cooperativity between subunits of the homodimer decreased, i.e. the Hill coefficient, n, decreased from 2.5 to 1.4 for acetyl phosphate in the presence of succinate. At low substrate concentrations hyperbolic kinetic data were observed with succinate. We have proposed a modified version of the concerted symmetry model to describe the kinetics observed with this enzyme. The primary differentiating feature of the proposed model is the requirement for activator ligand binding for catalysis. In the absence of succinate, the substrate (acetate or acetyl phosphate) also functions as an activating ligand.     

Acetate kinase from Veillonella alcalescens. Regulation of enzyme activity by succinate and substrates.

Acetate kinase of Veillonella alcalescens has been shown to be highly regulated enzyme exhibiting two levels of control: the requirement for succinate as a heterotropic allosteric effector, and cooperative binding at the substrate level. Succinate addition was necessary for enzymatic activity in both the direction of acyl phosphate synthesis and that of ATP synthesis. Control at the substrate level was apparent in the cooperative binding (Hill coefficients of 2) of acetyl phosphate, ATP, and ADP. Typical Michaelis kinetic data were observed for succinate (Ka = 20 mM for acetyl phosphate synthesis, 0.4 mM for ATP synthesis), acetate, and propionate. The primary effect of succinate was to increase the apparent Vmax of the enzymatic reaction for the variable substrates, ATP, ADP, and acetyl phosphate. The results are interpreted as evidence that, as a heterotropic effector of the acetate kinase reaction, succinate may regulate levels of propionyl-CoA (produced from propionyl phosphate by action of phosphotransacetylase), a compound required for the conversion of succinate to propionate. Acetase kinase has been shown to be a probable dimeric protein composed of two subunits of molecular weight 44,000 each.     

Synthesis of alpha-ketoglutarate by reductive carboxylation of succinate in Veillonella, Selenomonas, and Bacteriodes species.

Evidence for reductive carboxylation of succinate to synthesize alpha-ketoglutarate was sought in anaerobic heterotrophs from the rumen and from other anaerobic habitats. Cultures were grown in media containing unlabeled energy substrates plus [14C]succinate, and synthesis of cellular glutamate with a much higher specific activity than that of cellular asparate was taken as evidence for alpha-ketoglutarate synthase activity. Our results indicate alpha-ketoglutarate synthase functions in Selenomonas ruminantium, Veillonella alcalescens, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides uniformis, Bacteroides distasonis, and Bacteroides multiacidus. Evidence for this carboxylation was not found in strains representative of 10 other species.     

Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system

Phosphorylated and nonphosphorylated forms of peptide substrates for protein kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were separated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillaries yielded asymmetric analyte peaks with substantial peak tailing. Some of the peptides also exhibited broad peaks with unstable migration times. To improve the electrophoretic separation of the peptides, several strategies were implemented: extensive washing of the capillary with a base, adding betaine to the electrophoretic buffer, and coating the capillaries with polydimethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base substantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both the migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvements in the resolving power of the separations. These modifications all utilized an electrophoretic buffer that was compatible with a living biologic cell. Consequently they should be adaptable for the new capillary electrophoresis-based methods to measure kinase activation in single cells.     

Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.     

Comparative study of Agrobacterium biotypes 1, 2 and 3 by electrophoresis and serological methods

A comparative study by electrophoresis and serology of strains representing the three Agrobacterium biotypes was carried out. Thirteen Spanish isolates and strains from international collections were included. Ten antisera were prepared by using strains from the three biotypes and different types of antigens. The strains were studied by immunodiffusion, indirect immunofluorescence and indirect ELISA. Serological relationship among all the strains was observed, although serological heterogeneity within each of the biotypes occurred. Biotype 3 appears as serologically related to biotypes 1 and 2, having an intermediate position. This observation is in agreement with their biochemical characteristics. Electrophoretic analysis of the three biotypes showed that there was high variability. Three main bands appeared in the six strains studied. One specific band occurred in the biotype 1 strains and another in the biotype 3 strains.     

On the separation, detection and quantification of pectin derived oligosaccharides by capillary electrophoresis

Having previously reported that capillary electrophoresis can be used as a tool for the analysis of partially methyl-esterified oligogalacturonides we now describe a method that improves the resolution of individual oligomers, and detail a more rigorous quantification scheme that uses an internal standard and takes into account the relative molecular absorbance of different partially methyl-esterified species. The internal consistency of the method is subsequently demonstrated by performing the quantification of an endo-polygalacturonase pectin digest before and after de-methylation of the resultant oligomers.     

Taxonomy of the genus Listeria by using multilocus enzyme electrophoresis

Seventy-three strains of the seven recognized Listeria species were studied by performing a multilocus enzyme electrophoresis analysis of 18 enzyme loci. The mean number of alleles per locus was 9.5 and all of the loci were polymorphic. A total of 56 electrophoretic types were distinguished. Cluster analysis of a matrix of the genetic distances between paired electrophoretic types revealed that there were six principal clusters at the species level (genetic distances between clusters greater than 0.8). Listeria monocytogenes, Listeria innocua, Listeria welshimeri, Listeria seeligeri, and Listeria ivanovii each corresponded to one of these clusters with no overlap. Our results are in agreement with those of previous DNA hybridization experiments (Rocourt et al., Curr. Microbiol. 7:383-388, 1982). Listeria grayi and Listeria murrayi electrophoretic types formed a unique cluster, thus reinforcing the suggestion of Wilkinson and Jones (J. Gen. Microbiol. 98:399-421, 1977) that these two species should be considered two biovars of a single species.     

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.     

The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis

Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new spots appeared at more acidic pIs. Mass spectral studies indicated that cysteine alkylation was relatively fast, with 70-95% of the cysteines in the keratin proteins being alkylated within the first 10 min, while in the case of the HGTPs there was evidence for noncysteine alkylation occurring within this period. Alkylation of proteins for periods of up to 6 h prior to electrofocusing is being promoted as a better alternative to the current 2-DE protocol of the inclusion of a reductant in the immobilized pH gradient rehydration solution. This study has clearly demonstrated that long alkylation times do not suit all protein types or classes.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.     

Phylogeny and evolution of the genus Barbus in the Iberian Peninsula as revealed by allozyme electrophoresis

Variation of 14 enzyme systems encoded by 31 presumptive loci in different barbel species was studied using starch gel electrophoresis. Eighteen samples representing 11 Barbus tetraploid taxa were taken, including 10 populations from the Iberian Peninsula, six from other parts of western and southern Europe, one from northern Africa and one diploid species as outgroup from Asia Minor. The genetic analysis reassessed of the taxonomic status of the Iberian barbels into two distinct species groups. The first group included B. bocagei, B. comiza, B. graellsii, B. gulraonis, B. microcephalus , and B. sclateri , that aligned with B. callensis from northern Africa and with B. apoensis from Asia Minor; the other group included B. haasi and B. merldlonahs that was related to the European species, B. barbus, B. plebejus and B. peloponnesius . These groups are probably not monophyletic. It is suggested that the isolation of the Iberian Peninsula from Europe since the Oligocene-Miocene may explain the genetic affinities of the Iberian barbels with those of North African rather than with the European group.     

Chemical and serological studies of liposaccharides of bacteria of the genus Azospirillum

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS(Cd), LPS(Sp59b), LPS(Sp7), LPS(S17), and LPS(KBC1) preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab(Sp59b) and Ab(Cd) were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.     

Taxonomy of the genus Megacyclops (Crustacea,Copepoda): morphometry and the use of enzyme electrophoresis

Morphometric investigations on the populations of Megacyclops in Lake Constance showed, that in a sublittoral region from about 20 to 50 m depth an intermediate type between the two species M. viridis (littoral) and M. gigas (benthal) could be distinguished. Using enzyme electrophoresis it was evident, that these specimens belonged to M. viridis.The annual range of water temperatures in the sub-littoral region corresponds to that found in the shallow areas of the lake or in ponds during winter and early spring, when the adults of M. viridis are markedly larger than in summer and fall (seasonal variation). Nevertheless the sublittoral population represents a different local type with special characteristics in morphometric details.     

Optimization of the separation lactic acid enantiomers in body fluids by capillary electrophoresis

The optimization of the separation conditions of the two optical isomers of lactic acid by a factorial design is reported. Initially, different chiral selectors were systematically investigated and then a experimental design with three quantitative factors (cyclodextrin concentration and background buffer pH and concentration) were evaluated. Optimal conditions for obtaining a resolution higher than 1.5 were: phosphate buffer 200 mM at pH=6.0 with 413 mM 2-hydroxypropyl-beta-cyclodextrin added (HP-beta-CD), 20 degrees C, -20 kV of applied potential and polyacrylamide-coated capillary. The method was validated for the measurement in plasma and it was applied to the identification of both isomers in body fluids such as urine, amniotic fluid and cerebrospinal fluid. Samples were centrifuged and diluted (1:4) prior to the analysis.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing*

Summary. Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe₀-transferrin, Fe₁-transferrin and Fe₂-transferrin region. At the level of Fe₀-transferrin and Fe₁-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.