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PubMed1994

Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria.

Two factors known to regulate plant mitochondrial cyanide-resistant alternative oxidase activity, pyruvate and the redox status of the enzyme's intermolecular disulfide bond, were shown to differently affect activity in isolated soybean seedling mitochondria. Pyruvate stimulated alternative oxidase activity at low levels of reduced ubiquinone, shifting the threshold level of ubiquinone reduction for enzyme activity to a lower value. The disulfide bond redox status determined the maximum enzyme activity obtainable in the presence of pyruvate, with the highest rates occurring when the bond was reduced. With variations in cellular pyruvate levels and in the proportion of reduced alternative oxidase protein, a wide range of enzyme activity is possible in vivo.

Disulfides↗
PubMed2005

Amperometric biosensor for determining human salivary phosphate.

An amperometric biosensor was constructed for analysis of human salivary phosphate without sample pretreatment. The biosensor was constructed by immobilizing pyruvate oxidase (PyOD) on a screen-printed electrode. The presence of phosphate in the sample causes the enzymatic generation of hydrogen peroxide (H(2)O(2)), which was monitored by a potentiostat and was in proportion to the concentration of human salivary phosphate. The sensor shows response within 2s after the addition of standard solution or sample and has a short recovery time (2 min). The time required for one measurement using this phosphate biosensor was 4 min, which was faster than the time required using a commercial phosphate testing kit (10 min). The sensor has a linear range from 7.5 to 625 microM phosphate with a detection limit of 3.6 microM. A total of 50 salivary samples were collected for the determination of phosphate. A good level of agreement (R(2)=0.9646) was found between a commercial phosphate testing kit and the phosphate sensor. This sensor maintained a high working stability (>85%) after 12h operation and required only a simple operation procedure. The amperometric biosensor using PyOD is a simple and accurate tool for rapid determinations of human salivary phosphate, and it explores the application of biosensors in oral and dental research and diagnosis.

Biosensing Techniques↗
PubMed2001

Sequence-structure analysis of FAD-containing proteins.

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.

Amino Acid Motifs↗
PubMed2004

Tissue-specific contributions of pneumococcal virulence factors to pathogenesis.

We assessed the ability of Streptococcus pneumoniae mutants deficient in either choline binding protein A (CbpA), pneumolysin (Pln), pyruvate oxidase (SpxB), autolysin (LytA), pneumococcal surface protein A, or neuraminidase A (NanA) to replicate in distinct anatomical sites and translocate from one site to the next. Intranasal, intratracheal, and intravenous models of disease were assessed in 4-week-old BALB/cJ mice by quantitation of bacterial titers in the relevant organs. Mice were also observed by use of real-time bioluminescent imaging (BLI). BLI allowed visualization of the bacteria in sites not tested by sampling. All mutants were created in D39 Xen7, a fully virulent derivative of capsular type 2 strain D39 that contains an optimized luxABCDE cassette. NanA, SpxB, and, to a lesser extent, CbpA contributed to prolonged nasopharyngeal colonization, whereas CbpA and NanA contributed to the transition to the lower respiratory tract. Once lung infection was established, Pln, SpxB, and LytA contributed to bacterial replication in the lungs and translocation to the bloodstream. In the bloodstream, only Pln and LytA were required for high-titer replication, whereas CbpA was required for invasion of the cerebrospinal fluid. We conclude that transitions between body sites require virulence determinants distinct from those involved in organ-specific replication.

Animals↗
PubMed1983

[Measurement of human seminal components and its medicolegal application].

Microdeterminations for low molecular compounds highly contained in human seminal plasma, i. e., dehydroepiandrosterone sulfate (DHEA-S), spermine (Sp) and citric acid (CA) have been established. Experimental studies were carried out on application of these methods to identification of semen from cadaveric vaginae more than a few days after death. DHEA-S was measured by radioimmunoassay using anti-DHEA-S-17-hydrazone conjugated bovine serum albumin rabbit antiserum. Sp and CA were determined spectrophotometrically with monoamine oxidase and with citrate lyase, oxalacetate decarboxylase and pyruvate oxidase, respectively. DHEA-S and its cross-reacting materials were contained little in saliva, moderately in semen and vaginal contents and largely in serum and urine. By the above method Sp was not detectable in other body fluids except semen. CA was contained not only largely in semen but also a little in other body fluids. DHEA-S and Sp in the seminal fluid allowed to stand at room temperature were relatively stable during a month, whereas CA became undetectable in 10 days, all of which being persistent in seminal stain during at least one month. From these results the colorimetry of Sp among the three methods above mentioned was found to be most reliable for detecting seminal fluid from vaginal contents of a putrefied cadaver.

Body Fluids↗
PubMed1994

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria.

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.

Journal Article↗
PubMed1985

Oxygen dependent lactate utilization by Lactobacillus plantarum.

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.

Energy Metabolism↗
PubMed2003

Design of luminescent biochips based on enzyme, antibody, or DNA composite layers.

The use of beads bearing bioactive molecules to develop generic biochips based on chemi- and electro-chemiluminescent detection was evaluated. The biochips were composed of arrayed biosensors, including enzyme-charged beads, antigen-charged beads, or oligonucleotide-charged beads, entrapped in poly(vinyl alcohol) (PVA-SbQ) photopolymer. In each case the sensing layers were spotted at the surface of a glassy carbon electrode as 0.3 microL drops, generating 500-800 microm spots. The luminescent reactions were either catalysed by horseradish peroxidase or triggered by application of a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference. Enzyme biochips were designed for the concomitant detection of choline, glucose, glutamate, lactate, lysine, and urate, based on the corresponding oxidase-charged beads and the electro-chemiluminescent (ECL) reaction with luminol-immobilised beads of the hydrogen peroxide produced. Limits of detection of 1 micromol L(-1) for glutamate, lysine and uric acid, 20 micromol L(-1) for glucose, and 2 micromol L(-1) for choline and lactate were found with detection ranging over three decades at least. Use of the electro-chemiluminescent biochip was extended to a tri-enzymatic sensing layer based on kinase-oxidase activity for detection of acetate. A reaction sequence using acetate kinase, pyruvate kinase, and pyruvate oxidase enabled the production of H2O2 in response to acetate injection in the range 10 micromol L(-1) to 100 mmol L(-1). Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realised for the model detection of human IgG. Biotin-labelled anti-human IgG were used in a competitive assay, in conjunction with peroxidase-labelled streptavidin. Free antigen could then be detected with a detection limit of 25 pg (10(8) molecules) and up to 15 ng. In a similar way, the use of oligonucleotide-immobilised beads enabled the realisation of DNA-sensitive biochips which could be used to detect a biotin-labelled sequence al a level of 5x10(8) molecules.

Antibodies↗
PubMed1994

Enzymes in diagnostics: achievements and possibilities of recombinant DNA technology.

We discuss, from an industrial point of view, the scope and possibilities of recombinant DNA technology for "diagnostic enzyme" production and application. We describe the construction of enzyme-overproducing strains and show how to simplify downstream processing, increase product quality and process profitability, improve diagnostic enzyme properties, and adjust enzymes to harsh assay conditions. We also consider some safety and environmental aspects of enzyme production. Other aspects of diagnostic enzymes that we cover are the facilitation of enzyme purification by attachment of short amino acid tails, the introduction of tails or tags for site-specific conjugation or oriented immobilization, the construction of bi- or multifunctional enzymes, and the production of enzyme-based diagnostic tests as demonstrated by the homogeneous immunoassay system of CEDIA tests. We use as examples of diagnostic enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucose oxidase (EC 1.1.3.4), alkaline phosphatase (EC 3.1.3.1), alpha-glucosidase (EC 3.2.1.20), pyruvate oxidase (EC 1.2.3.3), creatinase (EC 3.5.3.3), and beta-galactosidase (EC 3.2.1.23).

Amino Acid Sequence↗
PubMed2005

Pyruvate fermentation by Oenococcus oeni and Leuconostoc mesenteroides and role of pyruvate dehydrogenase in anaerobic fermentation.

The heterofermentative lactic acid bacteria Oenococcus oeni and Leuconostoc mesenteroides are able to grow by fermentation of pyruvate as the carbon source (2 pyruvate --> 1 lactate + 1 acetate + 1 CO(2)). The growth yields amount to 4.0 and 5.3 g (dry weight)/mol of pyruvate, respectively, suggesting formation of 0.5 mol ATP/mol pyruvate. Pyruvate is oxidatively decarboxylated by pyruvate dehydrogenase to acetyl coenzyme A, which is then converted to acetate, yielding 1 mol of ATP. For NADH reoxidation, one further pyruvate molecule is reduced to lactate. The enzymes of the pathway were present after growth on pyruvate, and genome analysis showed the presence of the corresponding structural genes. The bacteria contain, in addition, pyruvate oxidase activity which is induced under microoxic conditions. Other homo- or heterofermentative lactic acid bacteria showed only low pyruvate fermentation activity.

Anaerobiosis↗
PubMed2003

The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli.

We compared pyruvate accumulation in six strains of Escherichia coli and their corresponding ppc mutants. Each strain contained a mutation of a gene involved in the pathway to acetate synthesis. Strains with mutations in genes encoding the pyruvate dehydrogenase complex generally exhibited the greatest pyruvate accumulation of which CGSC6162 (an aceF mutant) and CGSC6162 Delta ppc were studied in greater detail in controlled fermenters. Both CGSC6162 and CGSC6162 Delta ppc accumulated greater than 35 g/l pyruvate in a medium supplemented with acetate. We observed pyruvate mass yields from glucose of 0.72 in CGSC6162, with volumetric productivities above 1.5 g l(-1) h(-1). For CGSC6162 Delta ppc, we observed pyruvate yields of 0.78 and volumetric productivities above 1.2 g l(-1) h(-1). CGSC6162 consumed all initially supplied acetate, while CGSC6162 Delta ppc first consumed and then generated acetate during the course of a 36 h fermentation. Acetate generation and pyruvate oxidase activity was pH- and temperature-dependent, with a pH of 7.0 and the lowest temperature studied (32 degrees C) favoring the greatest pyruvate generation. Lactate was an unexpected by-product even though measured lactate dehydrogenase (LDH) activity was very low.

Acetates↗
PubMed1981

Influence of oxygen on respiration and glucose catabolism by Treponema pallidum.

Oxygen consumption by Treponema pallidum was observed to be glucose dependent. Treponemes consumed O2 to a low dissolved O2 concentration of 0.01 mumol of O2 per ml when respiration ceased. A fermentative degradation of glucose occurred during the maintenance of treponemes at 0.01 mumol of O2 per ml. Although the initial rate of respiration was independent of the dissolved O2 concentration, the respiration rate decreased upon prolonged incubation at 0.20 mumol of O2 per ml as compared with the respiration rate at 0.7 mumol of O2 per ml. Oxygen was determined to be the major electron acceptor during the oxidative metabolism of glucose. Acetate formation paralleled the respiration rate of the treponemes, whereas lactate production appeared to be independent of respiration. Accumulation of pyruvate occurred at 0.20 mumol of O2 per ml but not at 0.07 mumol/ml. Analyses of enzyme activities in cell-free extracts from treponemes maintained at either 0.07 or 0.20 mumol of O2 per ml for 4 h revealed a loss of pyruvate decarboxylase activity at the higher dissolved O2 concentration, suggesting the inactivation of pyruvate oxidase.

Animals↗
PubMed1968

Isolation and role of nonheme iron protein in Clostridium botulinum.

A type of iron-bound protein was isolated from Clostridium botulinum by a modification of the method used for isolating ferredoxin from C. pasteurianum. This method involved acetone and diethylaminoethyl cellulose treatments followed by ammonium sulfate fractionation. The protein exhibited maximal absorption in the ultraviolet region near 260 mmu. Portions of the isolated iron protein were separated by disc electrophoresis and, following specific iron-bound protein staining, showed a positive reaction in the same position on the gel column as was first demonstrated by use of cell-free extract. Evidence accumulated by use of a cell-free extract of C. botulinum suggests that pyruvate is metabolized through a phosphoroclastic system as demonstrated in other clostridia. It is probable that ferredoxin is an electron mediator between pyruvic oxidase and hydrogenase for hydrogen evolution and acetyl phosphate formation.

Bacterial Proteins↗
PubMed1993

Serum sialic acid enzymatic assay based on microtitre plates: application for measuring capillary serum sialic acid concentrations.

We have adapted a commercially-available kit for the enzymatic determination of sialic acid so that the reaction can be carried out in microtitre wells and the coloured end product quantitated on an ELISA plate reader. The small volume of serum used allows sialic acid to be measured in capillary blood samples. The assay was based on release of sialic acid from glycoconjugates by neuraminidase, cleavage of sialic acid by N-acetyl neuraminic acid aldolase to pyruvate, and then oxidation of pyruvate to hydrogen peroxide by pyruvate oxidase. Hydrogen peroxide was determined by the red product formed in the presence of peroxidase, 4-aminoantipyrine and N-ethyl-N-2-hydroxyethyl-3-toluidine. The assay was linear to at least 10 mmol/l and unaffected by haemolysis, and by the addition of glucose, 3-hydroxybutyrate, bilirubin and pyruvate. Capillary serum sialic acid concentrations were not significantly different from simultaneously measured venous serum sialic acid levels. Self-collected capillary blood samples were obtained from healthy subjects over 6 h during the day. No variations in serum sialic acid concentrations were found in response to a meal. We conclude that this micro-adaptation of a specific sialic acid assay will be suitable for epidemiological surveys of serum sialic acid collected by patients and normal subjects.

Biomarkers↗
PubMed1980

Enzymatic assay of serum sialic acid.

Serum sialic acid has been assayed enzymatically. The reaction includes neuraminidase hydrolysis of glycoprotein, cleavage of sialic acid to pyruvate by N-acetyl neuraminic acid (NANA)-aldolase, oxidation of pyruvate by pyruvate oxidase which produces hydrogen peroxide, and colorimetry of hydrogen peroxide using the peroxidase-p-chlorophenol-4-amino-antipyrine method. This method showed good correlation between a chemical method (r = 0.984), good recovery (98.8%) and good reproducibility (within-run-precision: 1.0% C.V.; day-to-day precision: 1.9% C.V). Intrinsic serum pyruvate produces an equimolar positive effect. The normal value range is 1.94 +/- 0.29 mmol/l (mean +/- S.D.,n = 24).

Chemical Phenomena↗
PubMed2004

Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor.

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD(+). SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.

Biosensing Techniques↗
PubMed1998

Is a hydrophobic amino acid required to maintain the reactive V conformation of thiamin at the active center of thiamin diphosphate-requiring enzymes? Experimental and computational studies of isoleucine 415 of yeast pyruvate decarboxylase.

The residue I415 in pyruvate decarboxylase from Saccharomyces cerevisiae was substituted with a variety of uncharged side chains of varying steric requirements to test the hypothesis that this residue is responsible for supporting the V coenzyme conformation reported for this enzyme [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. Changing the isoleucine to valine and threonine decreased the kcat value and shifted the kcat-pH profile to more alkaline values progressively, indicating that the residue at position 415 not only is important for providing the optimal transition state stabilization but also ensures correct alignment of the ionizable groups participating in catalysis. Substitutions to methionine (the residue used in pyruvate oxidase for this purpose) or leucine (the corresponding residue in transketolase) led to greatly diminished kcat values, showing that for each thiamin diphosphate-dependent enzyme an optimal hydrophobic side chain evolved to occupy this key position. Computational studies were carried out on the wild-type enzyme and the I415V, I415G, and I415A variants in both the absence and the presence of pyruvate covalently bound to C2 of the thiazolium ring (the latter is a model for the decarboxylation transition state) to determine whether the size of the side chain is critically required to maintain the V conformation. Briefly, there are sufficient conformational constraints from the binding of the diphosphate side chain and three conserved hydrogen bonds to the 4'-aminopyrimidine ring to enforce the V conformation, even in the absence of a large side chain at position 415. There appears to be increased coenzyme flexibility on substitution of Ile415 to Gly in the absence compared with the presence of bound pyruvate, suggesting that entropy contributes to the rate acceleration. The additional CH3 group in Ile compared to Val also provides increased hydrophobicity at the active center, likely contributing to the rate acceleration. The computational studies suggest that direct proton transfer to the 4'-imino nitrogen from the thiazolium C2H is eminently plausible.

Amino Acid Substitution↗