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PubMed2001

Xyloside transport by XylP, a member of the galactoside-pentoside-hexuronide family.

This paper describes the functional characterization of the xyloside transporter, XylP, of Lactobacillus pentosus with the aid of a spectroscopy-based assay system. In order to monitor the transport reaction, the natural xyloside isoprimeverose, a building block of hemicellulose, and the analogue methyl-isoprimeverose were chemically synthesized by a new and efficient procedure. The XylP protein was purified by metal affinity chromatography, following high level expression in Lactococcus lactis from the nisin-inducible promoter. The purified XylP protein was incorporated into liposomes, in which the glucose dehydrogenase from Acinetobacter calcoaceticus (sGDH) was entrapped. sGDH can oxidize aldose sugars in the presence of dichlorophenol-indophenol as electron acceptor. The coupled assay thus involves XylP-mediated isoprimeverose uptake followed by internal oxidation of the sugar by sGDH, which can be monitored from the reduction of 2,6-dichlorophenol-indophenol at 600 nm. The uptake of isoprimeverose was stimulated by the presence of the non-oxidizable methyl-isoprimeverose on the trans-side of the membrane, indicating that exchange transport is faster than unidirectional downhill uptake. Unlike other members of the galactoside-pentoside-hexuronide family, XylP does not transport monosaccharides (xylose) but requires a glycosidic linkage at the anomeric carbon position. Consistent with a proton motive force-driven mechanism, the uptake was stimulated by a membrane potential (inside negative relative to outside) and inhibited by a pH gradient (inside acidic relative to outside). The advantages of the here-described transport assay for studies of carbohydrate transport are discussed.

Bacterial Proteins↗
PubMed1968

Isolation of mutants of Euglena gracilis with impaired photosynthesis.

Four mutant strains of Euglena gracilis have been isolated after treatment of wild type cells with ultraviolet light or the chemical mutagen nitrosoguanidine. None of the mutants is capable of autotrophic growth or photosynthetic carbon dioxide fixation. The mutant strains contain normal amounts of the enzymes of the reductive pentose phosphate cycle and are qualitatively similar to the wild type in pigment composition, but are unable to carry out the Hill reaction (light induced reduction of 2,6-dichlorophenol indophenol). Isolated mutant plastids cannot photoreduce NADP with water as the electron donor but can carry out this reaction when the electron donating system is ascorbate and 2,6-dichlorophenol indophenol. Whole cells of the mutants show the light induced oxidation of cytochrome f by light reaction I but are unable to bring about cytochrome f reduction by light reaction II. The mutants appear to be blocked at or near light reaction II in the photosynthetic electron transport chain. The mutants may represent alterations of the chloroplast genome since the mutation isolation was carried out under conditions where chloroplast viability was severely impaired, but cell viability was unaffected.

Euglena↗
PubMed1973

Bicarbonate ion as a critical factor in photosynthetic oxygen evolution.

Bicarbonate ion, not dissolved CO(2) gas, is shown to increase 4- to 5-fold the rate of dichlorophenol indophenol reduction by isolated maize (Zea mays) chloroplasts. Glutaraldehyde fixed chloroplasts continue to exhibit bicarbonate-dependent 2,6-dichlorophenol indophenol reduction. Bicarbonate is shown to act close to the oxygen-evolving site, i.e. prior to the electron donation site of diphenyl carbazide to photosystem II. Dark incubation and light pretreatment of chloroplasts in various concentrations of bicarbonate, just prior to assay, indicate that bicarbonate binds to chloroplasts in the dark and is released again as the Hill reaction proceeds in the light. It is suggested that bicarbonate ions may play a critical role in the oxygen-evolving process in photosynthesis.

Journal Article↗
PubMed1976

Cyclic Photophosphorylation in the Mykotrophic Orhid Neottia nidus-avis.

The mykotrophic orchid Neottia nidus-avis (L.) Rich. is not able to evolve oxygen in the light. Plastid preparations from the lip (labellum) of the orchid perform a photosystem I-dependent photoreduction of methylviologen with the artificial electron donor couple 2,6-dichlorophenol indophenol ascorbate. Photosystem II reactions such as the ferricyanide Hill reaction or the photoreduction of 2,6-dichlorophenol indophenol with diphenylcarbazide as the electron donor are not functioning. The plastids exhibit phenazine methosulfate-mediated cyclic photophosphorylation. After infiltration with (32)P-labeled phosphate the labellum forms (32)P-ATP in the light. This rate of ATP formation is enhanced by additional infiltration of phenazine methosulfate prior to illumination. The brown color of the plant is caused by an absorption shift of carotenoids to longer wavelength. By comparison of absorption spectra with the fluorescence excitation spectra of plastid preparations and of the extracted pigments we show that no appreciable energy transfer from carotenoids to chlorophyll occurs.

Journal Article↗
PubMed1979

Regulation of the Photosynthesis Rhythm in Euglena gracilis: II. Involvement of Electron Flow through Both Photosystems.

Rhythmic changes in the light reactions of Euglena gracilis have been found which help to explain the basic reactions effected in the circadian rhythm of O(2) evolution. Diurnal changes in the slope of light intensity plots indicated that the maximal rate of photosynthesis changed throughout the circadian cycle. No evidence was obtained consistent with the premise that changes in chlorophyll content, as measured by total chlorophyll or chlorophyll a/b ratio, or photosynthetic unit size are responsible for this rhythim.The rate of light-induced electron flow through the entire electron chain (H(2)O to methyl viologen) was rhythmic both in whole cells and in isolated chloroplasts, and the highest rate of electron flow coincided with the highest rate of O(2) evolution. The individual activities of photosystem I (reduced from 2,6-dichlorophenol-indophenol to methyl viologen) and photosystem II (H(2)O to 2,6-dichlorophenol-indophenol) did not, however, change significantly with time of day, suggesting that the coordination of the two photosystems may be the site of circadian control. Evidence consistent with this concept was obtained from studies of low temperature emission from systems I and II following preillumination with system I or II light.

Journal Article↗
PubMed1985

Characterization of a Purified Photosystem II-Phycobilisome Particle Preparation from Porphyridium cruentum.

Detergent preparations isolated from thylakoids of the red alga Porphyridium cruentum, in a sucrose, phosphate, citrate, magnesium chloride medium consist of phycobilisomes and possess high rates of photosystem II activity. Characterization of these particles shows that the O(2)-evolving activity is stable for several hours and the pH optimum is about 6.5 to 7.2. Response of the system to light, electron donors and acceptors, and inhibitors verify that the observed activity, measured both as O(2) evolution and 2,6-dichlorophenol-indophenol reduction, is due to photosystem II. Furthermore, photosystem II is functionally coupled to the phycobilisome in this preparation since green light, absorbed by phycobilisomes of P. cruentum, is effective in promoting both O(2) evolution and 2,6-dichlorophenol-indophenol reduction. Photosystem II activity declines when light with wavelengths shorter than 665 nm is removed. Both 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine inhibit photosystem II activity in this preparation, indicating that the herbicide binding site is a component of the photosystem II-phycobilisome particle.

Journal Article↗
PubMed1987

Control of the Photosynthetic Apparatus of Acetabularia mediterranea by Blue Light : Analysis by Light-Saturation Curves.

During growth, Acetabularia mediterranea requires the action of blue light to maintain high rates of photosynthesis. In the present study, blue light-dependent alterations of the photosynthetic apparatus, which can be detected by analysis of light-saturation curves and by measurements of partial reactions of the photosynthetic electron transport chain, are described. Light-saturation curves of photosynthesis in vivo were measured with a new closed oxygen electrode system after culture of Acetabularia in continuous red or blue light. These curves were compared to those of 2,6-dichlorophenol-indophenol reduction by isolated chloroplast membranes. The analysis lead to the following statements: (a) only one reaction limits electron transport rates in vitro (dichlorophenol-indophenol reduction) at all light intensities irrespective of the light quality during growth, and (b) the limiting step is light driven and located in the reaction center of photosystem II. Presumably, this same reaction determines the flow of electrons under low light intensities in vivo in cells from white, blue, and red light. In addition to photosynthesis, the rates of dark respiration changed due to the action of blue light. Concomitantly, the light compensation point of apparent photosynthesis was shifted during monochromatic irradiations.

Journal Article↗
PubMed1992

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves.

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O(2) or H(2)O(2) and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K(m) 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O(2) as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.

Journal Article↗
PubMed1992

Inhibition of Water Splitting Increases the Susceptibility of Photosystem II to Photoinhibition.

Photosystem II (PSII)-enriched membrane particles were isolated from peas (Pisum sativum L.) and treated in several different ways to inhibit the water oxidation reactions, but not reaction center function itself, as judged by the light-induced rate of reduction of 2,6-dichlorophenol indophenol with and without the artificial electron donor, diphenyl carbazide. It was shown that such treatments increased the susceptibility of the PSII-enriched membranes to photoinhibition. This trend was further observed if 2,6-dichlorophenol indophenol was present during the illumination with photoinhibitory light. On the other hand, protection against the enhanced photoinhibition was found when the water-splitting activity was reconstituted or when the artificial electron donor diphenyl carbazide was present during the preillumination. The results indicate that irreversible photodamage occurred within the PSII reaction center as a consequence of illumination with strong light and that the rate of this damage was enhanced under conditions that are expected to give rise to a photoaccumulation of oxidizing species such as P680(+) on the donor side of PSII. This mechanism of photoinhibitory damage occurred under both aerobic and anaerobic conditions.

Journal Article↗
PubMed1983

Temperature dependence of growth and membrane-bound activities of Chloroflexus aurantiacus energy metabolism.

The temperature dependence of various activities related to the energy metabolism of isolated membranes and whole cells of the thermophilic bacterium Chloroflexus aurantiacus was determined after phototrophic growth at either 40, 50, or 60 degrees C. The data obtained were expressed by use of Arrhenius plots. Maximum activities were determined at about 65 degrees C for succinate 2,4-dichlorophenol-indophenol reductase as well as NADH oxidase and at about 70 degrees C for Mg-ATPase and for light-induced proton extrusion by cells. Activation energies for Mg-ATPase and light-induced proton extrusion were about 40 kJ mol-1 from 30 degrees C to about 50 degrees C and they increased significantly at higher temperatures. Essentially the same dependency was detectable with NADH oxidase, except for an increase in activation energy below 41 degrees C. All of these responses were independent of growth temperature. Succinate-2,4-dichlorophenol-indophenol reductase showed a change in activation energy around 41 degrees C only with cells grown at 60 degrees C. Differences in the responses of cells grown at different temperatures were identified on the basis of changes from sigmoidal to hyperbolic kinetics for light saturation of proton extrusion. Moreover, the thermostability of proton extrusion was maximal when assayed at the corresponding growth temperatures. In any case, thermostability was lowest at the 65 and 68 degrees C assay temperatures. Differential scanning calorimetry with membranes revealed irreversible heat uptake from about 60 to 72 degrees C. The results are discussed in light of the activation energy for the specific growth rate, which is lowest at temperatures from 40 degrees C to the optimum at 60 degrees C.

Bacteria↗
PubMed1987

Purification and properties of protoporphyrinogen oxidase from an anaerobic bacterium, Desulfovibrio gigas.

Protoporphyrinogen oxidase has been solubilized from plasma membranes of Desulfovibrio gigas. The enzyme was purified to apparent homogeneity with single silver-stained protein bands on isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. This protoporphyrinogen oxidase has a molecular weight (Mr) of 148,000 and is composed of three dissimilar subunits of Mrs 12,000, 18,500, and 57,000, which are held together by sulfhydryl bonds. Unlike other protoporphyrinogen oxidases, which use molecular oxygen as an electron acceptor, this enzyme does not couple to oxygen. The protoporphyrinogen oxidase donates electrons to 2,6-dichlorophenol-indophenol but not to NAD+, NADP+, flavin adenine dinucleotide, or flavin mononucleotide. The natural physiological electron acceptor of the protoporphyrinogen oxidase from D. gigas is unknown. By using 2,6-dichlorophenol-indophenol as the electron acceptor, the Km and Vmax values for oxidation of protoporphyrinogen were determined to be 21 microM and 8.38 nmol/min per 70 micrograms of protein, respectively. The catalytic rate constant, Kcat, was calculated to be 17.7 mol of protoporphyrin formed per mole of enzyme per min of incubation, and the Kcat/Km was 0.84. Energies of activation were calculated from Arrhenius plots with 7,429 cal (ca. 31,080 J)/mol per degree below 10 degrees C and 1,455 cal (ca. 6,088, J)/mol per degree above 10 degrees C. Optimum enzyme activity was at 23 degrees C, and inhibition was observed with both N-ethylmaleimide and iodoacetamide.

Anaerobiosis↗
PubMed1961

Glucose dehydrogenation in bacteria: a comparative study.

Hauge, Jens G. (National Institute for Public Health, Oslo, Norway). Glucose dehydrogenation in bacteria: a comparative study. J. Bacteriol. 82:609-614. 1961.-Extracts of a series of bacteria reported to convert glucose to gluconic acid via particulate enzyme systems were fractionated by differential centrifugation into heavy particles, light particles, and particle-free supernatant. Particles from Acetobacter suboxydans, Pseudomonas fluorescens, and Bacterium anitratum had several features in common, notably a high activity with indophenol as acceptor and no significant activity with methylene blue, tetrazolium, diphosphopyridine nucleotide, or triphosphopyridine nucleotide. The pH optima with indophenol were lower than those with oxygen as acceptor. These features are in line with the hypothesis that the particles of these organisms oxidize glucose with a tightly bound niacinamide coenzyme as primary acceptor.A triphosphopyridine nucleotide-linked glucose dehydrogenase was found present in the supernatant fraction of P. fluorescens.Azotobacter vinelandii and Aerobacter aerogenes particles oxidized glucose to a measurable degree with oxygen as acceptor only.

Bacteria↗
PubMedDate not supplied

Heat-stress stimulation of electron flow in a photosystem I submembrane fraction.

Oxygen uptake using methyl viologen as the terminal electron acceptor was recorded in digitonin-derived photosystem I submembrane fractions incubated at either 25 or 50 degrees C. A two- to four-fold heat-stress stimulation of electron flow was detected at 50 degrees C when reduced 2,6-dichlorophenol-indophenol was used as the primary electron donor. However, no stimulation was seen with N,N,N',N'-tetramethylphenylenediamine as the donor. The stimulation was enhanced by specific cations (Mg2+, Na+, K+), but not by Mn2 or Ca2+. The enhancement obtained with Mg2+ could be eliminated by incubating for a prolonged period. It is proposed that the observed heat-stress stimulation is due to a conformational change at the level of the cytochrome b6-f complex. This change increased the affinity of the protein complex for 2,6-dichlorophenol-indophenol at its oxidation sites. The involvement of a conformational modification is demonstrated by the absence of heat-stress stimulation in submembrane fractions immobilized in an albumin-glutaraldehyde cross-linked matrix.

2,6-Dichloroindophenol↗
PubMed1985

Formation of 4-aminophenoxyl free radical from the acetaminophen metabolite N-acetyl-p-benzoquinone imine.

N-Acetyl-p-benzoquinone imine, a hepatic metabolite of acetaminophen, and its analogue, N-acetyl-3,5-dimethyl-p-benzoquinone imine, were metabolized by rat liver microsomes and NADPH to their corresponding 4-aminophenoxyl free radicals. ESR spectra were recorded and unambiguously identified. As indicated by the purple color and confirmed by UV and mass spectroscopy, indophenols were formed as final products. The 4-aminophenoxyl free radical formation could be suppressed by the deacetylase inhibitors, sodium fluoride and paraoxon. Microsomal incubations of N-acetyl-2,6-dimethyl-p-benzoquinone imine and NADPH do not result in a detectable radical concentration; in addition, no indophenol was found. Substitution of NADPH-cytochrome P-450 reductase for rat liver microsomes eliminates the deacetylase activity and results in direct reduction of N-acetyl-3,5-dimethyl-p-benzoquinone imine to the N-acetyl-2,6-dimethyl-4-aminophenoxyl free radical. Neither the incubation of N-acetyl-p-benzoquinone imine nor that of N-acetyl-2,6-dimethyl-p-benzoquinone imine with NADPH-cytochrome P-450 reductase yielded a detectable concentration of the corresponding phenoxyl free radical. When starting material that had been exposed to the atmosphere was used, a previously reported free radical with a splitting constant of approximately 2 G was formed. This spectrum is identical with that of the 2,6-dimethyl-p-benzosemiquinone free radical, implying hydrolysis of the starting material. Neither the N-acetyl-4-aminophenoxyl nor the N-acetyl-2,6-dimethyl-4-aminophenoxyl radical reduces oxygen to form superoxide or react with oxygen in any other detectable way.

Animals↗
PubMed1982

[In vitro tumor sensitivity tests to chemotherapeutic agents by the suppression of dehydrogenase activity].

A test system using dehydrogenase activity for predicting the response to chemotherapeutic agents against cancer cells was introduced. Agar plate assay, INK which test, and SDI test commonly employed in the clinical study were also reviewed. Agar plate assay resembles antibiotic disc sensitivity test. The cancer uniformly suspended in the agar medium was exposed to drugs on paper discs for few hours. After removal of the disc, methylene blue or 2, 6-dichlorophenol indophenol was applied as a dehydrogenase indicator. INK test was introduced by Nishioka et al. in 1957. Several fragments of fresh cancer tissue were incubated with chemotherapeutic agents in roller test tubes. Twenty-four hours later, 2, 6-dichlorophenol indophenol was applied as a dehydrogenase indicator. To develop a simple, rapid, and comparable test, SDI test was introduced by us in 1964. A fresh cancer tissue was minced and made into the cell suspension. After cancer cells were exposed to chemotherapeutic agents, the activities of succinic dehydrogenase of the treated cells were measured by the reduction of 2, 3, 5-triphenyl tetrazolium chloride. Some points to be improved were investigated and discussed.

Antineoplastic Agents↗
PubMed1978

A rapid and sensitive assay for monoamine oxidase activity.

Based on the Berthelot reaction, a new sensitive and rapid procedure for monoamine oxidase (MAO) determination with substrate tyramine has been developed. The saturation with oxygen and the separation of ammonia from the substrate were omitted. At the end of incubation the samples were deproteinized with ethanol and consecutive centrifugation. The newly-formed ammonia is converted into the coloured compound indophenol, using the procedure of Fenton (1962). The indophenol concentration, respectively NH3 is determined by spectrophotometry at 625 nm, and calculated by comparison with a set of standard amounts of NH3. The enzyme activity is expressed as nanomoles ammonia, formed by 1 mg protein for 1 min. The method was tested for studying the relationship between the enzyme activity, time of incubation and protein concentration, as well as the effect of pargyline. The kinetics of MAO, using both liver and brain mitochondria as enzyme material was also studied. Considering the specificity, simplicity, versatility and rapid performance, the new method showed several advantages in comparison with the other known methods for MAO determination.

Animals↗
PubMed1963

GLYCOLIC ACID OXIDATION BY ESCHERICHIA COLI ADAPTED TO GLYCOLATE.

Furuya, Akira (University of Illinois College of Medicine, Chicago) and James A. Hayashi. Glycolic acid oxidation by Escherichia coli adapted to glycolate. J. Bacteriol. 85:1124-1131. 1963.-A procedure is described for extraction and partial purification of glycolic acid oxidase from Escherichia coli adapted to grow on glycolate as the sole carbon source. Enzyme activity was assayed by oxygen uptake and by reduction of 2,6-dichlorophenol-indophenol. Glyoxylic acid was the product of glycolate oxidation by the enzyme. Enzyme activity, which diminishes rapidly on storage, shows a maximum at pH 6 to 7. We were unable to show any cofactor requirement. Compounds which inhibited glycolate oxidation and their order of inhibitory activity were: p-hydroxymercuribenzoate > sodium azide > iodoacetate and o-phenanthroline > ethylenediaminetetraacetic acid. Tests of enzyme specificity showed that the following compounds were oxidized, but at different rates: glycolate, d-lactate, l-lactate, dl-alpha-hydroxybutyrate, dl-malate, and dl-glycerate. Citrate, tartrate, and dl-beta-hydroxybutyrate were not oxidized. Potassium cyanide stimulated oxygen uptake when glycolate and lactate were oxidized. Whether the oxidations were due to different oxidases or to a single oxidase with a wide range of specificities was tested by observing the oxidation of glycolate, d-lactate, and l-lactate under various conditions. Ammonium sulfate fractionation of a crude extract did not change the relative ability to oxidize the three acids. However, the three oxidative capacities diminished at different rates during storage at 0 C for 6 days. The partially purified glycolic oxidase preparations were probably mixtures of several different oxidases.

Azides↗
PubMed1999

Helicobacter pylori and ammonia concentrations of whole, parotid and submandibular/sublingual saliva.

The aim of this study was to determine the ammonia concentration in whole, parotid and submandibular/sublingual saliva of healthy volunteers using the indophenol direct method. It also investigated the hypothesis that higher saliva ammonia concentrations are associated with the presence of Helicobacter pylori (H. pylori) in the oral cavity. In healthy volunteers, the mean ammonia concentration of whole saliva (2574 mumol/l) was significantly higher (P < 0.0001) than the mean ammonia concentration of both parotid (238 mumol/l) and submandibular/sublingual (355 mumol/l) saliva. In whole saliva, no difference in ammonia concentration was found between healthy controls and dyspeptic patients (mean ammonia values 2574 and 2489 mumol/l respectively, P = 0.7). In addition, no significant differences were observed in the salivary ammonia concentration between dyspeptic patients with and without H. pylori carriage. It is concluded that the ammonia concentration in parotid and submandibular/sublingual saliva does not differ, but is significantly lower than the ammonia concentration of whole saliva. This difference is not due to carriage of H. pylori with its strong urease activity. Therefore, the determination of ammonia in whole saliva is an inappropriate screening test for patients being at risk for (chronic) gastritis and peptic ulcer disease.

Adult↗