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PubMed1979

Measurement of ribulose 1,5-bisphosphate from spinach chloroplasts.

A technique has been developed for the rapid and simple measurement of ribulose 1,5-bisphosphate from isolated spinach chloroplasts. The endogenous ribulose bisphosphate was detected enzymically using (14)CO(2) and ribulose bisphosphate carboxylase/oxygenase released from the chloroplasts. Ribulose 5-phosphate kinase was inhibited with 0.4 to 0.6 millimolar 2,6-dichlorophenol-indophenol and 4 micromolar carbonyl cyanide m-chlorophenylhydrazone. Phosphoenolpyruvate carboxylase activity was low with washed chloroplasts and its labeled product, [(14)C]oxalacetate, was destroyed by heating with 1.0 n HCl at 90 C. The assay method was linear from 0.05 to 0.87 nanomoles ribulose bisphosphate per milliliter. The latter value was determined with chloroplast material having 44 micrograms of chlorophyll per milliliter. This technique was simple and direct, used less chloroplast material, yet provided results comparable to a previously described enzymic technique in which ribulose bisphosphate was determined after the precipitation of chloroplast proteins by perchloric acid.

Journal Article↗
PubMed1980

A Light-dependent Protein Kinase Activity of Chloroplasts.

A protein kinase activity from spinach chloroplasts, tightly associated with the thylakoid membranes, has been solubilized and partially characterized. This membrane-bound protein kinase is stimulated by light and electron transport activity through photosystem II appears to be required for stimulation.Electron transport inhibitors like 3,4-dichlorophenyl-1,1-dimethylurea, Tris, and NH(2)OH treatments, inhibit the light activation process. Furthermore, after Tris inhibition, the protein kinase activity is restored by washing the Tris-treated chloroplasts with dichlorophenol indophenol plus ascorbate. The protein kinase remains active in the dark after short illumination periods, suggesting that a product of electron transport may be involved in light activation.Two endogenous substrates of the protein kinase in thylakoid membranes are the N,N'-dicyclohexylcarbodiimide-reactive proteolipid and the light-harvesting chlorophyll-protein complex. The membrane-bound protein kinase also phosphorylates externally added histone.

Journal Article↗
PubMed1981

Stimulation of Photosystem I Electron Transport by High Concentration of 3-(3,4-Dichlorophenyl)-1,1-dimethyl Urea in Uncoupled Chloroplasts.

The light saturated rate of photosystem I-dependent electron transport (ascorbate/dichlorophenol-indophenol --> methyl vilogen in presence of 1 micromolar 3-[3,4-dichlorophenyl]-1,1-dimethyl urea [DCMU]) was increased by a high concentration of DCMU added to broken and uncoupled chloroplasts isolated from pea (Pisum sativum). At 50 micromolar DCMU, the increase was around 50%. No stimulation was observed under limiting intensity of illumination, indicating that the relative quantum yield of electron transport was not affected by high DCMU. The light-saturated rate in coupled (to proton gradient formation) chloroplasts was unchanged by 50 micromolar DCMU, suggesting that the rate-limitation imposed by energy coupling was not affected. Using N,N,N',N'-tetramethyl-p-phenylene diamine as electron donor, essentially no DCMU stimulation of the rate was observed, indicating further that the electron donation at a site close to P700 was not affected by high DCMU. It is concluded that DCMU, in the range of 10 to 50 micromolar, affected the thylakoid membranes in such a way that the rate constant of electron donation by dichlorophenol-indophenol at the site prior to the site of energy coupling increased. Further observations that DCMU at 100 micromolar stimulated the rate in coupled chloroplasts indicated an additional DCMU action, presumably by uncoupling the chloroplasts from phosphorylation, as suggested by Izawa (Shibata et al., eds, Comprehensive Biochemistry and Biophysics of Photosynthesis, University Press, State College, Pennsylvania, pp 140-147, 1968). A scheme has been proposed for multiple sites of DCMU action on the electron transport system in chloroplasts.

Journal Article↗
PubMed1983

Light Activation of Purified Aconitase by Washed Thylakoid Membranes of Pea (Pisum sativum L.).

Purified aconitase, an iron-sulfur protein, from either beef heart mitochondria or pig heart can be activated fully by light when combined with washed thylakoid membranes from pea (Pisum sativum L.) chloroplasts. The light activation of the enzyme does not require any other additive or cofactor and is sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, 2,6-dichlorophenol-indophenol, ferricyanide, and methyl viologen, indicating that the photoelectron transport system of the thylakoid membranes, and in particular, photosystem I, is involved in the process of activation. Light activation of the enzyme is also markedly inhibited when the thylakoid membranes are treated with sulfite or arsenite, and abolished totally when the membranes are treated with Zwittergent, suggesting that the light effect mediator involved in the light modulation of chloroplastic enzymes mediates the activation of purified aconitase also.

Journal Article↗
PubMed1983

Effects of Photosystem II Herbicides on the Photosynthetic Membranes of the Cyanobacterium Aphanocapsa 6308.

The effects of the photosystem II herbicides diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) and atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on the photosynthetic membranes of a cyanobacterium, Aphanocapsa 6308, were compared to the effects on a higher plant, Spinacia oleracea. The inhibition of photosystem II electron transport by these herbicides was investigated by measuring the photoreduction of the dye 2,6-dichlorophenol-indophenol spectrophotometrically using isolated membranes. The concentration of herbicide that caused 50% inhibition of electron transport (I(50) value) in Aphanocapsa membranes for diuron was 6.8 x 10(-9) molar and the I(50) value for atrazine was 8.8 x 10(-8) molar. (14)C-labeled diuron and atrazine were used to investigate herbicide binding with calculated binding constants (K) being 8.2 x 10(-8) molar for atrazine and 1.7 x 10(-7) molar for diuron. Competitive binding studies carried out on Aphanocapsa membranes using radiolabeled [(14)C]atrazine and unlabeled diuron revealed that diuron competed with atrazine for the herbicide-binding site. Experiments involving the photoaffinity label [(14)C]azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-2-triazine) and autoradiography of polyacrylamide gels indicated that the herbicide atrazine binds to a 32-kilodalton protein in Aphanocapsa 6308 cell extracts.

Journal Article↗
PubMed1983

Changes in Photosystem II Account for the Circadian Rhythm in Photosynthesis in Gonyaulax polyedra.

Cell-free extracts that show activity in photosynthetic electron flow have been prepared from the unicellular dinoflagellate, Gonyaulax polyedra. Electron flow, as O(2) uptake, was measured through both photo-system I and II from water to methyl viologen, through photosystem I alone from reduced 2,6-dichlorophenol indophenol to methyl viologen which does not include the plastoquinone pool or from duroquinol to methyl viologen which includes the plastoquinone pool. Electron flow principally through photosystem II was measured from water to diaminodurene and ferricyanide, as O(2) evolution. Cultures of Gonyaulax were grown on a 12-hour light:12 hour dark cycle to late log phase, then transferred to constant light at the beginning of a light period. After 3 days, measurements of electron flow were made at the maximum and minimum of the photosynthetic rhythm, as determined from measurements of the rhythm of bioluminescence. Photosynthesis was also measured in whole cells, either as (14)C fixation or O(2) evolution. Electron flow through both photosystems and through photosystem II alone were clearly rhythmic, while electron flow through photosystem I, including or excluding the plastoquinone pool, was constant with time in the circadian cycle. Thus, only changes in photosystem II account for the photosynthesis rhythm in Gonyaulax.

Journal Article↗
PubMed1985

Photoinhibition of Photosynthesis in Broken Chloroplasts as a Function of Electron Transfer Rates during Light Treatment.

Photoinhibition was studied in osmotically broken chloroplasts isolated from spinach leaves (Spinacia oleracea L.). Both whole chain electron transport (measured as ferricyanide-dependent O(2) evolution in the presence of NH(4)Cl) and photosystem II activity (measured as O(2) evolution in the presence of either silicomolybdate plus 3-(3,4-diphenyl)-1,1 dimethylurea or parabenzoquinone) showed similar decreases in activity in response to a photoinhibitory treatment (8 minutes of high light given in the absence of an electron acceptor other than O(2)). Photosystem I activity was less affected. Photoinhibition of silicomolybdate reduction was largely reversible by an 8 minute dark incubation following the light treatment. Decreasing the O(2) concentration during photoinhibition below 2% increased photoinhibition of whole chain electron transport. Addition of superoxide dismutase to the reaction medium did not affect photoinhibition. Photoinhibition of both photosystem I and photosystem II activity increased as the rate of electron transfer during the treatment increased, and was largely prevented when 3-(3,4-diphenyl)-1,1-dimethylurea was present during the photoinhibition period. Noncyclic photophosphorylation was decreased as a consequence of whole chain electron transfer photoinhibition. Since diphenyl carbazide added after light treatment did not relieve photoinhibition of dichlorophenol indophenol reduction, we conclude that the site of inhibition is located within or near the photosystem II reaction center.

Journal Article↗
PubMed1986

Temperature Dependence of Photosynthetic Activities in Wheat Seedlings Grown in the Presence of BASF 13.338 (4-Chloro-5-Dimethylamino-2-Phenyl-3(2H)Pyridazinone).

When Triticum vulgare cv HD 2189 seedlings were grown in the presence of 125 micromolar BASF 13.338 (4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone), the rate of electron transport (H(2)O --> methyl viologen) in chloroplast thylakoids isolated from the treated seedlings was higher (by 50%) as compared to the control at assay temperatures above 30 degrees C. Below 30 degrees C, however, the rate with the treated seedlings was lower than the control rate. The temperature dependence of the rate of photosystem I electron transport (2-6-dichlorophenol indophenol-reduced --> methyl viologen) in the treated system was similar to that in the control. At high temperatures (>30 degrees C), with diphenyl carabazide as electron donor, the rates of electron transfer (diphenyl carbazide --> methyl viologen) were similar in the treated and in the control thylakoids. Direct addition of BASF 13.338 to the assay mixture for the measurement of rate of electron transport (H(2)O --> methyl viologen) in the thylakoids isolated from the control plants did not cause any change in the temperature dependence of photosynthetic electron transport. These results suggested that the donor side of photosystem II became tolerant to heat in the treated plants. Chlorophyll a fluorescence emission was monitored continuously in the leaves of control and BASF 13.338 treated wheat seedlings during continuous increase in temperature (1 degrees C per minute). The fluorescence-temperature profile showed a decrease in the fluorescence yield above 55 degrees C; this decrease was biphasic in the control and monophasic in the treated plants.

Journal Article↗
PubMed1986

Stability of chloroplastic triazine resistance in rutabaga backcross generations.

Triazine resistance originally observed in a weed biotype of birdsrape (Brassica campestris L.) has been transferred through cytoplasmic substitution into rutabaga (Brassica napus ssp. Rapifera [Metzg.] Minsk.) by conventional backcrossing. Photosynthetic function and resistance to triazines were examined in six backcross generations of rutabaga as well as in the original parents. Chloroplast thylakoid membranes were isolated and their sensitivity to atrazine, metribuzin, and diuron assayed by measuring the inhibition of photoreduction of 1,6-dichlorophenol indophenol as well as the alteration of in vitro chlorophyll fluorescence rise characteristics. Both assay methods indicated that triazine resistance persisted in all rutabaga backcross generations, and that it involved triazine binding sites in chloroplasts. There was little resistance to diuron. In vivo chlorophyll fluorescence was also monitored, in the absence of herbicides, as an indicator of the electron transfer properties of the chloroplast photosystem II complex. The results indicated that electron transport from Q(A) to Q(B) was slower (as indicated by a larger intermediate level fluorescence during the transient rise) in the triazine resistant parents as well as in all the rutabaga backcross generations.

Journal Article↗
PubMed1989

Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide.

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca(2+) gradients, induced by the toxin in T corn was prevented by N,N'-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N'-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site.

Journal Article↗
PubMed1992

Effect of Cold Treatments on the Binding Stability of Photosystem II Extrinsic Proteins and an Associated Increase in Susceptibility to Photoinhibition.

When pea plants (Pisum sativum L. cv Feltham First) are subjected to freezing conditions (-18 degrees C) followed by a thaw to 18 degrees C, there is a significant inhibition of water-splitting capacity judged by the rate of light-induced reduction of 2,6-dichlorophenol indophenol using isolated thylakoid membrane fragments enriched in photosystem II (PSII). The freeze-thaw-induced inhibition of water-splitting activity has been correlated with the loss of the 17- and 23-kilodalton extrinsic protein of PSII and with a weakening of the binding of the 33-kilodalton protein. There was no apparent loss of bound manganese. Addition of 10 millimolar CaCl(2), however, allowed a full recovery of the water-splitting activity of these modified PSII-enriched particles. The freeze-thaw-induced changes in the organization and functional capacity of PSII was found to increase its susceptibility to photoinhibition in agreement with the concepts presented in the accompanying paper, that oxidative damage can occur within the PSII reaction center as a consequence of extending the lifetime of P680(+).

Journal Article↗
PubMed2005

Utilization of the metal-cyano complex tetracyanonickelate (II) by Azotobacter vinelandii.

AIMS: The ability of Azotobacter vinelandii, a N(2)-fixing bacterium, to biodegrade tetracyanonickelate (TCN) was evaluated. METHODS AND RESULTS: The amounts of TCN were measured spectrophotometrically. Ammonia was determined colorimetrically by the indophenol method. The produced methane from TCN conversion by A. vinelandii was detected by gas chromatography. Results showed that A. vinelandii was able to biodegrade 1 mmol l(-1) of TCN. Ammonia and methane were detected during the process of TCN degradation. Effects of exogenous nitrogen sources on TCN degradation were addressed in this study. Results revealed that the addition of ammonia (1, 5 and 10 mmol l(-1)) into the reaction mixtures caused decrease of TCN degradation rate during a 24-h incubation period. This inhibition was also observed when nitrite (5 and 10 mmol l(-1)) was added, whereas TCN degradation still proceeded after the addition of nitrate at the same concentrations. Furthermore, the rate of TCN utilization was strikingly enhanced when 0.8% of glucose was added. CONCLUSIONS: Azotobacter vinelandii can degrade 1 mmol l(-1) of TCN into ammonia and methane. However, the inhibitory effects of exogenous ammonia and nitrite on TCN degradation by this bacterium were found in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report defining the capability of A. vinelandii to degrade TCN. This bacterium might have potential value in applied strategies for removing metal-cyano wastes. Furthermore, these findings would be helpful in designing a practical system inoculated with A. vinelandii for the treatment of TCN.

Ammonia↗
PubMed1974

Effect of the systemic fungicide carboxin on electron transport function in membranes of Micrococcus denitrificans.

The systemic fungicide carboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide) inhibited oxidation of succinate by membranes prepared from Micrococcus denitrificans, the K(i) being 16 muM. Oxycarboxin (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4,4-dioxide), F831 (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide-4-oxide), and another succinate oxidase inhibitor, 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTB) were less effective inhibitors of succinate oxidation by membranes of M. denitrificans. Oxidation of other substrates (nicotinamide adenine dinucleotide, reduced form, d-lactate, l-lactate, malate, and d,l-alpha-hydroxybutyrate) was inhibited to a lesser degree by carboxin, and formate oxidation was entirely resistant. With all substrates tested, oxycarboxin, the dioxide analogue of carboxin, was less effective than carboxin. Carboxin also inhibited dichlorophenol indophenol (DCIP) reductase activities by these membranes in a manner both qualitatively and quantitatively similar to the inhibition of oxidation of the various substrates. The inhibition of DCIP reductase activities by TTB was qualitatively similar to carboxin, but TTB was a less effective inhibitor with all substrates tested. The inhibition of DCIP reductase by carboxin could be relieved by phenazine methosulfate with all substrates except d-lactate. Only slight inhibition of d-lactate-stimulated uptake of [(14)C]glycine by these membrane vesicles was seen with carboxin. Uptake of [(14)C]glycine could be stimulated to varying degrees with the other substrates tested, but in no case did carboxin cause significant inhibition. Membranes isolated from M. denitrificans are a useful system for investigating the mechanism of inhibition of electron transport function by carboxin, and the use of this system for evaluations of carboxin and its metabolites is suggested.

Carboxin↗
PubMed1982

Dye-coupled electrode system for the rapid determination of cell populations in polluted water.

We determined cell populations in polluted waters by using a fuel cell-type electrode. The electrode was constructed from a platinum anode, a silver peroxide cathode, and a membrane filter for retaining microorganisms. The principle of cell number determination is based on sensing a redox dye reduced by the microorganisms with the electrode. Sample solutions containing microorganisms were membrane filtered, and the resulting filter containing microbial cells was attached to the surface of a platinum anode. The electrode was immersed in phosphate buffer solution (0.05 M, pH 7) containing a redox dye (2,4-dichlorophenol-indophenol), and the current generated was measured. The response time of the electrode system was 10 to 20 min, and the current generated was proportional to cell populations above 10(4) cells/ml.

Bacillus subtilis↗
PubMed1986

New procedure for extraction of ammonium from natural waters for N isotopic ratio determinations.

A new method for extracting ammonium from natural waters for N isotopic ratio determination is described. The method employs the conversion of the ammonium nitrogen into indophenol, which is then concentrated onto an octadecylsilane column. The method shows accuracy and precision comparable to those of other methods described in the literature. Some results from field experiments on the Swedish west coast are presented.

Journal Article↗
PubMed2001

Purification and characterization of cellobiose dehydrogenase from the plant pathogen Sclerotium (Athelia) rolfsii.

Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by several wood-degrading fungi. In the presence of a suitable electron acceptor, e.g., 2,6-dichloro-indophenol (DCIP), cytochrome c, or metal ions, CDH oxidizes cellobiose to cellobionolactone. The phytopathogenic fungus Sclerotium rolfsii (teleomorph: Athelia rolfsii) strain CBS 191.62 produces remarkably high levels of CDH activity when grown on a cellulose-containing medium. Of the 7,500 U of extracellular enzyme activity formed per liter, less than 10% can be attributed to the proteolytic product cellobiose:quinone oxidoreductase. As with CDH from wood-rotting fungi, the intact, monomeric enzyme from S. rolfsii contains one heme b and one flavin adenine dinucleotide cofactor per molecule. It has a molecular size of 101 kDa, of which 15% is glycosylation, and a pI value of 4.2. The preferred substrates are cellobiose and cellooligosaccharides; additionally, beta-lactose, thiocellobiose, and xylobiose are efficiently oxidized. Cytochrome c (equine) and the azino-di-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical were the best electron acceptors, while DCIP, 1,4-benzoquinone, phenothiazine dyes such as methylene blue, phenoxazine dyes such as Meldola's blue, and ferricyanide were also excellent acceptors. In addition, electrons can be transferred to oxygen. Limited in vitro proteolysis with papain resulted in the formation of several protein fragments that are active with DCIP but not with cytochrome c. Such a flavin-containing fragment, with a mass of 75 kDa and a pI of 5.1 and lacking the heme domain, was isolated and partially characterized.

Basidiomycota↗
PubMed2005

Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems.

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.

Comamonadaceae↗
PubMed1969

Alpha-hydroxyglutarate oxidoreductase of Pseudomonas putida.

Oxidation of d-alpha-hydroxyglutarate to alpha-ketoglutarate is catalyzed by d-alpha-hydroxyglutarate oxidoreductase, an inducible membrane-bound enzyme of the electron transport particle [ETP; a comminuted cytoplasmic membrane preparation with enzymic properties and chemical composition resembling beef heart mitochondrial ETP (1)] of Pseudomonas putida P2 (P2-ETP). Treatment of P2-ETP with a nonionic detergent yields a preparation with the sedimentation characteristics of a soluble enzyme, but which retains an intact electron transport chain. Oxygen acts solely as a terminal electron acceptor and may be replaced by ferricyanide, 2,6-dichlorophenol indophenol, or mammalian cytochrome c. The oxidoreductase is specific for the d-isomer (K(m) = 4.0 x 10(-4)m for dl-alpha-hydroxyglutarate) and is distinct both from l- and d-malate dehydrogenases. Spectral studies suggest that the carrier sequence is substrate --> flavine or nonheme iron --> cyt b --> [cyt c] --> oxygen.

Alcohol Oxidoreductases↗