11 results on '"Green, T."'
Search Results
2. Myeloperoxidase-dependent fluorescein chlorination by stimulated neutrophils.
- Author
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Hurst, J K, Albrich, J M, Green, T R, Rosen, H, and Klebanoff, S
- Abstract
Hypochlorous acid (HOC1) rapidly chlorinates fluorescein compounds forming, sequentially, the corresponding 4'-chlorofluorescein and 4',5'-dichlorofluoresceins. Chlorination by cell-free myeloperoxidase-catalyzed chloride peroxidation systems gives rise to these compounds as well as variable amounts of isomeric compounds chlorinated in the 2'- and 2',7'-positions. The fluorescence intensity of the dianionic form of the dye is partially quenched upon chlorination, and its proton equilibrium constants are shifted to more acidic values. Fluorescein covalently bound to zymosan (5-isothiocyanatofluorescein-zymosan) also formed these products when the unopsonized particles were incubated with phorbol myristate acetate- or N-formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils. This reaction was associated with a fall in fluorescence intensity, which was not observed when cells from individuals with chronic granulomatous disease or myeloperoxidase deficiency were used or when azide or catalase were added to the reaction medium. Fluorescent changes accompanying phagocytosis of serum-opsonized 5-isothiocyanatofluorescein-zymosan were also consistent with chlorination of the label; the changes were shown to be myeloperoxidase-dependent by use of myeloperoxidase-deficient or azide-treated cells. Oxidative bleaching of the structurally similar sulfonphthalein dyes by HOCl also occurs at rates which parallel the dye basicities. Results are discussed in relation to the use of fluoresceinated particles and sulfonphthalein dyes in the measurement of intraphagosomal acidification.
- Published
- 1984
- Full Text
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3. Purification of the solubilized NADPH:O2 oxidoreductase of human neutrophils. Isolation of its catalytically inactive cytochrome b and flavoprotein redox centers.
- Author
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Green, T R and Pratt, K L
- Abstract
The membrane-bound NADPH:O2 oxidoreductase of human neutrophils has been solubilized in approximately 70% yield and purified on concanavalin A-Sepharose and gel sieving columns of varying bed volumes and sieving ranges. The half-life of the solubilized oxidoreductase stored at 2-4 degrees C in the presence of 25% glycerol at pH 8.6 is approximately 30 h. The oxidoreductase contains a flavoprotein identifiable by its fluorescence spectrum for FAD which binds weakly to concanavalin A-Sepharose and elutes from gel sieving columns at a molecular weight range of approximately 51,000. This flavoprotein accounts for approximately 70% of the total FAD content found in granular membrane fractions recovered from activated neutrophils. Recovery of oxidoreductase activity from both concanavalin A-Sepharose affinity and gel sieving columns is affected by the resolution of the flavoprotein free of the cytochrome b component of the oxidoreductase. The resolved flavoprotein and cytochrome b appear unable to catalyze either NADH nor NADPH oxidase activities with O2, ferricyanide, or nitroblue tetrazolium salt serving as electron acceptors.
- Published
- 1988
- Full Text
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4. Expression of recombinant homo-oligomeric 5-hydroxytryptamine3 receptors provides new insights into their maturation and structure.
- Author
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Green, T, Stauffer, K A, and Lummis, S C
- Abstract
A recombinant baculovirus containing a mouse 5-hydroxytryptamine3 (5-HT3) receptor subunit cDNA under the control of the polyhedrin promoter was shown to direct the production of large amounts of functional 5-HT3 receptor in insect cells, as assayed by Western blotting and ligand binding. After solubilization, the receptor was purified to homogeneity by affinity chromatography and characterized pharmacologically. The ligand binding characteristics of the recombinant receptor were essentially identical to those of the native receptor, both before and after purification. Only fully glycosylated receptors bound to the ligand affinity resin, although subsequent removal of the sugar did not affect ligand binding. Visualization of the purified receptor using electron microscopy showed that the receptor preparation contained a homogeneous population of pentameric doughnut-shaped particles. The general appearance of the recombinant homo-oligomeric channels was indistinguishable from that of native 5-HT3 receptors. Yields of purified receptors were of the order of 200 micrograms/3 liters of original culture. The amount and homogeneity of the purified receptor are sufficient to begin preliminary crystallization trials.
- Published
- 1995
5. The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration.
- Author
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Wilson, D F, Rumsey, W L, Green, T J, and Vanderkooi, J M
- Abstract
Oxygen-dependent quenching of phosphorescence has been used to measure the dependence of mitochondrial oxidative phosphorylation on oxygen concentration in suspensions of isolated rat liver mitochondria. An instrument has been designed which simultaneously monitors the phosphorescence lifetime of a fluorophor and the reduction of cytochrome c by dual wavelength spectrophotometry. The phosphorescence lifetime method gives very rapid (less than 100 ms) measure of the oxygen concentration (Vanderkooi, J. M., Maniara, G., Green, T. J., and Wilson, D. F. (1987) J. Biol. Chem. 262, 5476-5482) from concentrations characteristic of air-saturated media to as low as 2 x 10(-8) M. The results may be summarized as follows. For well coupled rat liver mitochondria at pH 7.0 and in the presence of ATP, as the oxygen concentration was lowered, increased cytochrome c reduction was observed to begin at oxygen concentrations greater than 20 microM. For mitochondria in the presence of uncoupler, cytochrome c reduction began at oxygen concentrations less than 1.0 microM. The oxygen dependence of reduction of cytochrome c in well coupled mitochondria treated with ATP was strongly dependent on the pH of the suspending medium. Reduction of cytochrome c began at higher oxygen concentrations as the pH was made more alkaline. The oxygen concentration for half-maximal respiratory rates was much larger for well coupled mitochondria treated with ATP (approximately 0.7 microM) than for mitochondria treated with uncoupler (less than 0.1 microM). It is concluded that the oxygen dependence of mitochondrial oxidative phosphorylation is such that mitochondria could function in their proposed role of tissue oxygen sensors for regulation of such diverse functions as local blood flow and electrical activity in the carotid body.
- Published
- 1988
- Full Text
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6. The NADPH:O2 oxidoreductase of human neutrophils. Stoichiometry of univalent and divalent reduction of O2.
- Author
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Green, T R and Wu, D E
- Abstract
The ratio of superoxide production to oxidation of NADPH affected by the NADPH:O2 oxidoreductase of human neutrophils is strongly influenced by pH, NADPH substrate concentration, aging of the enzyme, or its exposure to excess deoxycholate. Freshly prepared enzyme exhibited a Km for NADPH of 52 microM as determined by assaying NADPH oxidase activity, or approximately 33 microM by measurement of superoxide formation. In the range of 100-150 microM NADPH at pH 7.6 and in the presence of 0.06% deoxycholate, the univalent flux of electron equivalents given up by NADPH to O2 was 99%. Following storage of the oxidoreductase overnight on ice, its Km for NADPH rose to 125 microM as determined by monitoring oxidation of NADPH but was unaltered when measured in terms of superoxide production. Concomitantly, its capacity to affect univalent reduction of O2 fell approximately 20-30% under the same assay conditions. Univalent flux rates of less than 40% were observed with exposure of the enzyme to concentrations of deoxycholate in excess of 0.1% or to pH values below 6.0 or above 8.0. The capacity of the enzyme to carry out univalent reduction fell with increasing NADPH concentrations in a manner resembling that previously reported with increasing concentrations of xanthine in the case of xanthine oxidase (Fridovich, I. (1970) J. Biol. Chem. 245, 4053-4057). The reduced form of the neutrophil oxidoreductase, like xanthine oxidase, thus appears to be capable of conducting both 1- and 2-electron transfer steps in reducing O2. Its capacity to affect univalent reduction of O2 depends upon the concentration of electron donor (NADPH) supplied as well as conditions of storage and assay of the native enzyme.
- Published
- 1986
- Full Text
- View/download PDF
7. The tumor suppressor activity of the transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) correlates with its ability to modulate sarcosine levels.
- Author
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Chen X, Overcash R, Green T, Hoffman D, Asch AS, and Ruiz-Echevarría MJ
- Subjects
- Animals, Cell Line, Tumor, Glycine genetics, Glycine metabolism, HEK293 Cells, Humans, Male, Membrane Proteins genetics, Mice, NIH 3T3 Cells, Neoplasm Proteins genetics, Protein Binding, Protein Structure, Tertiary, Sarcosine genetics, Sarcosine Dehydrogenase genetics, Tumor Suppressor Proteins genetics, Membrane Proteins metabolism, Neoplasm Proteins metabolism, Sarcosine metabolism, Sarcosine Dehydrogenase metabolism, Tumor Suppressor Proteins metabolism
- Abstract
The type I transmembrane protein with epidermal growth factor and two follistatin motifs 2 (TMEFF2) is expressed in brain and prostate and overexpressed in prostate cancer, but its role in this disease is unclear. Several studies have suggested that TMEFF2 plays a role in suppressing the growth and invasive potential of human cancer cells, whereas others suggest that the shed portion of TMEFF2, which lacks the cytoplasmic region, has a growth-promoting activity. Here we show that TMEFF2 has a dual mode of action. Ectopic expression of wild-type full-length TMEFF2 inhibits soft agar colony formation, cellular invasion, and migration and increases cellular sensitivity to apoptosis. However, expression of the ectodomain portion of TMEFF2 increases cell proliferation. Using affinity chromatography and mass spectrometry, we identify sarcosine dehydrogenase (SARDH), the enzyme that converts sarcosine to glycine, as a TMEFF2-interacting protein. Co-immunoprecipitation and immunofluorescence analysis confirms the interaction of SARDH with full-length TMEFF2. The ectodomain does not bind to SARDH. Moreover, expression of the full-length TMEFF2 but not the ectodomain results in a decreased level of sarcosine in the cells. These results suggest that the tumor suppressor activity of TMEFF2 requires the cytoplasmic/transmembrane portion of the protein and correlates with its ability to bind to SARDH and to modulate the level of sarcosine.
- Published
- 2011
- Full Text
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8. Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2.
- Author
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Green T, Grigorian A, Klyuyeva A, Tuganova A, Luo M, and Popov KM
- Subjects
- Binding Sites physiology, Crystallography, X-Ray, Humans, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Phosphorylation, Potassium chemistry, Potassium metabolism, Protein Binding physiology, Protein Serine-Threonine Kinases genetics, Protein Serine-Threonine Kinases metabolism, Protein Structure, Tertiary physiology, Pyruvate Dehydrogenase Acetyl-Transferring Kinase, Pyruvate Dehydrogenase Complex chemistry, Pyruvate Dehydrogenase Complex genetics, Pyruvate Dehydrogenase Complex metabolism, Structure-Activity Relationship, Mitochondrial Proteins chemistry, Protein Serine-Threonine Kinases chemistry
- Abstract
PDHK2 is a mitochondrial protein kinase that phosphorylates pyruvate dehydrogenase complex, thereby down-regulating the oxidation of pyruvate. Here, we present the crystal structure of PDHK2 bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase (L2) determined with or without bound adenylyl imidodiphosphate. Both structures reveal a PDHK2 dimer complexed with two L2 domains. Comparison with apo-PDHK2 shows that L2 binding causes rearrangements in PDHK2 structure that affect the L2- and E1-binding sites. Significant differences are found between PDHK2 and PDHK3 with respect to the structure of their lipoyllysine-binding cavities, providing the first structural support to a number of studies showing that these isozymes are markedly different with respect to their affinity for the L2 domain. Both structures display a novel type II potassium-binding site located on the PDHK2 interface with the L2 domain. Binding of potassium ion at this site rigidifies the interface and appears to be critical in determining the strength of L2 binding. Evidence is also presented that potassium ions are indispensable for the cross-talk between the nucleotide- and L2-binding sites of PDHK2. The latter is believed to be essential for the movement of PDHK2 along the surface of the transacetylase scaffold.
- Published
- 2008
- Full Text
- View/download PDF
9. Stoichiometry of O2 metabolism and NADPH oxidation of the cell-free latent oxidase reconstituted from cytosol and solubilized membrane from resting human neutrophils.
- Author
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Green TR and Shangguan X
- Subjects
- Cell Membrane enzymology, Cell-Free System, Cytochrome c Group metabolism, Cytosol enzymology, Humans, Kinetics, Oxidation-Reduction, Superoxides blood, Xanthine Oxidase pharmacology, NADH, NADPH Oxidoreductases blood, NADP blood, NADPH Oxidases, Neutrophils enzymology, Oxygen Consumption
- Abstract
The stoichiometry of NADPH-dependent O2 consumption expressed by reconstituted latent oxidase obtained by combining cytosol and membrane fractions from resting human neutrophils with GTP gamma S and SDS in a cell-free assay was evaluated with regard to NADPH consumed and superoxide and H2O2 production. Oxidase activity monitored simultaneously by O2 uptake analysis using a Clark cell and O2 electrode for O2 consumption, and spectrally at 340 nm for NADPH oxidation, in the presence of excess superoxide dismutase and catalase, yielded an O2 uptake: NADPH consumption ratio of 0.51 +/- 0.04 (+/- 1 S.D., n = 6). In the presence of varying concentrations of ferricytochrome c in excess of 100 microM, and with exclusion of superoxide dismutase, the net rate of O2 consumption plateaued at approximately 6% of the rate seen with exclusion of ferricytochrome c from final assay mixtures. Cytosol and solubilized membrane fractions employed in these assays were devoid of endogenous superoxide trapping, or dismutase-like, activities. These results indicate that of the total O2 consumed, 94% is associated with direct univalent generation of superoxide. The remaining albeit low level of O2 consumption appears to be recovered in the form of H2O2 indicating that the cell-free oxidase reconstituted with SDS is capable of channeling electron equivalents through its redox sites in a highly controlled manner in ensuring that superoxide is its principal O2 reduction product concomitant with oxidation of NADPH.
- Published
- 1993
10. Detection and isolation of the NADPH-binding protein of the NADPH:O2 oxidoreductase complex of human neutrophils.
- Author
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Green TR and Pratt KL
- Subjects
- Carrier Proteins isolation & purification, Cell Membrane enzymology, Chromatography, Gel, Chromatography, Ion Exchange, Dithionite pharmacology, Humans, Kinetics, Macromolecular Substances, NADH, NADPH Oxidoreductases isolation & purification, NADP metabolism, Oxidation-Reduction, Protein Precursors metabolism, Spectrophotometry, Superoxides blood, Ubiquitins metabolism, Carrier Proteins blood, NADH, NADPH Oxidoreductases blood, NADPH Oxidases, Neutrophils enzymology, Protein Precursors genetics, Ubiquitins genetics
- Abstract
Neutrophils assayed with nitro blue tetrazolium (NBT) exhibit intracellular rather than extracellular superoxide-generating activity when stimulated with phorbol myristate acetate. Enzyme activity is stimulated by anionic detergents, reversibly inhibited by 2',3'-NADPH dialdehyde, and present in equal levels in membrane fractions obtained from phorbol myristate acetate-stimulated and resting cell suspensions. Solubilized membrane shows enzyme activity co-eluting on molecular sieving columns with the cytochrome b redox component of the oxidoreductase complex. Enzyme activity was resolved free of the cytochrome b component following passage of solubilized membrane extracts through QAE-Sephadex anion exchange columns. Enzyme activity measured by the NBT assay appears to be that associated with the NADPH binding protein of the oxidoreductase complex. When exposed to NBT and NADPH this component of the oxidoreductase generates superoxide independent of cytochrome b.
- Published
- 1990
11. An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence.
- Author
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Vanderkooi JM, Maniara G, Green TJ, and Wilson DF
- Subjects
- Animals, Catalase, Fluoresceins, Glucose Oxidase, Indicators and Reagents, Kinetics, Luminescent Measurements, Mitochondria, Liver metabolism, Oxygen Consumption, Rats, Spectrometry, Fluorescence instrumentation, Spectrometry, Fluorescence methods, Spectrophotometry methods, Oxygen analysis
- Abstract
An optical method for measuring oxygen concentrations in aqueous solutions is described. This method is based upon the oxygen-dependent quenching of phosphorescence. Phosphorescence excitation and emission spectra and lifetimes of some of the probe molecules suitable for measurement of oxygen in aqueous solutions are given. The probes include fluorescein derivatives, 4'5'-diiodofluorescein, eosin Y, 5(and 6)-carboxyeosin, erythrosin, and 5(and 6)-carboxyerythrosin as well as the Zn(II), Y(III), Sn(IV), Lu(III), and Pd(II) derivatives of meso-tetra-(4-sulfonatophenyl)-porphine, meso-tetra-(N-methyl-4-pyridyl)-porphine and coproporphyrin. The phosphorescence lifetimes of the given probes were found to depend upon the oxygen concentration by a simple Stern-Volmer relationship with a quenching constant of approximately 10(9) M-1 S-1. Binding of the molecules to bovine serum albumin decreased the quenching constant for oxygen by approximately an order of magnitude and also inhibited probe self-quenching, indicating that at the protein binding site the probes are somewhat protected from collision with quenchers. The use of this optical method for measuring oxygen is demonstrated for reactions catalyzed by glucose oxidase and by cytochrome c oxidase. It is shown that, using this method oxygen concentrations can be measured from approximately 250 microM (air saturation) down to the nanomolar range.
- Published
- 1987
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