Optical Multichannel Analysis of Protochlorophyllide Phototransformation in Detergent-Solubilized Etioplast Membranes of Wheat

1994 ◽  
Vol 49 (1-2) ◽  
pp. 125-131 ◽  
Author(s):  
Fabrice Franck ◽  
Mohammed Aziz Ouazzani ◽  
Esther Dujardin ◽  
Radovan Popovic
Keyword(s):  
Continuous Light ◽  
Multichannel Analysis ◽  
Triton X ◽  
Kinetics Of ◽  
Etioplast Membranes ◽  
Protochlorophyllide Phototransformation ◽  
Absorbance Spectra

Extracts of wheat etioplast membranes obtained after treatment with 7 mᴍ n-octyl-β-ᴅ-glucopyranoside (OG), n-dodecyl-β-ᴅ-maltoside (DM) or Triton X-100 contained the three spectral forms of Pchlide (the photoactive Pchlide638 and Pchlide650 and the inactive Pchlide630) in various relative amounts . The OG extract had a Pchlide composition close to that of the intact membranes whereas the DM extract was enriched in Pchlide638 and the Triton extract was enriched in Pchlide630. Measurements of the kinetics of phototransformation and of timeresolved absorbance spectra during phototransformation in continuous light shows that the inactive Pchlide630 is in fact slowly transformed to Chlide, especially in the Triton extract where this form is more abundant. Addition of NADPH favours the phototransformation of Pchlide630 and the slow regeneration of Pchlide638 and Pchlide650 from Pchlide630 in darkness after illumination. No such regeneration was however observed in the Triton extract. NADPH had only slight effects on the Chlide shift towards shorter wavelengths after phototransformation in solubilized membranes.

Kinetic of oxidation of methyl orange by vanadium(V) under conditions VO2+ and decavanadates coexist: Catalysis by Triton X-100 micellar medium

2019 ◽  
Author(s):  
Chem Int
Keyword(s):  
Methyl Orange ◽  
Solution Ph ◽  
Vanadium Concentration ◽  
Limiting Behavior ◽  
First Order ◽  
First Order Kinetics ◽  
Ph Range ◽  
Kinetic Pattern ◽  
Triton X ◽  
Kinetics Of

The kinetics of oxidation of methyl orange by vanadium(V) {V(V)} has been investigated in the pH range 2.3-3.79. In this pH range V(V) exists both in the form of decavanadates and VO2+. The kinetic results are distinctly different from the results obtained for the same reaction in highly acidic solution (pH < 1) where V(V) exists only in the form of VO2+. The reaction obeys first order kinetics with respect to methyl orange but the rate has very little dependence on total vanadium concentration. The reaction is accelerated by H+ ion but the dependence of rate on [H+] is less than that corresponding to first order dependence. The equilibrium between decavanadates and VO2+ explains the different kinetic pattern observed in this pH range. The reaction is markedly accelerated by Triton X-100 micelles. The rate-[surfactant] profile shows a limiting behavior indicative of a unimolecular pathway in the micellar pseudophase.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Conformational Changes of the Membrane-Bound ATPase of Bacterial Chromatophores Revealed by Fluorescence Changes of Fluorescamine-Labelled Coupling Factors

1978 ◽  
Vol 33 (5-6) ◽  
pp. 421-427
Author(s):  
Peter Gräber ◽  
Sathamm Saphon
Keyword(s):  
Conformational Changes ◽  
Fluorescence Emission ◽  
Continuous Light ◽  
Coupling Factor ◽  
Single Turnover ◽  
Fluorescence Change ◽  
Transfer Inhibitor ◽  
Membrane Bound ◽  
Coupling Factors ◽  
Kinetics Of

Abstract The solubilized coupling factor (F1) of Rps. sphaeroides chromatophores was allowed to react with fiuorescamine which led to a fluorescence labelled F1 . After reconstitution with the depleted membranes the fluorescence-labelled F1 was shown to restore photophosphorylation in continuous light in a similar way to the non-labelled F1 . In parallel, a decrease of the fluorescence emission of the labelled and reconstituted coupling factor was observed. The solubilized and labelled F1 showed also a fluorescence decrease as the polarity of the medium was increased. In single turnover flashes the fluorescence change was found to be inhibited by an uncoupling agent such as FCCP. The kinetics of the change were sensitive to phosphorylating agents and to an “energy transfer inhibitor” such as venturicidin. It is suggested that the observed fluorescence changes reflect conformational changes of the ATPase enzyme complex.

Kinetics of micellization of Triton X-100 in aqueous solutions

1980 ◽  
Vol 84 (12) ◽  
pp. 1536-1540 ◽  
Author(s):  
C. U. Herrmann ◽  
M. Kahlweit
Keyword(s):  
Aqueous Solutions ◽  
Triton X ◽  
Kinetics Of

scholarly journals Studies of the acetylcholinesterase from houseflies (Musca domestica L.) resistant and susceptible to organophosphorus insecticides

1975 ◽  
Vol 149 (2) ◽  
pp. 463-469 ◽  
Author(s):  
A L Devonshire
Keyword(s):  
Musca Domestica ◽  
Rate Constants ◽  
Gel Electrophoresis ◽  
Organophosphorus Compounds ◽  
Organophosphorus Insecticides ◽  
Triton X ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Single Enzyme

Acetylcholinesterase from the heads of insecticide-resistant and -susceptible houseflies (Musca domestica L.) was studied in vitro. The enzymes could not be distinguished electrophoretically, and their behaviour on polyacrylamide-disc-gel electrophoresis was influenced by the presence of Triton X-100 in both the homogenate and the gels. In the absence of detergent, the acetylcholinesterase was heterogeneous, but behaved as a single enzyme when it was present. By analogy with studies of acetylcholinesterase from other sources, these observations were attributed to aggregation of the enzyme when not bound by membranes. The enzyme from resistant flies was more slowly inhibited than the susceptible enzyme, bimolecular rate constants (ki) differing by approx. 4-20-fold for a range of organophosphorus compounds. The kinetics of inhibition of acetylcholinesterase were consistent with the results of electrophoresis, i.e. they corresponded to those of a single enzyme in the presence of Triton X-100, but a mixture of enzymes in its absence. The susceptibility of the more sensitive components in these mixtures was determined.

scholarly journals Unveiling the multi-step solubilization mechanism of single vesicles by detergents

2019 ◽  
Author(s):  
Paul A. Dalgarno ◽  
José Juan-Colás ◽  
Gordon J. Hedley ◽  
Lucas Piñeiro ◽  
Mercedes Novo ◽  
...  
Keyword(s):  
Real Time ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Optical Methods ◽  
Fluorescence Correlation ◽  
Ionic Detergent ◽  
Triton X ◽  
Kinetics Of ◽  
Single Vesicle

AbstractThe solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

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