1,721,848 research outputs found
Flexibility of helix 2 in the human glutathione transferase P1-1. time-resolved fluorescence spectroscopy
No full textTime-resolved fluorescence spectroscopy and site-directed mutagenesis have been used to probe the flexibility of alpha-helix 2 (residues 35-46) in the apo structure of the human glutathione transferase P1-1 (EC 2.5.1.18) as well as in the binary complex with the natural substrate glutathione. Trp-38, which resides on helix 2, has been exploited as an intrinsic fluorescent probe of the dynamics of this region. A Trp-28 mutant enzyme was studied in which the second tryptophan of glutathione transferase P1-1 is replaced by histidine. Time-resolved fluorescence data indicate that, in the absence of glutathione, the apoenzyme exists in at least two different families of conformational states. The first one (38% of the total population) corresponds to a number of slightly different conformations of helix 2, in which Trp-38 resides in a polar environment showing an average emission wavelength of 350 nm. The second one (62% of the total population) displays an emission centered at 320 nm, thus suggesting a quite apolar environment near Trp-38. The interconversion between these two conformations is much slower than 1 ns. In the presence of saturating glutathione concentrations, the equilibrium is shifted toward the apolar component, which is now 83% of the total population. The polar conformers, on the other hand, do not change their average decay lifetime, but the distribution becomes wider, indicating a slightly increased rigidity. These data suggest a central role of conformational transitions in the binding mechanism, and are consistent with NMR data (Nicotra, M., Paci, M., Sette, M., Oakley, A. J., Parker, M. W., Lo Bello, M., Caccuri, A. M., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3020-3027) and pre-steady state kinetic experiments (Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034) indicating the existence of a pre-complex in which GSH is not firmly bound to the active site
Site-directed mutagenesis of human glutathione transferase P1-1. Spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants
No full textIn the human placental glutathione transferase, Cys 47 possesses, at physiological pH values, a pK(a) value of 4.2 and may exist as an ion pair with the protonated epsilon-amino group of Lys-54, Using site directed mutagenesis we investigate spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants. The results shown indicate that the thiolate ion detected at 229 nm should be assigned exclusively to Cys-47. The contribution of Lys-54 to the activation of Cys-47 is assessed by the spectral properties of the K54A mutant enzyme. The induced cooperativity toward glutathione, as a consequence of mutation of Lys-54 to alanine, clearly parallels that observed for the Cys-47 mutant enzymes (see the preceding paper (Ricci, G., Lo Bello, M., Caccuri, A. M., Pastore, A., Nuccetelli, M., Parker, M. W,, and Federici, G, (1995) J. Biol. Chem. 270, 1243-1248) and points out the importance of this electrostatic interaction in shaping the correct spatial arrangement for the binding of glutathione and in anchoring the flexible helix alpha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing helix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperativity
Alkaline hydrolysis of N-ethylmaleimide allows a rapid assay of glutathione disulfide in biological samples
No full textGoing Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
ELECTROCHEMICAL SENSORS FOR THE DETERMINATION OF METAL-IONS
No full textAn electrochemical method for the determination of the metal ions Fe3+, Cu2+ and Hg2+ has been developed. It is based on organic chelators changing their electrochemical behaviour as a function of metal ion concentration in solution. Amperometric measurements of the metal ions are performed by cyclic voltammetry using a glassy carbon or a modified carbon paste as working electrodes. Fe3+ is determined in the range 6-60 ppb using two chelators synthesized by the British Technology Group (UK Patent Nos. GB 2118176B and GB 2136807B), Cu2+ is determined in the range 0.35-4 ppm with bis-cyclohexanone oxalyldihydrazone and Hg2+ is determined in the range 20-200 ppm using glyoxal dithiosemicarbazone.[...
A new conducting polymer glucose sensor based on polythianaphthene
No full textA glucose biosensor has been developed by entrapment of glucose oxidase in a polythianaphthene film formed onto a platinum surface. Glucose has been measured by H2O2 detection at +400 mV (vs. SCE). Electrochemical kinetic constants for the enzyme electrode have been calculated and indicated that in the unsaturated region the rate limiting step is the diffusion of the substrate through the polymer. The biosensor exhibited a linear response in the range 0.05-7.4 mmol/L with a response time of 30-60 s and a relative standard deviation of 5.4%. The presence of the film onto the electrode prevents the oxidation of interfering agents such as ascorbic and uric acid. The use of additional polycarbonate membranes onto the electrode surface extended the linearity of the response allowing the probe to be used in blood glucose analysis. The amount of glucose in two control sera was measured using the biosensor. Results correlated well with the glucose nominal values.[...
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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