1,720,967 research outputs found
Stereospecificity of sodium borohydride reduction of pig kidney dopa decarboxylase
No full textSodium boro[3H]hydride reduction of pig kidney 3,4 dihydroxyphenylalanine decarboxylase followed by complete hydrolysis of the enzyme produced epsilon-[3H]pyridoxyllysine. Degradation of this material to 4'-[3H]pyridoxamine and stereochemical analysis with apoaspartate aminotransferase showed that the re side at C-4' of the coenzyme is exposed to solvent. In order to determine the face exposed to the solvent in the external Schiff's base, attempts to trap reaction intermediates were made by reduction with sodium boro [3H]hydride of the holoenzyme in the presence of various substrates or substrate analogs. In all cases, covalently bound radioactive material was found which was identified as epsilon-N-pyridoxyllysine. These results suggest that the internal Schiff's base is in mobile equilibrium with the external Schiff's base and that sodium borohydride reduction displaces this equilibrium, resulting in complete reduction of the internal Schiff's base
Chemical modification of pig kidney 3,4-dihydroxyphenylalanine decarboxylase with diethyl pyrocarbonate. Evidence for an essential histidyl residue
No full textDiethyl pyrocarbonate inhibits pig kidney holo-3,4-dihydroxyphenylalanine decarboxylase with a second-order rate constant of 1170 M-1 min-1 at pH 6.8 and 25 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity can be restored by hydroxylamine, and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.03. Complete inactivation of 3,4-dihydroxyphenylalanine decarboxylase requires the modification of 6 histidine residues/mol of enzyme. Statistical analysis of the residual enzyme activity and of the extent of modification shows that, among 6 modifiable residues, only one is critical for activity. Protection exerted by substrate analogues, which bind to the active site of the enzyme, suggests that the modification occurs at or near the active site. The modified inactivated 3,4-dihydroxyphenylalanine decarboxylase still retains most of its ability to bind substrates. Thus, it may be suggested that the inactivation of enzyme by diethyl pyrocarbonate is not due to nonspecific steric or conformational changes which prevent substrate binding. However, the modified enzyme fails to produce at high pH either an enzyme-substrate complex or an enzyme-product complex absorbing at 390 nm. Considerations on this peculiar feature of the modified enzyme consistent with a catalytic role for the modified histidyl residue are discussed. The overall conclusion of this study may be that the modification of only one histidyl residue of 3,4-dihydroxyphenylalanine decarboxylase inactivates the enzyme and that this residue plays an essential role in the mechanism of action of the enzyme
Purification and characterization of rat-liver 3,4-dihydroxyphenylalanine decarboxylase
No full textA simple and rapid procedure, which takes advantage of the effectiveness of conventional and HPLC hydrophobic interaction, for the isolation of highly purified rat liver 3,4-dihydroxyphenylalanine decarboxylase is described in detail. Some of its structural and functional properties are reported and discussed in comparison with those of pig kidney 3,4-dihydroxyphenylalanine decarboxylase
An essential arginine residue at the binding site of pig kidney 3,4-dihydroxyphenylalanine decarboxylase
No full textPig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase is inactivated by the arginine-specific reagent phenylglyoxal. Under these experimental conditions, the reaction follows pseudo-first-order kinetics with a second-order rate constant of 25 m-1 min-1. Holo- and apo-enzyme were inactivated at the same rate. However, inactivation seems to be related to modification of 1 and 2 arginyl residues per mol of holo- and apo-enzyme, respectively. Only one of these two residues was essential to decarboxylase activity of the enzyme. Phenylglyoxal-modified apo-Dopa decarboxylase retained the capacity to bind pyridoxal-P. Neither this reconstituted species nor the phenylglyoxal-modified holoenzyme were able to form Schiff base intermediates with aromatic amino acids in L and D forms. These data together with protection experiments suggest that the susceptible arginine residue in holoenzyme may somehow perturb the substrate binding site. However, unlike in other pyridoxal-P enzymes, this critical arginine in Dopa decarboxylase does not seem to behave as an anionic recognition site for the phosphate group of the coenzyme or the carboxy group of the substrate. It is speculated that this guanidyl group could function in hydrogen bonding of substrate side chain
Going 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
Time-resolved extrinsic fluorescence of aromatic-L-amino-acid decarboxylase
No full textThe coenzyme-linked fluorescence of aromatic-L-amino-acid decarboxylase decays non-exponentially. The decay of both native and NaBH4 reduced samples can only be fitted by two exponentials each roughly accounting for about half of the total fluorescence. Denaturation of the reduced protein with 8 M urea makes the fluorescence decay mono-exponential, like that observed for the reference compound pyridoxamine-5-phosphate. An extra pyridoxyl moiety can be bound to the enzyme after incubation with excess pyridoxal phosphate and reduction with NaBH4. This sample is almost twice as fluorescent and shows also two lifetimes. After denaturation only one fluorescence lifetime is observed. The presence of two non-equivalent pyridoxal sites in the native enzyme can be postulated. The heterogeneous decay behaviour of the pyridoxyl moiety in the enzyme together with the variability of lifetime shown, makes this fluorophore an even more interesting fluorescent probe for proteins
Absence of metabolic cross-correction in Tay-Sachs cells. Implications for gene therapy
No full textWe have investigated the ability of a receptor-mediated gene transfer strategy (cross-correction) to restore ganglioside metabolism in fibroblasts from Tay-Sachs (TS) patients in vitro. TS disease is a GM2 gangliosidosis attributed to the deficiency of the lysosomal enzyme beta-hexosaminidase A (HexA) (beta-N-acetylhexosaminidase, EC 3.2.1.52). The hypothesis is that transduced cells overexpressing and secreting large amounts of the enzyme would lead to a measurable activity in defective cells via a secretion-recapture mechanism. We transduced NIH3T3 murine fibroblasts with the LalphaHexTN retroviral vector carrying the cDNA encoding for the human Hex a-subunit. The Hex activity in the medium from transduced cells was approximately 10-fold higher (up to 75 milliunits) than observed in non-transduced cells. TS cells were cultured for 72 h in the presence of the cell medium derived from the transduced NIH3T3 cells, and they were analyzed for the presence and catalytic activity of the enzyme. Although TS cells were able to efficiently uptake a large amount of the soluble enzyme, the enzyme failed to reach the lysosomes in a sufficient quantity to hydrolyze the GM2 ganglioside to GM3 ganglioside. Thus, our results showed that delivery of the therapeutic HexA was not sufficient to correct the phenotype of TS cells
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
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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