1,721,136 research outputs found
Fluorescent Assay to Demonstrate the Interaction of Thioredoxin Reductase and Protein Disulfide Isomerase.
No full textFluorescent Assay to Demonstrate the Interaction of Thioredoxin Reductase and Protein Disulfide Isomerase Tomazzolli R.1, Guella G.2, Bellisola G.3, Colombatti M.3 and Menestrina G.1 1: ITC-CNR IBF Unit at Trento, Via Sommarive 18, 38050 Povo (TN), Italy 2: Lab Bioorg Chem, Dep Physics, Uni Trento, Via Sommarive 14, 38050 Povo, Italy 3: Dep Pathology, Sect of Immunol, Uni Verona, L.go Scuro 10, 37134 Verona, Italy In eukaryotic cells, the ubiquitous thioredoxin reductase-thioredoxin system (TrxR-Trx) and the glutathione reductase-glutathione system (GSHR-GSSG) catalyse substrate disulfide reduction using NADPH as a source of reducing equivalents. TrxR reduces the redox protein Trx as well as other endogenous and exogenous compounds, while the glutathione system plays a key role in protecting cellular macromolecules from damage caused by reactive oxygen species. Due to a second redox-active site, mammalian TrxR has a broader substrate specificity than glutathione reductase and Escherichia coli TrxR [1]. Thioredoxin forms a superfamily with several other proteins sharing little sequence similarity but possessing a common active site [2]. Among these, protein disulfide isomerase (PDI) is colocalized, in cells, with TrxR. In vitro there are now some evidences that PDI can be a substrate of TrxR [3, 4]. To further demonstrate this interaction we have produced a fluorogenic substrate suitable to test TrxR-dependent PDI activity. The substrate is constituted by two identical cys-containing tri-peptides linked at the N-terminus to a molecule of fluorescein-5-isothiocyanate (FITC) and held together by a disulfide bridge between the cysteines. The preparation follows a few steps: the tri-peptide is labelled with FITC; it is separated from FITC excess by two reverse phase liquid chromatography elutions and analysed by electrospray ionization mass spectrometry (ESI-MS); oxidation occurs spontaneously or is induced by DMSO. The final substrate, named pep-FITC, is characterized by NMR and ESI-MS. The reduction of pep-FITC was measured at 25°C in a Fluoromax spectrofluorimeter and was indicated by an increase of the FITC fluorescence at 520nm with excitation at 494nm. All the fluorescence measurements were taken in NaP 0.1M, EDTA 2mM pH 7.5. We demonstrated that the TrxR (both mammalian and bacterial)-PDI system successfully reduced pep-FITC while the single components didn’t. GSHR didn’t reduce pep-FITC, in spite of its aminoacidic sequence similarity to GSSG but was active on a fluorogenic GSSG, labelled at N-termini with FITC, thus demonstrating the narrow substrate specificity of GSHR. Further proofs of the interaction between PDI and TrxR we obtained by fluorescence resonant energy transfer (FRET). In this case we labelled the enzymes with Alexa Fluor 546 and Alexa Fluor 488 respectively, two dyes forming a donor/acceptor pair with a Forster distance of 58 Å [5]. A FRET was observed under suitable conditions confirming tight interaction. (1) Mustacich D, Powis G. 2000. Biochem. J., 346: 1-8. (2). Hirota K, Nakamura H, Masutani H, Yodoi J. 2002. Ann. NY Acad. Sci., 957:189-99. (3). Lundstrom J, Holmgren A. 1990. J. Biol. Chem., 265: 9114-9120. (4). Bellisola G, Fracasso G, Ippoliti R, Menestrina G, Rosen A, Solda S, Udali S, Tomazzolli R, Tridente G, Colombatti M. 2004. Biochem. Pharmacol., 67: 1721-1731. (5). Epe B, Steinhauser KG, Woolley P. 1983. Proc. Natl. Acad. Sci. USA 80 (May), 2579-2583
Rarisetenolide, Epoxyrarisetenolide, and Epirarisetenolide, new-skeleton sesquiterpene lactones as taxonomic markers and defensive agents of the marine ciliated morphospecies Euplotes rariseta
No full textHydrolytic breakdown of the euplotins, highly strained, adaptive, hemiacetal esters of the marine ciliate Euplotes crassus. A mimic of degradative pathway in nature and a trick for the assignment of the absolute configuration
No full textChanges in the chloroplast lipid composition during grapevine berry ripening through an LC-MS based lipidomics approach
No full textGrapevine berry ripening induces peculiar changes of the lipid composition of the
chloroplast membranes . By exploiting our lipidomics approach based on HPLC-MS/MS
techniques we were able to detect a significant increase of oxidation products deriving from MGDG and DGDG galactolipids. This oxidation process implies the addition of an hydroperoxyl group (-OOH) to the a-linolenic acyl chains of the chloroplast galactolipids. Since the addition occurs as a regio-(position 13) and stereospecific (S absolute configuration) reaction, it implies the involvement of a specific lipooxygenase enzym
Epoxyfocardin and its Putative biogenetic Precursor, Focardin, Bioactive, New-Skeleton, Diterpenoids of the Marine Ciliate Euplotes focardii from Antarctica
No full textRogioldiol A, a New Obtusane Diterpene, and Rogiolal, a Degraded Derivative, of the Red Seaweed Laurencia Microcladia from Il Rogiolo along the Coast of Tuscany: a Synergism in Structural Elucidation
No full textThe structure of rogioldiol A ((-)-1), isolated from the red seaweed Laurencia microcladia, was determined. Employing the exciton-coupling technique for rogioldiol A p-bromobenzoate (2), the absolute configuration at C(9) of (-)-1 was assigned, and, together with extensive NMR experiments, the absolute configuration at C(10) and preferred conformations of (-)-1 were determined. The absolute configuration of the hetero-substituted cyclohexane ring was deduced in analogy from the X-ray structure of 4. a derivative of the aldehyde 3, which was isolated from the same seaweed and is believed to be a degradation product of (-)-1
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