Publications

FLUORESCENT ASSAY TO TEST PROTEIN DISULFIDE ISOMERASE ACTIVITY INDUCED BY THIOREDOXIN REDUCTASE.  (2004)

Authors:
R. Tomazzolli; G. Guella; G. Bellisola; M. Colombatti; G. Menestrina.
Title:
FLUORESCENT ASSAY TO TEST PROTEIN DISULFIDE ISOMERASE ACTIVITY INDUCED BY THIOREDOXIN REDUCTASE.
Year:
2004
Type of item:
Contributo in atti di convegno
Tipologia ANVUR:
Contributo in Atti di convegno
Language:
Inglese
Format:
A Stampa
Congresso:
SIBPA 2004. XVii Congresso Nazionale della Società di Biofisica Pura ed Applicata
Place:
Pisa - Italia
Period:
23-25 Settembre 2004
Page numbers:
20-null
Keyword:
thioredoxin, thioredoxin reductase, PDI, FRET
Short description of contents:
In eukaryotic cells the ubiquitous thioredoxin reductase-thioredoxin system (TrxR-Trx) catalyses substrate disulfide reduction using NADPH as a source of reducing equivalents. Thioredoxin forms a superfamily with several other proteins sharing little sequence similarity but possessing a common active site. Among these, protein disulfide isomerase (PDI) is colocalized, in cells,with TrxR and there are now some in vitro evidences that PDI can be a substrate of TrxR1. To further demonstrate this interaction we have now produced a fluorogenic substrate suitable to test TrxR-dependent PDI activity. The substrate was formed 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; it was called pep-FITC. The reduction of pep-FITC was measured at 25°C in a Fluoromax spectrofluorimeter and was demonstrated by an increase of the FITC fluorescence at 520nm with excitation at 494nm. We could show that the TrxR-PDI system successfully reduced pep-FITC, while the isolated components did not. Further proofs of the interaction between PDI and TrxR were 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.A FRET was observed under suitable conditions confirming tight interaction.
Product ID:
20854
Handle IRIS:
11562/20854
Deposited On:
September 9, 2009
Last Modified:
November 2, 2016
Bibliographic citation:
R. Tomazzolli; G. Guella; G. Bellisola; M. Colombatti; G. Menestrina., FLUORESCENT ASSAY TO TEST PROTEIN DISULFIDE ISOMERASE ACTIVITY INDUCED BY THIOREDOXIN REDUCTASE.Proceedings of "SIBPA 2004. XVii Congresso Nazionale della Società di Biofisica Pura ed Applicata" , Pisa - Italia , 23-25 Settembre 2004 , 2004pp. 20-null

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