Publications

Authors:

R., Tomazzolli; G., Guella; G., Bellisola; Colombatti, Marco; 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

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 3, 2022

Bibliographic citation:

R., Tomazzolli; G., Guella; G., Bellisola; Colombatti, Marco; 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 , 2004 , pp. 20-null

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