Redox properties and cross-linking of the dithiol disulfide active-sites of mammalian protein disulfide-isomerase

Hawkins, H.C. and Denardi, M. and Freedman, R.B. (1991) Redox properties and cross-linking of the dithiol disulfide active-sites of mammalian protein disulfide-isomerase. Biochemical Journal, 275 . pp. 341-348. ISSN 0264-6021. (The full text of this publication is not available from this repository)

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Abstract

1. The redox properties of the active-site dithiol/disulphide groups of PDI were determined by equilibrating the enzyme with an excess of GSH + GSSG, rapidly alkylating the dithiol form of the enzyme to inactivate it irreversibly, and determining the proportion of the disulphide form by measuring the residual activity under standard conditions. 2. The extent of reduction varied with the applied redox potential; to a first approximation, the data fitted a model in which all the enzyme dithiol/disulphide groups are independent and equivalent and the equilibrium constant between these sites and the GSH/GSSG redox couple is 42-mu-M at pH 7.5. 3. The standard redox potential for PDI active-site dithiol/disulphide couples was calculated from this result and found to be -0.11 V; hence PDI is a stronger oxidant and weaker reductant than GSH, nicotinamide cofactors, thioredoxin and dithiothreitol. 4. The redox equilibrium data for PDI with the GSH/GSSG redox couple showed sigmoidal deviations from linearity. The sigmoidicity could be modelled closely by assuming a Hill coefficient of 1.5. 5.This evidence of co-operative interactions between the four active sites in a PDI dimer was extended by studying the reaction between PDI and homobifunctional alkylating agents with various lengths between the reactive groups. A species whose electrophoretic mobility suggested it contained an intrachain cross-link was observed in all cases, whereas there was no evidence for cross-linking between the chains of the PDI homodimer. Most effective cross-linking was achieved with reagents containingfive or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 angstrom) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.

Item Type: Article
Subjects: Q Science > QP Physiology (Living systems)
Divisions: Faculties > Science Technology and Medical Studies > School of Biosciences
Depositing User: O.O. Odanye
Date Deposited: 04 Nov 2009 11:40
Last Modified: 04 Nov 2009 11:40
Resource URI: http://kar.kent.ac.uk/id/eprint/22971 (The current URI for this page, for reference purposes)
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