There is growing evidence that protein disulphide isomerase (PDI) has a common chaperone function in the endoplasmic reticulum. To characterise this function, we investigated the interaction of purified PDI with radiolabelled model peptides, somatostatin and mastoparan, by cross-linking. The interaction between the peptides and PDI was specific, for it showed saturation and was abolished by denaturation of PDI. The interaction between a hydrophobic peptide without cysteine residues was much more sensitive to Triton X-100 than the interaction between PDI and a more hydrophilic peptide with or without cysteine residues. We therefore propose that hydrophobic interactions between protein disulphide isomerase and peptides play an important role in the binding process. The interaction between PDI and the bound peptide therefore is enhanced by the formation of mixed disulphide bonds.

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

1. The activities of protein disulphide-isomerase (PDI) and thioredoxin in catalysing disulphide bond isomerization in a protein substrate were compared by using the standard assay, namely the re-activation of 'scrambled' RNAase. 2. The specific activity of PDI was 25-fold greater than that of thioredoxin. 3. The greater efficiency of PDI compared with thioredoxin is considered to be due more to the presence of multiple catalytic domains in PDI than to differences in their active-site sequences. 4. Data and procedures were defined for expressing enzyme activity in standard units, i.e. mumol of active RNAase generated/min.

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 microM 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 containing five or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 A) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.

The high resolution structure of full-length protein disulphide-isomerase (PDI) has not been determined, but the polypeptide is generally assumed to comprise a series of consecutive domains. Models of its domain organisation have been proposed on the basis of various sequence-based criteria and, more recently, from structural studies on recombinant fragments corresponding to putative domains. We here describe direct studies of the domain architecture of full-length mammalian PDI based on limited proteolysis of the native enzyme. The results are consistent with an emerging model based on the existence of 4 consecutive domains each with the thioredoxin fold. The model was further tested by expressing recombinant fragments corresponding to alternative domain models and to truncated domains; the observed properties of these purified fragments supported the 4-domain model. A multiple alignment of many PDI-like sequences was generated to test whether domain boundaries could be predicted from any features of the alignment, such as sequence variability or hydrophilicity; neither of these parameters reliably predicted the domain boundaries determined by experiment.