DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase. However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding. Carboxymethylation of DsbC at Cys 98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation. Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH. The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10-and 14-mer peptides and S-carboxymethylated insulin A chain. In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI. DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer. Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI.

Protein disulfide-isomerase (PDI) in near stoichiometric concentrations promotes reactivation and prevents aggregation of guanidine-hydrochloride-denatured rhodanese during refolding upon dilution. PDI also suppresses aggregation of rhodanese during thermal inactivation. The above-mentioned properties displayed by PDI completely satisfy the definition of chaperone and provide additional evidence to confirm the hypothesis proposed previously [Wang, C. C. & Tsou, C. L. (1993) FASEB J. 7, 1515-1517] that PDI is both an enzyme and a chaperone. Since rhodanese contains no disulfide bonds, the chaperone-like activity of PDI acting on rhodanese is independent of its disulfide-isomerase activity.

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Protein disulfide-isomerase (PDI) in near stoichiometric concentrations promotes reactivation and prevents aggregation of guanidine-hydrochloride-denatured rhodanese during refolding upon dilution. PDI also suppresses aggregation of rhodanese during thermal inactivation. The above-mentioned properties displayed by PDI completely satisfy the definition of chaperone and provide additional evidence to confirm the hypothesis proposed previously [Wang, C. C. & Tsou, C. L. (1993) FASEB J. 7, 1515-15171 that PDI is both an enzyme and a chaperone. Since rhodanese contains no disulfide bonds, the chaperonelike activity of PDI acting on rhodanese is independent of its disulfide-isomerase activity.Keywords. Protein disulfide-isomerase ; chaperone ; rhodanese ; refolding ; aggregation.Protein disulfide isomerase (PDI) is one of the two characterized foldases [I] and is believed to assist nascent peptide folding by catalyzing the formation of disulfide bonds during protein biogenesis [2]. In view of its catalysis of the joining of thiols distantly situated in the peptide sequences and its peptide-binding property with low specificity [3], we have hypothesized that PDI may act both as an enzyme and a chaperone in protein folding [4]. Recently, we have shown that PDI assists the refolding of Gdn . HC1 (guanidine hydrochloride)-denatured GraP-DH (~-glyceraldehyde-3-phosphate dehydrogenase), a protein containing no disulfide bonds, to its native state and increases its reactivation yield by suppressing aggregation in a way closely similar to the action of a chaperone [5]. It has also been suggested by a few authors that PDI may have chaperone function reported the chaperone and antichaperone activity of PDI in the oxidative refolding of lysozyme. However, Lilie et al. [lo] found no chaperone-like effect of PDI on the refolding of the antibody Fab (antigen-binding antibody fragment consisting of the entire light chain and the two N-terminal domains of the heavy chain) with intact disulfide bonds at pH 7.0 under oxidizing conditions. LaMantia and Lennarz [I I] indicated that the isomerase activity is not essential for cell viability, which may require instead some other functions of PDI.In order to provide more evidence on the chaperone activity of PDI with a target protein that has been well studied in its chaperone-assisted folding 112, 131, we have studied the PDIassisted refolding of Gdn . HC1-denatured rhodanese, which consists of a single chain folded into two domains of equal size containing four cysteine residues [14]. The sequence retains all the information required for targeting, transport, folding, and activity [15], which makes rhodanese a valuable model to study the folding processes of single chain and multidomain proteins. As rhodanese has no disulfide bonds, it is possible to analyze the chaperone activity of PDI independent of its isomerase activity. With target proteins containing disulfide bonds like lysozyme or immunoglobulin, it is difficult to exclude the possibility of disulfide isomerization as the prim...

Protein disulphide isomerase (PDI) shows chaperone and anti-chaperone activities in assisting refolding of denatured and reduced lysozyme in redox Hepes buffer, but only chaperone activity in phosphate buffer and redox Hepes buffer containing 0.1 M NaCl. In non-redox Hepes buffer its anti-chaperone activity is very weak. PDI displays its anti-chaperone activity only for those substrates showing relatively low aggregation during refolding, and is strongly dependent on refolding conditions, of which ionic strength appears to be an important factor. The S-methylated PDI, fully active as a chaperone but devoid of isomerase activity, by itself shows only anti-chaperone activity, but reinforces rather than suppresses the chaperone activity of native PDI in the refolding of lysozyme. A fragment of PDI with the C-terminal peptide-binding sequence removed and devoid of chaperone activity does not show anti-chaperone activity in lysozyme refolding. It appears that the anti-chaperone activity of PDI is dependent on its chaperone activity.