Tài liệu Báo cáo khoa học: Bacitracin is not a specific inhibitor of protein disulfide isomerase pptx

Bacitracin is not a specific inhibitor of protein disulfide isomerase Anna-Riikka Karala and Lloyd W. Ruddock Biocenter Oulu and Department of Biochemistry, University of Oulu, Finland Introduction Protein disulfide isomerase (PDI) is an endoplasmic reticulum (ER)-resident protein catalyst that helps newly translated polypeptide chains to fold and form native disulfide bonds [1]. PDI can catalyze the oxida- tion of two cysteines to form a disulfide bond, as well as the reduction and isomerization of disulfide bonds in peptides and proteins. PDI has four structural thior- edoxin-like domains, a, b, b¢, and a¢, a linker region x between the b¢ and a¢ domains, and a C-terminal acidic extension. The a and a¢ domains contain the CGHC active site motif, and are sufficient alone to perform thiol–disulfide exchange reactions in simple substrates [2]. The b¢ domain has the principal peptide and non- native protein-binding site, and is required for isomeri- zation reactions [2–4], whereas the b domain is of unknown function. PDI is one of a family of  20 PDI-like proteins identified in the ER [1]. These proteins contain one or more domains that are similar to the domains of PDI, and many have been shown to catalyze thiol–disulfide exchange reactions. However, their specific roles, substrate specificities and mechanisms of cooperation with other catalysts and chaperones in the cell are not yet clear. Besides PDI being abundant in the ER, several stud- ies have shown non-ER locations for PDI family mem- bers [5]. PDI inhibitors and specific antibodies have often been used to discover the function of PDI-like proteins, especially outside the ER. Bacitracin is a commonly used inhibitor in these studies, and it is usu- ally considered to be a specific inhibitor of PDI activ- ity [6–8]. However, in vitro evidence for the action of bacitracin as an inhibitor of PDI is scarce, and evi- dence of its specificity for PDI is nonexistent. Bacitracin is also used medicinally to prevent infections in small cuts and burns and to treat gastrointestinal infections. In addition, it is used as an animal feed additive for disease prevention and growth promotion in farm animals. For all of these functions, the effects are unrelated to PDI inhibition. Commercially available Keywords bacitracin; chaperone; protein disulfide isomerase; protein folding; thiol–disulfide exchange Correspondence L. W. Ruddock, University of Oulu, Department of Biochemistry, PO Box 3000, University of Oulu, Oulu 90014, Finland Fax: +358 8 5531141 Tel: +358 8 5531683 E-mail: lloyd.ruddock@oulu.fi (Received 12 August 2009, revised 3 March 2010, accepted 19 March 2010) doi:10.1111/j.1742-4658.2010.07660.x To successfully dissect molecular pathways in vivo, there is often a need to use specific inhibitors. Bacitracin is very widely used as an inhibitor of pro- tein disulfide isomerase (PDI) in vivo. However, the specificity of action of an inhibitor for a protein-folding catalyst cannot be determined in vivo. Furthermore, in vitro evidence for the specificity of bacitracin for PDI is scarce, and the mechanism of inhibition is unknown. Here, we present in vitro data showing that 1 mm bacitracin has no significant effect on the ability of PDI to introduce or isomerize disulfide bonds in a folding protein or on its ability to act as a chaperone. Where bacitracin has an effect on PDI activity, the effect is relatively minor and appears to be via competition of substrate binding. Whereas 1 mm bacitracin has minimal effects on PDI, it has significant effects on both noncatalyzed protein folding and on other molecular chaperones. These results suggest that the use of bacitracin as a specific inhibitor of PDI in cellular systems requires urgent re-evaluation. Abbreviations BPTI, bovine pancreatic trypsin inhibitor; CM, carboxymethyl; ER, endoplasmic reticulum; PDI, protein disulfide isomerase. 2454 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS bacitracin contains at least nine different peptides, of which bacitracin A is the most abundant, and it is mainly used as an antibiotic against infections caused by Gram-positive bacteria [9]. The antibiotic effect is based on the inhibition of bacterial cell wall synthesis by a variety of mechanisms. Bacitracin has been used as a specific PDI inhibitor in a very wide range of studies. These include studying the mechanisms of virus entry [10–12], the reductive activa- tion of diphtheria and cholera toxins [7,13], gamete fusion [14], platelet adhesion [15,16], melanoma cell death [17], glioma cell invasion [18], the regulation of transcriptional activity of nuclear factor kappaB [19], the regulation of NAD(P)H oxidase [20], the shedding of human thyrotropin receptor ectodomain [21], the aggregation of Cu ⁄ Zn superoxide dismutase in motor neurons [22], the operation of the vitamin K cycle [23], protection against stroke [24] and thrombus formation [25], and the functions of coagulation factor XIII [26] and tissue factor [27–29]. Although it is very commonly used in cell biological studies, the mechanism of PDI inhibition by bacitracin is unknown. We have recently speculated that inhibition could arise because of one of two effects [30]. First, bacitracin could inhibit PDI by competing with substrate binding, especially by compet- ing for the substrate-binding site on the b¢ domain. Sec- ond, PDI activity could be inhibited by the metal ions that bacitracin is known to bind. These metal ions could be coordinated by the active site cysteines of the catalytic domains of PDI, decreasing their activity. In addition, other thiols present in the studied system could bind metal ions, and their reactivity could be changed. It has also been shown that some commercially available baci- tracin preparations contain proteases, which could also explain some of the inhibitory effects against PDI [31]. In the present study, we studied the effect of bacitra- cin on PDI activity in a variety of in vitro assays. Our results show that 1 mm bacitracin can partially inhibit the reductive activity of PDI, but it has no significant influence on other in vitro functions of PDI. However, bacitracin has effects on other proteins involved in protein folding and on noncatalyzed systems, with the effects on these systems being larger than the maximal effect seen on PDI. Hence, we propose that bacitracin should not be regarded as a specific inhibitor of PDI. Results Bacitracin does not inhibit the catalysis of disulfide bond formation and isomerization by PDI PDI is a catalyst of thiol–disulfide exchange reactions, including oxidation, reduction and isomerization [1]. The simplest in vitro assays for catalysis of thiol–disul- fide exchange are based on small peptides. To examine whether bacitracin is able to inhibit the ability of PDI to introduce disulfide bonds into a substrate in the absence of the concomitant formation of secondary structure, a fluorescent decapeptide PDI substrate [32] was used. In a glutathione buffer at pH 7.0, a time- dependent decrease in fluorescence was observed that could be fitted to a first-order process (Fig. 1A), con- sistent with the formation of a disulfide bond in the substrate [32]. The rate constant for oxidation of 3.4 lm peptide in the presence of 0.7 lm PDI was 0.85 ± 0.05 min )1 (n = 6). Bacitracin contains a mix- ture of peptides, with the most abundant, bacitracin A, containing an aromatic phenylalanine moiety. Hence, at 1 mm, there are two opposing effects on the fluores- cence of the system in the presence of bacitracin. First, there is a net increase in fluorescence due to the baci- tracin. However, with excitation at 280 nm and emis- sion at 350 nm, bacitracin is much less fluorescent on a per molar basis than the PDI peptide substrate, which contains a tryptophan. Second, there is a net decrease in the fluorescence due to the inner filter effect, whereupon if the sample absorbs strongly at the excitation and ⁄ or emission wavelength, the fluores- cence signal decreases. However, this effect was mini- mized by using a cuvette with an excitation pathlength of 4 mm. Because of these opposing effects, the fluo- rescence of the peptide is quenched, and it contributes a smaller proportion of the total fluorescence of the system. However, in the presence of 1 mm bacitracin, the catalyzed formation of a disulfide bond in the decapeptide PDI substrate can still be observed through a decrease in its fluorescence, and this could be fitted to a first-order process (Fig. 1A). The rate constant for PDI-catalyzed oxidation of the peptide substrate with 1 mm bacitracin present was 0.73 ± 0.09 min )1 (n = 6), or 86% ± 11% of that in the absence of bacitracin. Higher concentrations of bacitracin could not be used, owing to the two effects outlined above, but these results suggest that bacitracin has minimal effects on the catalysis of oxidation by PDI. The ability of PDI to introduce and isomerize disul- fide bonds can be also be analyzed in folding proteins, e.g. in the bovine pancreatic trypsin inhibitor (BPTI) refolding assay. BPTI is a widely studied protein con- taining three disulfides in the native form. In a gluta- thione-based refolding buffer, BPTI becomes kinetically trapped in states containing two disulfide bonds (2S), and in order to reach the native 3S state, BPTI has to undergo isomerization reactions. Noncat- alyzed glutathione-based refolding of BPTI is slow, A R. Karala and L. W. Ruddock Bacitracin is not specific for PDI FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS 2455 with only around one-quarter of the BPTI being able to achieve the native 3S state within 2 h [33]. However, all the steps of BPTI refolding are catalyzed by PDI, and within 40 min BPTI was refolded to 94% ± 3% native 3S form (Fig. 1B). When 1 mm bacitracin is added to the PDI-catalyzed BPTI refolding system, the MS analysis becomes significantly less accurate, so an additional step to remove excess bacitracin after quenching of the reaction but prior to analysis is required. With this, the refolding of BPTI followed very similar kinetics in the presence or absence of baci- tracin, and after 40 min of refolding with 1 mm baci- tracin present, 90% ± 5% of BPTI was in the native 3S state (Fig. 1C). These results imply that bacitracin does not inhibit the ability of PDI to introduce or isomerize disulfide bonds in a folding protein. Bacitracin inhibits rhodanese aggregation and the chaperone activity of BiP In addition to disulfide bond formation, PDI has been shown to have chaperone-like activity [34]. As rhoda- nese contains no disulfide bonds, and is prone to aggre- gation during refolding, it can be used as a model with which to study chaperone activity in folding. Analysis of the nonassisted refolding of rhodanese showed the expected aggregation of the folding intermediates. The addition of PDI or the noncatalytic PDI family member ERp27 to the refolding system decreased the aggrega- tion rate (Table 1), with ERp27 showing a greater effect (29% decrease in aggregation rate) than PDI (18% decrease in rate). When 1mm bacitracin was added to the PDI-catalyzed reaction the rate of aggregation of rhodanese was significantly reduced. This is unexpected as inhibition of PDI activity would be expected to increase the rate of aggregation. However, the rate of aggregation in the noncatalyzed refolding of rhodanese was also decreased by bacitracin. The decrease in the noncatalyzed rate (31%) was similar to that of the PDI-catalyzed reaction (a 32% decrease). These results Fig. 1. Bacitracin has minimal effects on the oxidation and isomeri- zation reactions of PDI. (A) Representative traces showing the fluo- rescence change associated with oxidation of the PDI substrate peptide NRCSQGSCWN in a glutathione-based buffer at pH 7.0. The upper trace shows the PDI-catalyzed reaction, and the lower trace the PDI-catalyzed reaction in the presence of 1 m M bacitracin. The lines of best fit are to first-order reactions. (B, C) Time course analysis of the oxidative refolding of BPTI. The refolding experi- ments were performed in a glutathione-based buffer at pH 7.0. The relative amounts of the folding species were analyzed by ESI-MS. (B) Representative trace for BPTI refolding in the presence of PDI. (C) Representative trace for BPTI refolding in the presence of PDI and 1 m M bacitracin. For clarity, the glutathionylated intermediates are not shown separately. The sum of all glutathionylated interme- diates never represents more than 10% of the total protein at any time point. Table 1. Analysis of the aggregation rate during rhodanese refold- ing at pH 7.2. The rate of aggregation relative to the negative con- trol in the absence of bacitracin is presented as mean ± standard deviation (number of samples). Statistical significance between each pair of samples with and without bacitracin present was determined using Student’s t-test (two-tailed, two-sample unequal variance). Note that the effects of bacitracin on PDI and ERp27 inhibition of aggregation are equivalent to those on the noncata- lyzed reaction. Sample No bacitracin 1m M bacitracin t-test for an effect of bacitracin Negative control 100 ± 19 (8) 69 ± 9 (8) P < 0.05 +4.5 l M PDI 82 ± 12 (6) 56 ± 15 (5) P < 0.05 +4.5 l M BiP 6 ± 11 (6) 18 ± 6 (5) P < 0.05 +4.5 l M ERp27 71 ± 11 (4) 54 ± 2 (3) P < 0.05 Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock 2456 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS imply that bacitracin interacts with rhodanese, decreas- ing the aggregation of its folding intermediates, and that it has no observable effect on the chaperone activity of PDI family members. In parallel studies, the aggrega- tion rate of rhodanese was reduced, on average, by 95% by the addition of the ER-resident molecular chaperone BiP (Table 1). However, the addition of 1 mm bacitra- cin to the BiP-assisted refolding reaction did not decrease the aggregation rate, as would have been expected from the previous results, but increased the rate of aggregation. This implies that bacitracin signifi- cantly inhibits the chaperone activity of BiP. Bacitracin inhibits the reductive activity of PDI by competing with substrate binding As well as having oxidase, isomerase and chaperone-like activity, PDI is also able to catalyze the reduction of disulfide bonds. The effects of bacitracin on this activity were examined using the insulin precipitation assay. In the noncatalyzed assay, the disulfides of insulin are reduced by dithiothreitol, causing the aggregation and precipitation of the B-chain of insulin, resulting in an increase in light scattering that can be monitored by increased absorbance, e.g. at 540 nm (Fig. 2A). Like many in vitro PDI assays, the assay is indirect, with complex kinetics. For this reason, we decided to mea- sure the lag-phase of the reaction, i.e. the time before an apparent increase in absorbance of 0.1 was recorded. The addition of PDI to the assay accelerated the reduc- tion and precipitation of the B-chain significantly, decreasing the lag-phase (Fig. 2A). When bacitracin was included with PDI, the lag-phase of the insulin precipita- tion increased (Fig. 2A), with the effects increasing with increasing concentration of bacitracin (Fig. 2B). With Student’s t-test (two-tailed, two-sample unequal vari- ance), this effect was found to be significant (P < 0.05), even with the addition of 0.1 mm bacitracin. Unlike in the rhodanese assay, in this assay 1 mm bacitracin had no significant effect on the lag-phase of the reaction or on the subsequent gradient for aggregation (Fig. 2A), implying that the effects of bacitracin addition observed on PDI were due directly to inhibition of PDI-catalyzed insulin reduction. Fig. 2. Effects of bacitracin and other compounds on the relative rate of reduction of the B-chain of bovine insulin. Insulin was reduced at 1mgÆmL )1 in the presence of 10 mM dithiothreitol and 1 mM EDTA at pH 7. When present, PDI, PDI a domain (PDIa), DsbA and DsbC were used at 1 l M. Bacitracin (Bac) was used at 1 mM, if not indicated otherwise in the figure. Triton X-100 (TX) was used at 0.05% (v ⁄ v) and 2- propylphenol (2PP) at 1 m M. The reduction of the B-chain of insulin causes precipitation that can be followed as an absorbance increase at 540 nm. (A) Representative changes in absorbance as a function of time. From left to right, the traces are: PDI, PDI + bacitracin, noncata- lyzed reaction, noncatalyzed reaction + bacitracin. (B–D) Lag times for precipitation of insulin under different conditions. The relative activity is presented as mean ± standard deviation; n = 2–7, with the value given in parentheses. (B) With PDI present. (C) Noncatalyzed reactions. (D) With PDI a domain, DsbA or DsbC present. A R. Karala and L. W. Ruddock Bacitracin is not specific for PDI FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS 2457 To study the potential mechanism of action of baci- tracin, the insulin reduction assay was also performed in the presence of other two other thiol–disulfide exchange enzymes, Escherichia coli DsbA and DsbC, as well as the isolated catalytic a domain of PDI. Both the PDI a domain and DsbA have a catalytic site, with an associated substrate-binding site, but lack an inde- pendent substrate-binding site, which is present in full- length PDI [4,35] and DsbC [36]. DsbC substantially increased the rate of insulin reduction, although to a lower level than that of PDI, and as with PDI, this catalysis was inhibited by the presence of bacitracin (Fig. 2D; P < 0.1). In contrast, DsbA and the PDI a domain exhibited significantly lower rates of cataly- sis, and neither showed any change in activity upon the addition of bacitracin (P > 0.5). For further study of the potential mechanism of action, the reaction conditions of the assay were var- ied. The removal of EDTA from the reaction signifi- cantly reduced the noncatalyzed rate (Fig. 2C; P < 0.05) and the catalysis of insulin reduction ⁄ aggre- gation by PDI (Fig. 2B; P < 0.05), presumably owing to the presence of trace amounts of metal ions in the buffer that would be able to coordinate to thiol groups (for example, see [37] for the effects of zinc on PDI activity). However, the effects of removal of EDTA and addition of bacitracin appeared to be independent of each other (Fig. 2B), implying that any metal ions bound by bacitracin and taken into the reaction are not causing the inhibition. It has previously been shown that PDI binds its sub- strates via hydrophobic interactions, and that substrate binding to the noncatalytic b¢ domain can be inhibited by low molecular mass compounds such as Triton X-100 and 2-propylphenol [3,38]. To study the effect of inhibitors of substrate binding on the reductive activity of PDI, the precipitation of insulin was followed in the presence of PDI and 0.05% (v ⁄ v) Triton X-100 or 1 mm 2-propylphenol. In the noncata- lyzed reaction, the addition of Triton X-100 had a minimal effect on the system, whereas the addition of 2-propylphenol increased the rate of insulin aggre- gation (Fig. 2C; P < 0.05). In contrast, the addition of either Triton X-100 or 2-propylphenol decreased the rate of PDI-catalyzed insulin aggregation (Fig. 2B; P < 0.05), showing that inhibition of the primary substrate-binding site in the b¢ domain decreases the insulin-reducing activity of PDI. Discussion Inhibitors are widely used to study the physiological functions of proteins in vivo. Bacitracin is a metallo- peptide antibiotic that has been widely used as a spe- cific PDI inhibitor [7,10–29]. However, neither the specificity of bacitracin for PDI nor the detailed mech- anisms of inhibition of PDI have been investigated. Furthermore, since the original reporting of the inhibi- tion of PDI by bacitracin [8], concerns have been raised about protease contamination of some commer- cially available bacitracin preparations [31]. Here, we have tested the effect of bacitracin in a variety of in vitro assays for PDI activity. On the basis of the BPTI refolding assay and peptide oxidation assay 1 mm bacitracin does not have a significant effect on the oxidative or isomerization activity of PDI. In addition, the chaperone activity of PDI in the rhodanese-refolding assay was not significantly chan- ged in the presence of bacitracin. In the insulin reduc- tion assay, bacitracin was able to decrease the activity of PDI in a concentration-dependent manner, but this effect was small, such that, in the presence of 1 mm bacitracin with 1 lm PDI, there was a decrease in the contribution of PDI to the lag-phase of the reaction by 30%. It is unclear why bacitracin had a significant effect on PDI activity in only one of the four assays examined. However, three of these assays are indirect measures of complex multistep kinetic processes. Fur- thermore, PDI has two active sites with concomitant substrate-binding ability and an additional substrate- binding site located in a noncatalytic domain [1], and the relative contributions of these to each of the assays is unclear. It should also be noted that the starting states of the proteins ⁄ peptides are very different, with insulin starting in the disulfide-linked folded state, and rhodanese and BPTI starting in the reduced and unfolded state. Despite this complication, these results imply that bacitracin, even at high concentrations, is ineffective at inhibiting the majority of the functions of PDI usually analyzed in vitro and that are thought to be representative of its in vivo functions [1]. Although 1 mm bacitracin had only a minor effect on PDI, it had more significant effects on other com- ponents in our assays. In the noncatalyzed refolding of rhodanese, bacitracin alone was able to decrease the aggregation of the folding intermediates more signifi- cantly than PDI itself. In addition, bacitracin signifi- cantly reduced the chaperone activity of BiP in this assay. Furthermore, in the insulin reduction assay, bacitracin inhibited the activity of DsbC. These results strongly imply that bacitracin is not a selective inhibi- tor of PDI. Instead, bacitracin can also interact with folding polypeptide chains and other molecular chaper- ones and folding catalysts. Bacitracin probably inter- acts with these, and with PDI, via its hydrophobic side chains, which could interact with exposed hydrophobic Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock 2458 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS side chains of a folding protein or with the hydropho- bic binding site of molecular chaperones such as BiP, PDI and DsbC. To further study the mechanism of action of bacitra- cin, the reduction of insulin was analyzed in the pres- ence of the PDI a domain and DsbA, which are capable of catalyzing the oxidation and reduction reac- tions, but lack the independent substrate-binding site that is present in PDI and DsbC. Bacitracin had no effect on the activity of the PDI a domain or DsbA, implying that the active site is probably not the target of inhibition by bacitracin. This result was confirmed by assays in which EDTA was omitted from the assay. Molecules that are known to interfere with substrate binding by PDI were also used in the insulin reduction assay. Triton X-100 at 0.05% (v ⁄ v) (equivalent to 0.8 mm), which is known to affect substrate binding by the noncatalytic b¢ domain [3], reduced PDI activity in the assay to a slightly greater extent than 1 mm bacitracin. Although pathways for cellular metabolism are unknown, and it is possible that in vivo processing of bacitracin may yield a product that can inhibit PDI activity, there is no published evidence for the in vivo specificity of bacitracin (or potential products) for PDI inhibition. Our results clearly show that bacitracin in vitro is not a specific inhibitor of PDI, but that it interacts with many other molecules present in the cell, including nonfolded proteins and other molecular chaperones, probably via hydrophobic interactions. As bacitracin, a peptide with a molecular mass of  1400 Da, is often used at millimolar concentrations to inhibit PDI in vivo, the risk of nonspecific effects on other systems increases even more. Furthermore, the mechanism of action of bacitracin is not very effective in inhibiting PDI in vitro, even at millimolar concen- trations, with only a 30% reduction in the insulin reduction assay and no significant changes in the ability of PDI to introduce or isomerize disulfide bonds or to act as a chaperone. Hence, the use of bacitracin as a specific inhibitor for studying the role and function of PDI in cellular systems requires urgent re-evaluation. Experimental procedures Bacitracin The bacitracin used in this study was from Sigma-Aldrich (Steinheim, Germany). Although there have been reports that there is protease contamination of some commercially available bacitracin preparations [31], we were loathe to fractionate the material to ensure that there was no prote- ase contamination, as bacitracin is a complex mixture of peptides, and we did not want to lose a potentially inhibi- tory subpopulation. To confirm that there was no signifi- cant protease activity in the material that we used, reduced, denatured BPTI was incubated with 1 mm bacitracin for 1 h at room temperature in 0.1 m sodium phosphate buffer (pH 7.0) containing 1 mm EDTA. Analysis by SDS ⁄ PAGE showed no evidence of degradation of the denatured BPTI over this time period. In addition, ESI-MS analysis of BPTI refolding (see below) showed no evidence of BPTI degrada- tion products. Generation of expression vectors N-terminally histidine-tagged mature PDI, PDI a domain and mature BPTI with an additional initiating methionine expression vector were generated for previous studies [39,40]. Mature human BiP (Glu19–Leu653) was generated by PCR from a human liver cDNA library (Clontech, Mountain View, CA, USA) in two parts. BiP Glu19– Arg323 was constructed as an NdeI–SacI fragment, and BiP Ala324–Leu653 as a SacI–XhoI fragment. Mature human ERp27 (Glu26–Leu273) was generated by PCR from IMAGE clone 5207225 as an NdeI–BamHI fragment. Mature E. coli DsbA (Ala20–Leu208) and mature E. coli DsbC (Asp21–Lys236) were constructed as NdeI–BamHI fragments by PCR from an E. coli colony. All inserts were cloned into a modified pET23b (Novagen, Madison, WI, USA), which codes for an N-terminal hexahistidine tag before the first amino acid of the protein sequence. Protein expression and purification PDI (EC 5.3.4.1; UniProt ID P07237), PDI a domain, BiP (EC 3.6.4.10; UniProt ID P11021), ERp27 (Uni- Prot ID Q96DN0), DsbA (UniProt ID P0AEG4) and DsbC (UniProt ID P0AEG6) were expressed in E. coli strain BL21(DE3) pLysS grown in LB medium at 37 °C and induced at a D 600 nm of 0.3 for 3 h with 1 mm isopro- pyl thio-b-d-galactoside. Lysis of bacteria was performed by freeze–thawing the samples twice. PDI, PDI a domain, BiP and DsbA were purified by immobilized metal affinity chromatography and anion exchange chromatography, as previously described for PDIs from Caenorhabditis elegans [40], except that for PDI a domain and DsbA, the anion exchange column was run in 20 mm Tris buffer (pH 8.6) instead of 20 mm sodium phosphate buffer (pH 7.2). DsbC was purified in the same way as PDIs from C. elegans [40], except that DsbC was applied to a Resource S (Amersham Biosciences, Uppsala, Sweden) cation exchanger instead of a Resource Q anion exchanger, and the column was run in 20 mm citric acid trisodium buffer (pH 5.5). ERp27 was purified in the same way as PDIs from C. elegans [40], except that ERp27 was eluted from the anion exchange column with a tripartite gradient (0–45% over one column volume, 45–70% over seven column volumes, and 60–100% A R. Karala and L. W. Ruddock Bacitracin is not specific for PDI FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS 2459 over two column volumes). BPTI (UniProt ID P00974) was expressed and purified as described previously [40]. Pure reduced BPTI was lyophilized and resuspended in 10 mm HCl (pH 2.0) to prevent oxidative refolding. The concentrations of proteins were determined spectrophoto- metrically, using a calculated absorption at 280 nm (PDI, 45 040 m )1 Æcm )1 , M r = 56386; BPTI, 5680 m )1 Æcm )1 , M r = 6648; BiP, 29 660 m )1 Æcm )1 , M r = 71 356; ERp27, 18 450 m )1 Æcm )1 , M r = 28 837; DsbA, 22 560 m )1 Æcm )1 , M r = 22 217; DsbC, 16 960 m )1 Æcm )1 , M r = 24 545). All proteins were stored in aliquots at – 20 °C. All purified proteins were analyzed for authenticity by MS, and all experimentally determined masses were the same as the expected masses (within the mass accuracy limit of the spectrometer). Peptide oxidase assay The method of Ruddock et al. [32], using a fluorescent decapeptide, was used to determine the oxidase activity of PDI. In brief, disulfide bond formation (oxidation) is moni- tored by the quenching of the intrinsic fluorescence of the single tryptophan in the peptide as the arginine is brought into close proximity upon disulfide formation. McIlvaine buffer (0.2 m disodium hydrogen phosphate, 0.1 m citric acid, pH 7.0), to give a final assay volume of 1 mL, was placed in a fluorescence cuvette. Except where noted in the text, to this was added 10 lL of oxidized glutathione (50 mm stock solution in 20 m m sodium phosphate buffer, pH 7.2), 20 lL of reduced glutathione (100 mm stock solu- tion in 20 mm sodium phosphate buffer, pH 7.2) and 0.7 lm enzyme. After mixing, the cuvette was placed in a Perkin-Elmer LS50 spectrophotometer for 5 min to allow thermal equilibration of the solution; 6.3 lL of substrate peptide (539 lm stock solution in 30% acetonitrile ⁄ 0.1% trifluoroacetic acid) was added and mixed, and the change in fluorescence intensity (excitation at 280 nm, emission at 350 nm, slits of 5 ⁄ 5 nm) was monitored over an appropri- ate time period (15 min–1 h), with 600–1800 data points being collected. Refolding of reduced and denatured BPTI A modified version of the methods that we have previously reported [40] was used to analyze BPTI refolding. In partic- ular, this method has additional steps to remove excess bac- itracin prior to the MS analysis. The refolding of BPTI was initiated by the addition of denatured reduced protein to the refolding buffer (2 mm reduced glutathione, 0.5 mm oxidized glutathione, 0.1 m sodium phosphate, 1 mm EDTA, pH 7.0). In the catalyzed refolding, PDI was pre- equilibrated in the refolding buffer for 5 min before BPTI was added. BPTI was used at 50 lm and, when present in the refolding reaction, PDI was used at 7 lm and bacitracin at 1 mm. The folding reaction was stopped by the addition of 1.1 m iodoacetamide (Sigma-Aldrich), and BPTI and its folding intermediates were purified with a PepClean C-18 spin column (Pierce, Rockford, IL, USA) before ESI-MS analysis (Micromass, Manchester, UK). Bacitracin-contain- ing BPTI samples were additionally purified by cation exchange chromatography and with a PepClean C-18 spin column. The cation exchange resin carboxymethyl (CM) cellulose 32 (Whatman, Maidstone, UK) was first pretreat- ed by suspending 3 g of resin in 30 mL of 0.5 m sodium hydroxide and stirring for 30 min. The cellulose was then allowed to settle, and washed with double-distilled water. After being washed with water, the cellulose was washed with 0.5 m HCl for 30 min, and then with double-distilled water until neutral pH was achieved. Before use, the cellu- lose was washed with 10 mm EDTA to remove metal ions. The eluants from the PepClean C-18 spin column were diluted nine-fold with equilibration buffer (50 mm Tris buf- fer, pH 8) before being mixed with pre-equilibrated CM cel- lulose (250 lL) and stirred for 30 min. The unbound sample was discarded by centrifugation (1500 g for 1 min). After washing of the CM cellulose three times with the equilibration buffer, BPTI and its folding intermediates were eluted with elution buffer (50 mm Tris, 1 m NaCl, pH 8). Before the ESI-MS analysis, eluted proteins were purified with a PepClean C-18 spin column. The kinetic traces with and without bacitracin were repeated in tripli- cate. It should be noted that different species may bias their detection by ESI-MS, and the results are therefore only semiquantitative. Inhibition of aggregation of denatured rhodanese The molecular chaperone-like activity of PDI was moni- tored using a slightly modified version of the rhodanese assay used by Song and Wang [34]. Rhodanese from bovine liver (Sigma-Aldrich) was denatured to a final concentra- tion of 45 lm in 0.2 m sodium phosphate buffer (pH 7.2) containing 6 m guanidine hydrochloride and 10 mm dith- iothreitol for 45 min at room temperature. The refolding was started by diluting denatured rhodanese to a final con- centration of 0.9 lm in the refolding buffer (0.1 m sodium phosphate, pH 7.2, 5 mm dithiothreitol, 50 mm sodium thiosulfate). The aggregation of rhodanese during refolding was followed spectrophotometrically at 320 nm over 5 min. PDI, BiP and ERp27 were used at 4.5 lm, and bacitracin at 1 mm, when present. ATP was used at 2 mm when BiP was present. Proteins were equilibrated in the refolding buffer for 3 min before the addition of rhodanese. Insulin precipitation assay A modified version of the insulin turbidity assay reported by Holmgren [41] was used. The precipitation reaction of the B-chain of bovine insulin (Sigma-Aldrich) was initiated by adding the insulin to 0.1 m sodium phosphate buffer Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock 2460 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS (pH 7.0) containing 1 mm EDTA and 10 mm dithiothreitol. 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J Biol Chem 254 , 9627–9632. Bacitracin is not specific for PDI A R. Karala and L. W. Ruddock 2462 FEBS Journal 277 (2010) 2454–2462 ª 2010 The Authors Journal compilation ª 2010 FEBS . Bacitracin is not a specific inhibitor of protein disulfide isomerase Anna-Riikka Karala and Lloyd W. Ruddock Biocenter Oulu and Department of Biochemistry,. in familial amyotrophic lateral sclerosis and association of protein- disulfide isomerase with A R. Karala and L. W. Ruddock Bacitracin is not specific for