- Email: [email protected]
[38] Protein disulfide-isomerase
[381
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fraction. Figure 2 shows the time course of incorporation of [3HIE-64 into serum and cytosolic and mitochondrial/lysosomal fractions of the rat liver. The radioactivity of the blood increases rapidly after the injection, reaching a maximum within 30-60 min, and then decreasing rapidly. Incorporation of the radioactivity into the cytosolic fraction in liver starts slightly later than that of the blood and then also decreases rapidly. By contrast, the radioactivity appears in the particulate fractions within 1 hr, retains the maximum plateau for 5-6 hr, and then decreases gradually over 12 hr. Figure 3 shows the correlation between cathepsin B activities and protein-bound [3H]E-64 in the mitochondrial/lysosomal fractions of liver. The highest radioactivity is found in the lysosomal fraction and the distribution of the radioactivities is the same as that of the lysosomal marker enzymes, such as cathepsin B and acid phosphatase. If protein-bound radioactivities in the lysosomal fraction represent E-64-sensitive cysteine proteases, there should be a reciprocal relationship between the inhibition of the activities of cathepsin B, a representative lysosomal cysteine protease, and the proteinbound radioactivities in the fraction. Inhibition of cathepsin B and the radioactivities of [3H]E-64 reach maximum levels within 1 hr after injection of [3H]E-64 and maintain the maximum level of the reciprocal relationship between these two activities, as shown in Fig. 3. Therefore, E-64 and CA-074 are incorporated into the liver cytosol in the free form. They permeate into the lysosomes, where they bind to and effectively inactivate the target cysteine proteases.
[38]
Protein Disulfide-Isomerase
By ROBERT B. FREEDMAN,HILARY C. HAWKINS,and STEPHEN
U.
McLAUGHLIN
Introduction Protein disulfide-isomerase (PDI, EC 5.3.4.1) is an abundant protein within the lumen of the endoplasmic reticulum of secretory cells, and functions as a catalyst in the formation of native disulfide bonds in nascent secretory and cell surface proteins. In its catalytic action in vitro, it facilitates the folding and assembly of a wide range of disulfide-bonded proteins, and individual thiol-disulfide interchange steps are accelerated by over 1000fold. The role of PDI in co- and posttranslational modification of proteins has been confirmed by its cross-linking to nascent immunoglobulins, by the requirement for PDI for efficient cotranslational disulfide formation in a METHODS IN ENZYMOLOGY, VOL. 251
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
398
PROTEIN THIOLS AND SULFIDES
[38]
reconstituted in vitro translation system, and by the phenotype of yeast lacking a functional PDI. In vertebrates, the protein is also a component of two other endoplasmic reticulum (ER) lumenal enzyme systems, prolyl4-hydroxylase and the microsomal triglyceride transfer protein. Chemical modification data indicated that PDI functions in thiol-disulfide interchange reactions through dithiol/disulfide active site groups that are in the disulfide form in the isolated enzyme. This was confirmed by sequencing of the enzyme, which indicated that it is a member of the thioredoxin superfamily. Protein disulfide-isomerase sequences now available from vertebrates, higher plants, and yeast all indicate a protein of approximately 500 residues with 2 regions homologous to thioredoxin, including a conserved active site motif (WCGHCK). The PDI from bacteria, generally known as DsbA, is considerably smaller and is a more remote member of the thioredoxin superfamily. The tertiary structure of DsbA has been determined, but not that of any eukaryotic PDI. The properties, purification, and assay of PD! were reviewed in a previous volume of this series. 1 More recent reviews2-6 have focused on its structural and functional properties.
Purification of Protein Disulfide-Isomerase from Bovine Liver Protein disulfide-isomerase cDNA sequences are known from a range of organisms, but the protein has been purified and characterized to any extent only from mammalian liver and yeast (Saccharomyces cerevisiae). The reported purification of yeast PDI yielded only 2 mg of purified enzyme from 1 kg of yeast cell pellet, 7 and most of the enzymatic characterization of PDI has been carried out on material purified from mammalian liver; this is therefore the focus of the present chapter. The first high-yielding purification of the enzyme8 used detergent solubilization of whole homogenate, avoiding losses incurred by subcellular fractionation to produce microsomes.9 The procedure modified by Lambert 1 D. A. Hillson, N. Lambert, and R. B. Freedman, this series, Vol. 107, p. 281. z R. B. Freedman, Cell (Cambridge, Mass.) 57, 1069 (1989). 3 R. B. Freedman, N. J. Bulleid, H. C. Hawkins, and J. L. Paver, Biochem. Soc. Syrup. 55, 167 (1989). 4 R. Noiva and W. J. Lennarz, J. Biol. Chem. 267, 3553 (1992). 5 T. E. Creighton and R. B. Freedman, Curr. Biol. 3, 790 (1993). 6 R. B. Freedman, T. R. Hirst, and M. F. Tuite, Trends Biochem. Sci. 19, 331 (1994). 7 T. Mizunaga, Y. Katakura, T. Miura, and Y. Maruyama, J. Biochem. (Tokyo) 108, 846 (1990). s D. E. Carmichael, J. E. Morin, and J. E. Dixon, J. Biol. Chem. 252, 7163 (1977). 9 p. j. E Rowling, S. H. McLaughlin, G. S. Pollock, and R. B. Freedman, Protein Expression Purif 5, 331 (1994).
[38]
PROTEIN DISULFIDE-ISOMERASE
399
and Freedman 1° utilizes the stability of the enzyme at 54 °, at which temperature only 20% of total activity is lost, and its very low p/, which facilitates purification by ion-exchange chromatography. This method was described previously in this series (1), but has now been updated using modern chromatographic material and FPLC (fast protein liquid chromatography), which greatly reduces the time for purification (see Fig. 1).
Procedure Homogenization in Triton. Bovine liver from freshly slaughtered animals is freed of connective tissue and stored at - 2 0 ° in 500-g aliquots. On demand 500 g is taken from a - 2 0 ° freezer and cut into small pieces (approximately 1-cm cubes) while it is still frozen. These pieces are washed in ice-cold saline [0.9% (w/v) NaC1]. The liver is finely chopped and added to a precooled Waring blender in the ratio of 1 vol of liver to 2 vol of homogenization buffer [1% (w/v) Triton X-100, 0.1 M sodium phosphate buffer-5 m M EDTA, pH 7.5, containing 1 m M phenylmethylsulfonyl fluoride (PMSF) and aprotinin (1/xg/ml)], homogenizing at full speed for four 30-sec bursts with 30-sec intervals. The homogenate is decanted into a beaker on ice and additional homogenization buffer is added to give a total volume of approximately 1.5 liters. The homogenate is filtered through a double layer of muslin and then centrifuged at 18,000 g for 30 min at 4°; the pellet is discarded. Heat Treatment. The supernatant is filtered through glass wool to remove floating fat and then transferred to a 70 ° water bath with constant stirring until the temperature reaches 54 °, which is maintained _+1° for 15 rain by alternately immersing the beaker in the bath and ice. The treated extract is transferred to an ice bath and cooled to <10 ° with stirring; it is then centrifuged at 18,000 g for 40 min at 4°. All subsequent steps are carried out at 4 °. Ammonium Sulfate Fractionation. Solid ammonium sulfate is gradually added to the supernatant with stirring to give a final saturation of 55% at 0 °. After stirring for a further 30 min, the material is centrifuged at 38,000 g for 30 rain at 4 ° and the supernatant decanted through fluted Whatman (Clifton, N J ) No. 1 filter paper to remove floating fat. Further ammonium sulfate is added to increase the saturation to 93% at 0 °, with stirring for 30 min and centrifugation as before. The supernatant is carefully removed and discarded, and a total of 15-20 ml of 25 m M citrate buffer (pH 5.3) is used to dissolve the pellet, with the aid of a hand homogenizer. The resuspended pellet is dialyzed overnight against the citrate buffer (2 × 5 liters) at 4 °. 10N. Lambert and R. B. Freedman, Biochem. J. 213, 225 (1983).
Bovine Liver Wash in saline Homogenize in 1% Triton X-100, pH 7.5
Homogenate Strain through muslin Centrifuge at 18,000 g, 30 min
18,000 g Supematant Filter through gla~ wool Heat to 54 ° for 15 min Cool, centrifuge at 18,000 g, 40 rain
Heat-treated supematant Ammonium sulfate fractionation Dissolve in citrate buffer, pH 5.3 Dialyze vs citrate buffer, pH 5.3
55-93% (NI-I4)2SOfraction CM-Sepharose chromatography Pool void
CM-Se ~harose eluate Ammonium sulfate precipitation Dialyze vs 20 mM piperazine, pH 5.2 Q-Sepharose chromatography, pH 5.2
Q-Sepharose 0.35M NaC1 eluate Dialyze vs 20 mM piperazine, pH 5.2 Mono Q chromatography, pH 5.2 using linear gradient
Mono Q eluate Pool PDI containin~ fractious Dialyze and lyophilize Store -20°
Purified PDI FI~. 1. F l o w c h a r t for the p u r i f i c a t i o n of p r o t e i n d i s u l f i d e - i s o m e r a s e .
[38]
PROTEINDISULFIDE-ISOMERASE
401
Cation-Exchange Chromatography. The dialysate is filtered through a 0.2-~m pore size membrane filter and applied to a CM-Sepharose Fast Flow (5 x 25 cm) column and eluted with 25 mM citrate buffer (pH 5.3) at 5 ml min -I. Fractions (15 ml) are collected. Void peak fractions are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% resolving gel and PDI-containing fractions, identified by the known Mr of PDI, are pooled and protein precipitated by 100% (NH4)2SO4 saturation (69.70 g/100 ml) at 0° as described above, centrifuging at 38,000 g for 30 rain at 4°. The pellets are resuspended in a total of 10-20 ml of 20 mM piperazine, pH 5.2, and dialyzed against this buffer (2 × 5 liters) overnight. Alternatively, depending on its volume, the void can be dialyzed overnight and loaded directly onto the anionexchange column. FPLC Anion-Exchange Chromatography. The dialysate is filtered through a 0.2-tzm pore size membrane filter and loaded at 3 ml rain -1 onto a Q-Sepharose Fast Flow column (2.6 x 5 cm) preequilibrated in piperazine, pH 5.2; 10-ml fractions are collected. When unbound material has washed through, bound material is eluted with steps of 0.10, 0.35, and 1 M NaC1. Peak fractions are analyzed by SDS-PAGE on a 10% resolving gel. Fractions from the 0.35 M NaC1 wash containing the most pure PDI are pooled and dialyzed overnight against 20 mM piperazine, pH 5.2 (1 x 2 liters) to remove salt. The dialysate is loaded onto a Mono Q HR10/10 (8-ml) column, equilibrated with 20 mM piperazine, pH 5.2, at 1 ml min-L Bound material is eluted with a linear salt gradient from 0 to 1 M NaC1 over 160 ml, collecting 2-ml fractions. Peak fractions are analyzed by SDS-PAGE, using a 10% gel, and PDI-containing fractions are combined. These fractions are dialyzed against double-distilled H20 for a maximum of 2 hr to remove salt. The enzyme is freeze-dried and stored at - 2 0 ° and retains more than 70% of initial activity after 9 months of storage.
Molecular Properties of Purified Protein Disulfide-Isomerase All eukaryotic PDIs are proteins of approximately 500 amino acids, which in solution under physiological conditions are found as noncovalent homodimers. Mature bovine PDI comprises 490 residues, has a relative molecular mass based on composition of 55,165, migrates on SDS-PAGE with an Mr of 57,000, and has a pI of 4.5. The active site dithiol-disulfide groups in each thioredoxin-like domain show a remarkably oxidizing standard redox potential of - 110 mV, corresponding to an equilibrium constant with reduced and oxidized glutathione of 5 x 10 s M. In the dithiol form
402
PROTEIN THIOLSAND SULFIDES
[38]
of each active site, the m o r e reactive thiol group (the m o r e N-terminal of the pair) has a pKa of 6.7.
A s s a y of P r o t e i n D i s u l f i d e - I s o m e r a s e Protein disulfide-isomerase catalyzes thiol-disulfide interchange reactions in proteins: depending on the substrates and conditions it can catalyze net formation, net reduction, or net isomerization of protein disulfides. Some work has been carried out with tow molecular weight substrates and with model peptides, but assays based on proteins are most common. Protein disulfide-isomerase shows an extremely b r o a d protein substrate specificity and can therefore be assayed by a wide range of approaches. Net disulfide formation has b e e n most extensively studied using reduced bovine pancreatic ribonuclease or trypsin inhibitor (BPTI) as substraten-14; in the case of BPTI, conversions between individual disulfidebonded isomers can be resolved and their rates determined, which enables the catalytic properties of P D I to be defined in relation to specific disulfide formation or isomerization reactions. Net disulfide reduction has been most c o m m o n l y characterized as a G S H - d e p e n d e n t reduction of the disulfides of insulin (glutathione-insulin transhydrogenase).15 Net disulfide isomerization can also be assayed with insulin; in the presence of a low concentration of thiol, isomerization of insulin disulfides leads to an accumulation of insulin B chain polymers that aggregate, and the process can be monitored turbidometrically. But the most sensitive and versatile assay of isomerization is that based on reactivation of " s c r a m b l e d " ribonuclease, that is, on the recovery of enzymatic activity in a sample of ribonclease that has b e e n reduced and reoxidized under denaturing conditions. The substrate initially has little enzymatic activity, but this appears as disulfide isomerizations lead to the accumulation of ribonuclease molecules with native disulfide pairing. The appearance of ribonuclease activity can be assayed with nucleotides n or high molecular weight R N A , I'16 by spectrophotometric I'I1 or radiochemica116 approaches, and the assay of P D I can either be continuous, with 11M. M. Lyles and H. F. Gilbert, Biochemistry 30, 613 (1991). 12A. Zapun, T. E. Creighton, P. J. E. Rowling, and R. B. Freedman, Proteins: Struct. Funcr Genet. 14, 10 (1992). 1~j. S. Weissman and P. S. Kim, Nature (London) 365, 185 (1993). 14T. E. Creighton, C. J. Bagley, L. Cooper, N. J. Darby, R. B. Freedman, J. Kemmink, and A. Sheikh, J. Mol. Biol. 232, 1176 (1993). is N. Lambert and R. B. Freedman, Biochem. J. 213, 235 (1983). 16R. Myllyla and J. Oikarinen, J. Biochem. Biophys. Methods 7, 115 (1983).
[~8]
PROTEIN DISULFIDE-ISOMERASE
403
the ribonuclease substrates present in the isomerase assay system,n or discontinuous, with samples withdrawn for assay of ribonuclease activityJ
Preparation of Substrate Randomly reoxidized ("scrambled") ribonuclease (RNase) is prepared by reducing the four disulfide bonds in RNase and then allowing them to reoxidize under denaturing conditions. This complex undefined mixture of the 105 possible disulfide-bonded isomers is essentially inactive with <1% of the activity of native RNase. Preparations, using our published method, 17 varied in their properties as a substrate for PDI, owing to uncontrolled chemistry of reoxidation that was due in turn to differences in the quality of the distilled water used. In preliminary studies, controlled reoxidation in the presence of copper ions TM or diamide 19 was less successful than in the presence of selenite,2° which gave scrambled RNase with optimum properties of low Km and high Vmax and was reproducible. The following revised procedure includes selenite-controlled reoxidation and other modifications2° to make it more simple. Bovine pancreatic RNase is fully reduced under denaturing conditions by incubation at a concentration of 25-30 mg/ml in 50 mM Tris-HC1, pH 8.5, containing 10 M urea and 130 mM dithiothreitol (DTT), that is, an approximately 15-fold molar excess of DTT over protein disulfide bonds. Reduction is complete at room temperature overnight or at 37° for 1 hr. It is not necessary to flush the solution with nitrogen gas. Ultrapure urea should be used throughout and solutions made up as fresh as possible to reduce to a minimum the concentration of cyanate ions formed by isomerization of urea. 21 The solution is acidified to pH 4.0-4.5 with glacial acetic acid and then eluted on a desalting column in 0.1 M acetic acid containing 8 M urea to remove DTT alone. A small prepacked desalting column (approximately 10-ml column volume) is quick and convenient, and a large bulk preparation can be eluted in aliquots (2.5 ml, 60-75 mg). The rate of elution is fast even with 8M urea, and a rate of 1-2 ml/min with a simple siphon is maintained during subsequent elutions. Eluted fractions are monitored at 280 nm, protein-containing fractions pooled, and the overall concentration of the eluate determined (A280 = 0.55 for a solution of 1 mg/ml). Overall 17 H. C. Hawkins, E. C. Blackburn, and R. B. Freedman, Biochem. J. 275, 349 (1991). 18 A. K. Ahmed, S. W. Schaffer, and D. B. Wetlaufer, J. Biol. Chem. 250, 8477 (1975). 19N. S. Kosower and E. M. Kosower, this series, Vol. 143, p. 264. 2o j. Lundstrom, G. Krause, and A. Holmgren, J. Biol. Chem. 267, 9047 (1992). 21 p. Dirnhuber and F. Schutz, Biochem. J. 42, 628 (1948).
404
PROTEIN THIOLS AND SULFIDES
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recovery is 50-70%. Elution without urea lowers the protein recovery considerably. Reduced RNase is reoxidized under denaturing conditions at a low concentration of 0.5-2 mg/ml to favor the formation of intramolecular disulfide bonds. The eluate is diluted with a concentrated solution of Tris/ sarcosine hydrochloride in 8 M urea to bring the buffer to a final concentration of 0.1 M Tris/0.1 M sarcosine in 8 M urea. The solution is adjusted to pH 8.5 and sodium selenite added to a final concentration of 6 /xM (25-fold molar excess of RNase over selenite). Sarcosine is included to react preferentially with cyanate ions, which are generated from the urea and would otherwise inactivate RNase by carbamoylation.22 Reoxidation in the dark at room temperature overnight is completed within 18 hr; assays with 5,5'-dithiobis(2-nitrobenzoic acid) 23 indicate <0.1 tool of free thiol per mole of RNase. The solution of scrambled RNase is acidified to pH 4.0-4.5 with glacial acetic acid and desalted by extensive dialysis against 10 mM acetic acid (narrow dialysis tubing, approximately 10 × 50 vol of acetic acid used over 2 days). Dialysis is considered complete when the presence of salts is undetectable on a refractometer. Low levels of residual salts do not interfere with the PDI assay. Overall recovery is 45-50%. The dialysate is freezedried in 0.5-mg aliquots and stored at 4°. Its effectiveness as substrate decreases with time when stored in solution or at a lower temperature.
Characterization of Substrate The effectiveness of scrambled RNase as a substrate for PDI varies with each preparation. An effective preparation assayed with purified bovine PDI has a Km of 2-5 /xM and a Vmax of approximately 5 /xmol of RNase reactivated per minute per gram of PDI. The substrate is used routinely at a concentration of 2Kin. The material is heterogeneous in disulfide bonding and molecular weight but is not routinely characterized in this respect.
Assay of Protein Disulfide-Isomerase Activity When PDI is incubated with scrambled RNase under mildly reducing conditions, it catalyzes the reactivation of the substrate specifically by isomerization of the incorrect disulfide bonds relative to those found in the native protein. RNase activity is in turn assayed by hydrolysis of high 22 G. R. Stark, W. H. Stein, and S. Moore, J. BioL Chem. 235, 3177 (1960). 23 G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
[381
PROTEINDISULFIDE-ISOMERASE
405
molecular weight RNA. The rate at which RNase activity increases is a measure of PDI activity. In the two-stage assay, PDI is incubated with scrambled RNase and samples removed at different time points to assay RNase activity. The following procedure revises the previous details 1 based on the original assay.15 Routinely, PDI is incubated with DTT (10/xM) and scrambled RNase (concentration, 2Kin; see above) in a total volume of 200 /xl. The stock solution of scrambled RNase contains freeze-dried protein dissolved at 10 times the required concentration in distilled water or 10 mM acetic acid to pH 3-4; it is stored at 4° and discarded after 1 week. The PDI is activated by preincubation with DTT in 180/xl of buffer (50 mM sodium phosphate or Tris-HC1, pH 7.5) for 2 min at 30°, and 20/~1 of scrambled RNase is then added. The incubation is continued and 10-/xl samples are withdrawn at five time points at invervals of 3 or 4 min. Each sample is added to a cuvette containing 250/xg of highly polymerized RNA in 3.0 ml of 50 mM Tris/25 mM KC1/5 mM MgCl2, pH 7.5, at 30°. RNase activity is assayed by the change in absorbance at 260 nm on a spectrophotometer capable of recording a small change in absorbance of 0.1 against a high background of 2 units. Assays of turbid samples with high background absorbance should use dual-wavelength mode, recording A260 relative to A280 with a narrow slit width of 2.5 nm. The background rate of reactivation of scrambled RNase by DTT alone is assayed in the absence of PDI and subtracted from each PDI assay. Calculation of Protein Disulfide-Isomerase Activity The RNase activity of each time-point sample is measured by the initial linear change in A260 per minute. The PDI activity is calculated as the increase in RNase activity per minute, that is, the change in A2a0 per minute; linear regression analysis with a correlation coefficient >0.99 is considered acceptable. This direct measurement of PDI activity in spectrophotometric units is then converted into standard units of activity, defined as the amount of PDI that regenerates 1 /xmol of RNase/min. 17 The quantity of RNase generated in the PDI incubation is related to the measurement of RNase activity in the assay by a conversion factor obtained from assays of pure RNase. The factor depends on the volumes used in the procedure: in the routine procedure described above, 1/xmol of RNase generated in the PDI incubation corresponds to RNase activity measured in the assay as a change in A260 of 920 A260 units/min. Hence, one standard unit of PDI activity corresponds to an increase in RNase activity of 920 A260 units/rain per minute.
406
PROTEIN THIOLSAND SULFIDES
[391
Previously, PDI activity was defined directly in spectrophotometric units and 1 unit of PDI activity generated RNase activity at the rate of 1 A260 unit/min per minute. 1 This definition was rigidly dependent on the volumes used in the procedure. Previous calculations of specific PDI activity were based on an incubation volume of 1000/xl; they can therefore be converted from 184 "old" units per gram of PDI to 1/xmol/min per gram.
[39] G l u c o c o r t i c o i d R e c e p t o r T h i o l s a n d Steroid-Binding Activity B y S. STONEY SIMONS, JR. and WILLIAM B. PRATT
Introduction The amino acids that determine the steroid-binding sites of steroid receptors are located in the COOH-terminal one-third to one-half of the receptors in a region referred to as the h o r o m e - b i n d i n g d o m a i n (HBD). The H B D must be properly folded for there to be a high-affinity steroidbinding pocket, and for some of the receptors (e.g., glucocorticoid and mineralocorticoid receptors), the H B D must be associated with the 90-kDa heat-shock protein (hsp90) component of the protein-folding system for there to be an appropriate steroid-binding site (see Fig. 1). That is, if hsp90 is dissociated from these receptors, the steroid-binding pocket apparently collapses and the H B D must be refolded by a multicomponent proteinfolding system composed of hsp70, hsp90, and other factors before it is in a conformation such that it can bind steroid. The protein-folding determinants for steroid binding have been reviewed by Pratt. ~ An additional determinant for steroid-binding activity of the glucocorticoid receptor (GR) is the presence or absence of an intramolecular disulfide between a vicinally spaced pair of cysteine SH groups lying in the H B D Y In the 1970s, it was observed that the addition of thiol reagents to cytosol preparations inactivated ligand binding by several steroid-receptors. Subsequent studies of redox manipulation of cytosolic steroid-binding activ1W. B. Pratt, J. Biol. Chem. 268, 21455 (1993). 2N. R. Miller and S. S. Simons, Jr., J. Biol. Chem. 263, 15217 (1988). 3p. K. Chakraborti,M. J. Garabedian, K. R. Yamamoto,and S. S. Simons,Jr., J. Biol. Chem. 267, 11366 (1992). METHODS IN ENZYMOLOGY, VOL. 251
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
PROTEINDISULFIDE-ISOMERASE
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fraction. Figure 2 shows the time course of incorporation of [3HIE-64 into serum and cytosolic and mitochondrial/lysosomal fractions of the rat liver. The radioactivity of the blood increases rapidly after the injection, reaching a maximum within 30-60 min, and then decreasing rapidly. Incorporation of the radioactivity into the cytosolic fraction in liver starts slightly later than that of the blood and then also decreases rapidly. By contrast, the radioactivity appears in the particulate fractions within 1 hr, retains the maximum plateau for 5-6 hr, and then decreases gradually over 12 hr. Figure 3 shows the correlation between cathepsin B activities and protein-bound [3H]E-64 in the mitochondrial/lysosomal fractions of liver. The highest radioactivity is found in the lysosomal fraction and the distribution of the radioactivities is the same as that of the lysosomal marker enzymes, such as cathepsin B and acid phosphatase. If protein-bound radioactivities in the lysosomal fraction represent E-64-sensitive cysteine proteases, there should be a reciprocal relationship between the inhibition of the activities of cathepsin B, a representative lysosomal cysteine protease, and the proteinbound radioactivities in the fraction. Inhibition of cathepsin B and the radioactivities of [3H]E-64 reach maximum levels within 1 hr after injection of [3H]E-64 and maintain the maximum level of the reciprocal relationship between these two activities, as shown in Fig. 3. Therefore, E-64 and CA-074 are incorporated into the liver cytosol in the free form. They permeate into the lysosomes, where they bind to and effectively inactivate the target cysteine proteases.
[38]
Protein Disulfide-Isomerase
By ROBERT B. FREEDMAN,HILARY C. HAWKINS,and STEPHEN
U.
McLAUGHLIN
Introduction Protein disulfide-isomerase (PDI, EC 5.3.4.1) is an abundant protein within the lumen of the endoplasmic reticulum of secretory cells, and functions as a catalyst in the formation of native disulfide bonds in nascent secretory and cell surface proteins. In its catalytic action in vitro, it facilitates the folding and assembly of a wide range of disulfide-bonded proteins, and individual thiol-disulfide interchange steps are accelerated by over 1000fold. The role of PDI in co- and posttranslational modification of proteins has been confirmed by its cross-linking to nascent immunoglobulins, by the requirement for PDI for efficient cotranslational disulfide formation in a METHODS IN ENZYMOLOGY, VOL. 251
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
398
PROTEIN THIOLS AND SULFIDES
[38]
reconstituted in vitro translation system, and by the phenotype of yeast lacking a functional PDI. In vertebrates, the protein is also a component of two other endoplasmic reticulum (ER) lumenal enzyme systems, prolyl4-hydroxylase and the microsomal triglyceride transfer protein. Chemical modification data indicated that PDI functions in thiol-disulfide interchange reactions through dithiol/disulfide active site groups that are in the disulfide form in the isolated enzyme. This was confirmed by sequencing of the enzyme, which indicated that it is a member of the thioredoxin superfamily. Protein disulfide-isomerase sequences now available from vertebrates, higher plants, and yeast all indicate a protein of approximately 500 residues with 2 regions homologous to thioredoxin, including a conserved active site motif (WCGHCK). The PDI from bacteria, generally known as DsbA, is considerably smaller and is a more remote member of the thioredoxin superfamily. The tertiary structure of DsbA has been determined, but not that of any eukaryotic PDI. The properties, purification, and assay of PD! were reviewed in a previous volume of this series. 1 More recent reviews2-6 have focused on its structural and functional properties.
Purification of Protein Disulfide-Isomerase from Bovine Liver Protein disulfide-isomerase cDNA sequences are known from a range of organisms, but the protein has been purified and characterized to any extent only from mammalian liver and yeast (Saccharomyces cerevisiae). The reported purification of yeast PDI yielded only 2 mg of purified enzyme from 1 kg of yeast cell pellet, 7 and most of the enzymatic characterization of PDI has been carried out on material purified from mammalian liver; this is therefore the focus of the present chapter. The first high-yielding purification of the enzyme8 used detergent solubilization of whole homogenate, avoiding losses incurred by subcellular fractionation to produce microsomes.9 The procedure modified by Lambert 1 D. A. Hillson, N. Lambert, and R. B. Freedman, this series, Vol. 107, p. 281. z R. B. Freedman, Cell (Cambridge, Mass.) 57, 1069 (1989). 3 R. B. Freedman, N. J. Bulleid, H. C. Hawkins, and J. L. Paver, Biochem. Soc. Syrup. 55, 167 (1989). 4 R. Noiva and W. J. Lennarz, J. Biol. Chem. 267, 3553 (1992). 5 T. E. Creighton and R. B. Freedman, Curr. Biol. 3, 790 (1993). 6 R. B. Freedman, T. R. Hirst, and M. F. Tuite, Trends Biochem. Sci. 19, 331 (1994). 7 T. Mizunaga, Y. Katakura, T. Miura, and Y. Maruyama, J. Biochem. (Tokyo) 108, 846 (1990). s D. E. Carmichael, J. E. Morin, and J. E. Dixon, J. Biol. Chem. 252, 7163 (1977). 9 p. j. E Rowling, S. H. McLaughlin, G. S. Pollock, and R. B. Freedman, Protein Expression Purif 5, 331 (1994).
[38]
PROTEIN DISULFIDE-ISOMERASE
399
and Freedman 1° utilizes the stability of the enzyme at 54 °, at which temperature only 20% of total activity is lost, and its very low p/, which facilitates purification by ion-exchange chromatography. This method was described previously in this series (1), but has now been updated using modern chromatographic material and FPLC (fast protein liquid chromatography), which greatly reduces the time for purification (see Fig. 1).
Procedure Homogenization in Triton. Bovine liver from freshly slaughtered animals is freed of connective tissue and stored at - 2 0 ° in 500-g aliquots. On demand 500 g is taken from a - 2 0 ° freezer and cut into small pieces (approximately 1-cm cubes) while it is still frozen. These pieces are washed in ice-cold saline [0.9% (w/v) NaC1]. The liver is finely chopped and added to a precooled Waring blender in the ratio of 1 vol of liver to 2 vol of homogenization buffer [1% (w/v) Triton X-100, 0.1 M sodium phosphate buffer-5 m M EDTA, pH 7.5, containing 1 m M phenylmethylsulfonyl fluoride (PMSF) and aprotinin (1/xg/ml)], homogenizing at full speed for four 30-sec bursts with 30-sec intervals. The homogenate is decanted into a beaker on ice and additional homogenization buffer is added to give a total volume of approximately 1.5 liters. The homogenate is filtered through a double layer of muslin and then centrifuged at 18,000 g for 30 min at 4°; the pellet is discarded. Heat Treatment. The supernatant is filtered through glass wool to remove floating fat and then transferred to a 70 ° water bath with constant stirring until the temperature reaches 54 °, which is maintained _+1° for 15 rain by alternately immersing the beaker in the bath and ice. The treated extract is transferred to an ice bath and cooled to <10 ° with stirring; it is then centrifuged at 18,000 g for 40 min at 4°. All subsequent steps are carried out at 4 °. Ammonium Sulfate Fractionation. Solid ammonium sulfate is gradually added to the supernatant with stirring to give a final saturation of 55% at 0 °. After stirring for a further 30 min, the material is centrifuged at 38,000 g for 30 rain at 4 ° and the supernatant decanted through fluted Whatman (Clifton, N J ) No. 1 filter paper to remove floating fat. Further ammonium sulfate is added to increase the saturation to 93% at 0 °, with stirring for 30 min and centrifugation as before. The supernatant is carefully removed and discarded, and a total of 15-20 ml of 25 m M citrate buffer (pH 5.3) is used to dissolve the pellet, with the aid of a hand homogenizer. The resuspended pellet is dialyzed overnight against the citrate buffer (2 × 5 liters) at 4 °. 10N. Lambert and R. B. Freedman, Biochem. J. 213, 225 (1983).
Bovine Liver Wash in saline Homogenize in 1% Triton X-100, pH 7.5
Homogenate Strain through muslin Centrifuge at 18,000 g, 30 min
18,000 g Supematant Filter through gla~ wool Heat to 54 ° for 15 min Cool, centrifuge at 18,000 g, 40 rain
Heat-treated supematant Ammonium sulfate fractionation Dissolve in citrate buffer, pH 5.3 Dialyze vs citrate buffer, pH 5.3
55-93% (NI-I4)2SOfraction CM-Sepharose chromatography Pool void
CM-Se ~harose eluate Ammonium sulfate precipitation Dialyze vs 20 mM piperazine, pH 5.2 Q-Sepharose chromatography, pH 5.2
Q-Sepharose 0.35M NaC1 eluate Dialyze vs 20 mM piperazine, pH 5.2 Mono Q chromatography, pH 5.2 using linear gradient
Mono Q eluate Pool PDI containin~ fractious Dialyze and lyophilize Store -20°
Purified PDI FI~. 1. F l o w c h a r t for the p u r i f i c a t i o n of p r o t e i n d i s u l f i d e - i s o m e r a s e .
[38]
PROTEINDISULFIDE-ISOMERASE
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Cation-Exchange Chromatography. The dialysate is filtered through a 0.2-~m pore size membrane filter and applied to a CM-Sepharose Fast Flow (5 x 25 cm) column and eluted with 25 mM citrate buffer (pH 5.3) at 5 ml min -I. Fractions (15 ml) are collected. Void peak fractions are analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% resolving gel and PDI-containing fractions, identified by the known Mr of PDI, are pooled and protein precipitated by 100% (NH4)2SO4 saturation (69.70 g/100 ml) at 0° as described above, centrifuging at 38,000 g for 30 rain at 4°. The pellets are resuspended in a total of 10-20 ml of 20 mM piperazine, pH 5.2, and dialyzed against this buffer (2 × 5 liters) overnight. Alternatively, depending on its volume, the void can be dialyzed overnight and loaded directly onto the anionexchange column. FPLC Anion-Exchange Chromatography. The dialysate is filtered through a 0.2-tzm pore size membrane filter and loaded at 3 ml rain -1 onto a Q-Sepharose Fast Flow column (2.6 x 5 cm) preequilibrated in piperazine, pH 5.2; 10-ml fractions are collected. When unbound material has washed through, bound material is eluted with steps of 0.10, 0.35, and 1 M NaC1. Peak fractions are analyzed by SDS-PAGE on a 10% resolving gel. Fractions from the 0.35 M NaC1 wash containing the most pure PDI are pooled and dialyzed overnight against 20 mM piperazine, pH 5.2 (1 x 2 liters) to remove salt. The dialysate is loaded onto a Mono Q HR10/10 (8-ml) column, equilibrated with 20 mM piperazine, pH 5.2, at 1 ml min-L Bound material is eluted with a linear salt gradient from 0 to 1 M NaC1 over 160 ml, collecting 2-ml fractions. Peak fractions are analyzed by SDS-PAGE, using a 10% gel, and PDI-containing fractions are combined. These fractions are dialyzed against double-distilled H20 for a maximum of 2 hr to remove salt. The enzyme is freeze-dried and stored at - 2 0 ° and retains more than 70% of initial activity after 9 months of storage.
Molecular Properties of Purified Protein Disulfide-Isomerase All eukaryotic PDIs are proteins of approximately 500 amino acids, which in solution under physiological conditions are found as noncovalent homodimers. Mature bovine PDI comprises 490 residues, has a relative molecular mass based on composition of 55,165, migrates on SDS-PAGE with an Mr of 57,000, and has a pI of 4.5. The active site dithiol-disulfide groups in each thioredoxin-like domain show a remarkably oxidizing standard redox potential of - 110 mV, corresponding to an equilibrium constant with reduced and oxidized glutathione of 5 x 10 s M. In the dithiol form
402
PROTEIN THIOLSAND SULFIDES
[38]
of each active site, the m o r e reactive thiol group (the m o r e N-terminal of the pair) has a pKa of 6.7.
A s s a y of P r o t e i n D i s u l f i d e - I s o m e r a s e Protein disulfide-isomerase catalyzes thiol-disulfide interchange reactions in proteins: depending on the substrates and conditions it can catalyze net formation, net reduction, or net isomerization of protein disulfides. Some work has been carried out with tow molecular weight substrates and with model peptides, but assays based on proteins are most common. Protein disulfide-isomerase shows an extremely b r o a d protein substrate specificity and can therefore be assayed by a wide range of approaches. Net disulfide formation has b e e n most extensively studied using reduced bovine pancreatic ribonuclease or trypsin inhibitor (BPTI) as substraten-14; in the case of BPTI, conversions between individual disulfidebonded isomers can be resolved and their rates determined, which enables the catalytic properties of P D I to be defined in relation to specific disulfide formation or isomerization reactions. Net disulfide reduction has been most c o m m o n l y characterized as a G S H - d e p e n d e n t reduction of the disulfides of insulin (glutathione-insulin transhydrogenase).15 Net disulfide isomerization can also be assayed with insulin; in the presence of a low concentration of thiol, isomerization of insulin disulfides leads to an accumulation of insulin B chain polymers that aggregate, and the process can be monitored turbidometrically. But the most sensitive and versatile assay of isomerization is that based on reactivation of " s c r a m b l e d " ribonuclease, that is, on the recovery of enzymatic activity in a sample of ribonclease that has b e e n reduced and reoxidized under denaturing conditions. The substrate initially has little enzymatic activity, but this appears as disulfide isomerizations lead to the accumulation of ribonuclease molecules with native disulfide pairing. The appearance of ribonuclease activity can be assayed with nucleotides n or high molecular weight R N A , I'16 by spectrophotometric I'I1 or radiochemica116 approaches, and the assay of P D I can either be continuous, with 11M. M. Lyles and H. F. Gilbert, Biochemistry 30, 613 (1991). 12A. Zapun, T. E. Creighton, P. J. E. Rowling, and R. B. Freedman, Proteins: Struct. Funcr Genet. 14, 10 (1992). 1~j. S. Weissman and P. S. Kim, Nature (London) 365, 185 (1993). 14T. E. Creighton, C. J. Bagley, L. Cooper, N. J. Darby, R. B. Freedman, J. Kemmink, and A. Sheikh, J. Mol. Biol. 232, 1176 (1993). is N. Lambert and R. B. Freedman, Biochem. J. 213, 235 (1983). 16R. Myllyla and J. Oikarinen, J. Biochem. Biophys. Methods 7, 115 (1983).
[~8]
PROTEIN DISULFIDE-ISOMERASE
403
the ribonuclease substrates present in the isomerase assay system,n or discontinuous, with samples withdrawn for assay of ribonuclease activityJ
Preparation of Substrate Randomly reoxidized ("scrambled") ribonuclease (RNase) is prepared by reducing the four disulfide bonds in RNase and then allowing them to reoxidize under denaturing conditions. This complex undefined mixture of the 105 possible disulfide-bonded isomers is essentially inactive with <1% of the activity of native RNase. Preparations, using our published method, 17 varied in their properties as a substrate for PDI, owing to uncontrolled chemistry of reoxidation that was due in turn to differences in the quality of the distilled water used. In preliminary studies, controlled reoxidation in the presence of copper ions TM or diamide 19 was less successful than in the presence of selenite,2° which gave scrambled RNase with optimum properties of low Km and high Vmax and was reproducible. The following revised procedure includes selenite-controlled reoxidation and other modifications2° to make it more simple. Bovine pancreatic RNase is fully reduced under denaturing conditions by incubation at a concentration of 25-30 mg/ml in 50 mM Tris-HC1, pH 8.5, containing 10 M urea and 130 mM dithiothreitol (DTT), that is, an approximately 15-fold molar excess of DTT over protein disulfide bonds. Reduction is complete at room temperature overnight or at 37° for 1 hr. It is not necessary to flush the solution with nitrogen gas. Ultrapure urea should be used throughout and solutions made up as fresh as possible to reduce to a minimum the concentration of cyanate ions formed by isomerization of urea. 21 The solution is acidified to pH 4.0-4.5 with glacial acetic acid and then eluted on a desalting column in 0.1 M acetic acid containing 8 M urea to remove DTT alone. A small prepacked desalting column (approximately 10-ml column volume) is quick and convenient, and a large bulk preparation can be eluted in aliquots (2.5 ml, 60-75 mg). The rate of elution is fast even with 8M urea, and a rate of 1-2 ml/min with a simple siphon is maintained during subsequent elutions. Eluted fractions are monitored at 280 nm, protein-containing fractions pooled, and the overall concentration of the eluate determined (A280 = 0.55 for a solution of 1 mg/ml). Overall 17 H. C. Hawkins, E. C. Blackburn, and R. B. Freedman, Biochem. J. 275, 349 (1991). 18 A. K. Ahmed, S. W. Schaffer, and D. B. Wetlaufer, J. Biol. Chem. 250, 8477 (1975). 19N. S. Kosower and E. M. Kosower, this series, Vol. 143, p. 264. 2o j. Lundstrom, G. Krause, and A. Holmgren, J. Biol. Chem. 267, 9047 (1992). 21 p. Dirnhuber and F. Schutz, Biochem. J. 42, 628 (1948).
404
PROTEIN THIOLS AND SULFIDES
[38]
recovery is 50-70%. Elution without urea lowers the protein recovery considerably. Reduced RNase is reoxidized under denaturing conditions at a low concentration of 0.5-2 mg/ml to favor the formation of intramolecular disulfide bonds. The eluate is diluted with a concentrated solution of Tris/ sarcosine hydrochloride in 8 M urea to bring the buffer to a final concentration of 0.1 M Tris/0.1 M sarcosine in 8 M urea. The solution is adjusted to pH 8.5 and sodium selenite added to a final concentration of 6 /xM (25-fold molar excess of RNase over selenite). Sarcosine is included to react preferentially with cyanate ions, which are generated from the urea and would otherwise inactivate RNase by carbamoylation.22 Reoxidation in the dark at room temperature overnight is completed within 18 hr; assays with 5,5'-dithiobis(2-nitrobenzoic acid) 23 indicate <0.1 tool of free thiol per mole of RNase. The solution of scrambled RNase is acidified to pH 4.0-4.5 with glacial acetic acid and desalted by extensive dialysis against 10 mM acetic acid (narrow dialysis tubing, approximately 10 × 50 vol of acetic acid used over 2 days). Dialysis is considered complete when the presence of salts is undetectable on a refractometer. Low levels of residual salts do not interfere with the PDI assay. Overall recovery is 45-50%. The dialysate is freezedried in 0.5-mg aliquots and stored at 4°. Its effectiveness as substrate decreases with time when stored in solution or at a lower temperature.
Characterization of Substrate The effectiveness of scrambled RNase as a substrate for PDI varies with each preparation. An effective preparation assayed with purified bovine PDI has a Km of 2-5 /xM and a Vmax of approximately 5 /xmol of RNase reactivated per minute per gram of PDI. The substrate is used routinely at a concentration of 2Kin. The material is heterogeneous in disulfide bonding and molecular weight but is not routinely characterized in this respect.
Assay of Protein Disulfide-Isomerase Activity When PDI is incubated with scrambled RNase under mildly reducing conditions, it catalyzes the reactivation of the substrate specifically by isomerization of the incorrect disulfide bonds relative to those found in the native protein. RNase activity is in turn assayed by hydrolysis of high 22 G. R. Stark, W. H. Stein, and S. Moore, J. BioL Chem. 235, 3177 (1960). 23 G. L. Ellman, Arch. Biochem. Biophys. 82, 70 (1959).
[381
PROTEINDISULFIDE-ISOMERASE
405
molecular weight RNA. The rate at which RNase activity increases is a measure of PDI activity. In the two-stage assay, PDI is incubated with scrambled RNase and samples removed at different time points to assay RNase activity. The following procedure revises the previous details 1 based on the original assay.15 Routinely, PDI is incubated with DTT (10/xM) and scrambled RNase (concentration, 2Kin; see above) in a total volume of 200 /xl. The stock solution of scrambled RNase contains freeze-dried protein dissolved at 10 times the required concentration in distilled water or 10 mM acetic acid to pH 3-4; it is stored at 4° and discarded after 1 week. The PDI is activated by preincubation with DTT in 180/xl of buffer (50 mM sodium phosphate or Tris-HC1, pH 7.5) for 2 min at 30°, and 20/~1 of scrambled RNase is then added. The incubation is continued and 10-/xl samples are withdrawn at five time points at invervals of 3 or 4 min. Each sample is added to a cuvette containing 250/xg of highly polymerized RNA in 3.0 ml of 50 mM Tris/25 mM KC1/5 mM MgCl2, pH 7.5, at 30°. RNase activity is assayed by the change in absorbance at 260 nm on a spectrophotometer capable of recording a small change in absorbance of 0.1 against a high background of 2 units. Assays of turbid samples with high background absorbance should use dual-wavelength mode, recording A260 relative to A280 with a narrow slit width of 2.5 nm. The background rate of reactivation of scrambled RNase by DTT alone is assayed in the absence of PDI and subtracted from each PDI assay. Calculation of Protein Disulfide-Isomerase Activity The RNase activity of each time-point sample is measured by the initial linear change in A260 per minute. The PDI activity is calculated as the increase in RNase activity per minute, that is, the change in A2a0 per minute; linear regression analysis with a correlation coefficient >0.99 is considered acceptable. This direct measurement of PDI activity in spectrophotometric units is then converted into standard units of activity, defined as the amount of PDI that regenerates 1 /xmol of RNase/min. 17 The quantity of RNase generated in the PDI incubation is related to the measurement of RNase activity in the assay by a conversion factor obtained from assays of pure RNase. The factor depends on the volumes used in the procedure: in the routine procedure described above, 1/xmol of RNase generated in the PDI incubation corresponds to RNase activity measured in the assay as a change in A260 of 920 A260 units/min. Hence, one standard unit of PDI activity corresponds to an increase in RNase activity of 920 A260 units/rain per minute.
406
PROTEIN THIOLSAND SULFIDES
[391
Previously, PDI activity was defined directly in spectrophotometric units and 1 unit of PDI activity generated RNase activity at the rate of 1 A260 unit/min per minute. 1 This definition was rigidly dependent on the volumes used in the procedure. Previous calculations of specific PDI activity were based on an incubation volume of 1000/xl; they can therefore be converted from 184 "old" units per gram of PDI to 1/xmol/min per gram.
[39] G l u c o c o r t i c o i d R e c e p t o r T h i o l s a n d Steroid-Binding Activity B y S. STONEY SIMONS, JR. and WILLIAM B. PRATT
Introduction The amino acids that determine the steroid-binding sites of steroid receptors are located in the COOH-terminal one-third to one-half of the receptors in a region referred to as the h o r o m e - b i n d i n g d o m a i n (HBD). The H B D must be properly folded for there to be a high-affinity steroidbinding pocket, and for some of the receptors (e.g., glucocorticoid and mineralocorticoid receptors), the H B D must be associated with the 90-kDa heat-shock protein (hsp90) component of the protein-folding system for there to be an appropriate steroid-binding site (see Fig. 1). That is, if hsp90 is dissociated from these receptors, the steroid-binding pocket apparently collapses and the H B D must be refolded by a multicomponent proteinfolding system composed of hsp70, hsp90, and other factors before it is in a conformation such that it can bind steroid. The protein-folding determinants for steroid binding have been reviewed by Pratt. ~ An additional determinant for steroid-binding activity of the glucocorticoid receptor (GR) is the presence or absence of an intramolecular disulfide between a vicinally spaced pair of cysteine SH groups lying in the H B D Y In the 1970s, it was observed that the addition of thiol reagents to cytosol preparations inactivated ligand binding by several steroid-receptors. Subsequent studies of redox manipulation of cytosolic steroid-binding activ1W. B. Pratt, J. Biol. Chem. 268, 21455 (1993). 2N. R. Miller and S. S. Simons, Jr., J. Biol. Chem. 263, 15217 (1988). 3p. K. Chakraborti,M. J. Garabedian, K. R. Yamamoto,and S. S. Simons,Jr., J. Biol. Chem. 267, 11366 (1992). METHODS IN ENZYMOLOGY, VOL. 251
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.