- Email: [email protected]
[29] Reconstitution of cell-free NADPH oxidase activity by purified components
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[29] R e c o n s t i t u t i o n o f C e l l - F r e e N A D P H O x i d a s e A c t i v i t y by Purified Components
By
ARIE ABO and ANTHONY W. SEGAL
Introduction The assembly of the N A D P H oxidase of phagocytes on activation is essential for the efficient killing of microorganisms. The components of this system are primarily expressed in professional phagocytes which include neutrophils, macrophages, monocytes, and eosinophils. The activated oxidase utilizes N A D P H as an electron donor to catalaze the reduction of oxygen to superoxide (O2- -) which serves as a precursor for the formation of reduced oxygen species, such as H20:, • OH, and HOC1.1 The electron-transporting component of the N A D P H oxidase is an unusual membrane-bound flavocytochrome b composed of two subunits: a 21-kDa ot subunit (also known as p21ph°x) and a 76- to 92-kDa heavily glycosylated/3 subunit (also known as gp91Ph°x). The /3 subunit contains the binding sites for N A D P H and FAD, the location of the heme(s) is as yet uncertain. The activation of the electron transport oxidase is dependent on the assembly of the cytosolic components p47ph°x and p67ph°x and the Rasrelated small GTP-binding protein p21 Rac on the membrane (Fig. 1). Impairment in several components of the oxidase complex is a direct cause of chronic granulomatous disease (CGD), a rare syndrome associated with chronic infections and impaired microbicidal activity. 2 A key to our understanding and identifying the N A D P H oxidase components emerged with the initial discovery of the cell-free system in which in vitro reconstitution was obtained by the combination of cytosol and membranes prepared from resting phagocytes together with optimal concentrations of amphiphiles like arachidonic acid or SDS. 3 Furthermore, by mixing normal membranes and cytosol with those prepared from CGD neutrophils, it was shown that most patients with autosomal recessive CGD exhibit deficiencies in the cytosolic fraction, whereas the membrane fraction is impaired in X-linked patients. 4 t A. W. Segal and A. Abo, Trends Biochem. Sci. 18, 43 (1993). 2 R. M. Smith and J. T. Curnutte, Blood 77, 673 (1991). 3 y . Bromberg and E. Pick, J. Biol. Chem. 260, 13539 (1985). 4 j. T. Curnutte, R. L. Berkow, R. L. Roberts, S. B. Shurin, and P. J. J. Scott, Clin. Invest.
81, 606 (1988). METHODSIN ENZYMOLOGY,VOL.256
Copyright © 1995 by AcademicPress,Inc. All rightsof reproductionin any formreserved.
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Various groups have shown that fractions containing p47 ph°x and p67 ph°x could not alone replace the cytosol in the cell-free assay. We and others then identified an additional cytosolic factor essential for oxidase activity which, on purification, resolved into a heterodimeric complex of proteins subsequently identified as Ras-related small GTP-binding proteins p21 Rac and G D P dissociation inhibitor for R h o ( R h o - G D I ) . 5 R e c o m b i n a n t p21 R a c l or p21 Rac2 loaded with G T P could replace the complex of Rac and G D I . 6-8 Subsequently, it was shown that the oxidase activity was fully reconstituted by purified recombinant p67 ph°x, p47 ph°x, and p21 Rac synthesized in Escherichia coli or Sf9 (Spodoptera frugiperda fall a r m y w o r m ovary) cells together with purified cytochrome b from h u m a n neutrophil or recombinant cytochrome b p r e p a r e d in Sf9 cells. 9-u This chapter describes the preparation of recombinant N A D P H oxidase cytosolic c o m p o n e n t s in E. coli and reconstitution of oxidase activity in the cell-free system with purified cytochrome b or solubilized neutrophil m e m branes.
P r e p a r a t i o n of N e u t r o p h i l M e m b r a n e s a n d Cytosol N o r m a l h u m a n blood is a good source of neutrophils (typical yield is 2 - 3 × 10 s neutrophils/100 ml blood) which are readily purified from the blood and can serve as a source of cytosol and m e m b r a n e fractions for optimization of the cell-free assay. However, this source does not generally provide enough neutrophils for purification of cytochrome b or the cytosolic components. Blood from patients with chronic myeloid leukemia contains up to approximately 100 times the normal neutrophil count and these patients are occasionally leukaphoresed to r e m o v e large n u m b e r of granulocytes. Additional sources of these ceils and purification methods are described in m o r e detail in Segal et al.12 5 A. Abo and E. Pick, J. BioL Chem. 266, 23577 (1991). 6 A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 7 U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 8 U. G. Knaus, P. G. Heyworth, B. T. Kinsella, J. T. Curnutte, and G. M. Bokoch, J. Biol. Chem. 267, 23575 (1992). 9 A. Abo, A. Boyhan, I. West, J. Adrian, Thrasher, and A. W. Segal, J. Biol. Chem. 24, 16767 (1992). l0 D. Rotrosen, C. L. Yeung, T. L. Leto, H. L. Malech, and C. H. Kwong, Science 256, 1512 (1992). 11D. Rotrosen, C. L. Yeung, and J. P. Katkin, J. Biol. Chem. 268, 14256 (1993). 12A. W. Segal, A. M. Harper, A. R. Cross, and O. T. G. Jones, in "Methods in Enzymology" (G. Di Sabato and J. Everse, eds.), Vol. 132, p. 378. Academic Press, San Diego, 1986.
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RECONSTITUTION OF NADPH OXIDASEACTIVITY
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Methods Isolation of Neutrophils from Human Peripheral Blood. This is a brief description of neutrophil isolation. More detailed descriptions can be found in Segal et aL t2 Human blood (100 ml) is drawn from a healthy volunteer into preservative-free heparin (5 U/ml) mixed with 0.15 M NaC1 and sedimented with 10% volume of 10% dextran (w/v, average molecular weight 242,000) for approximately 30 min until erythrocytes have sedimented. The supernatant plasma containing the leukocytes is layered on Ficoll/Hypaque (Lymphoprep, Nycomed, Pharma, 5 vol of plasma on 1 vol of gradient) and is centrifuged at 400g for 15 min. The pellet contains the neutrophils and residual erythrocytes. The erythrocytes are lysed by hypotonic lysis. The pellet, up to 0.2 ml in volume, is resuspended in 20 ml of water and is vigorously mixed on a vortex mixer or pipetted up and down for 10 sec. Osmolarity is restored by the addition of 20 ml 0.31 M NaCI (1.8%) containing 5 IU/ml heparin. Preparation of Membranes and Cytosol. The neutrophils are resuspended at a concentration of 2 × 108 cells/ml in a break buffer consisting of 6 mM PIPES, pH 7.3, 6% (w/w) sucrose, 60 mM KC1, 1.8 mM NaC1, 2.3 mM MgCI2, IIzM diisopropyl fluorophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 /xg/ml TLCK. The cells are sonicated three times at 4° for 15 sec, overlaid on a discontinuous sucrose gradient (in 10 mM Tris, pH 7.4) of 15% (w/w) on top of 34% (w/w) on top of 45% (w/w), and centrifuged at 200,000g at 4°C for 15 rain in a TLX Beckman ultracentrifuge using a TLS55 swing-out rotor. The cytosol is collected above the 15% sucrose, the plasma membranes at the 15/34% interface, and the specific granules at the 34/45% interface. The specific granules and plasma membrane are diluted 1 : 4 with cold break buffer lacking sucrose and are pelleted by centrifugation as described earlier.
F~G. 1. Model of the role p21 Rac might play in the activation complex of the NADPH oxidase. The specialized components of the NADPH oxidase include the a and/3 subunits of flavocytochrome b and the two cytosolic proteins, p47ph°x and p67ph°x. p21 Rac is present in the GDP-bound form in a complex with GDI (GDP dissociation inhibition factor), an association that requires its modification by isoprenylation near its C terminus that could also be required for attachment to the membrane. Activation is associated with separation of p21 Rac from GDI, the exchange of GTP for GDP, and its movement with p47ph°x and p67ph°X in an activation complex into association with the flavocytochrome in the membranes. The bound GTP is then hydrolyzed to GDP as a consequence of endogenous GTPase activity of the rac or through modulation by GAPs that accelerate GTPase activity. The function of a third specialized cytosolic protein, p40ph°x, which is associated with p67ph°x but is not required in the cell-free system,15 remains to be determined.
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M e m b r a n e Solubilization and Reconstitution. The membrane and specific granules serve as a good source of cytochrome b for cell-free activation; however, better activity can be obtained if the membrane is solubilized with a detergent such as octylglucoside or deoxycholate which is then removed by dialysis or diluted below the critical micellar concentration to allow reconstitution of the proteins with the lipid vesicles. The pelleted membranes are resuspended to a concentration of 1-2 × 108 cells equivalents/ml in a cold solubilization buffer consisting of 120 mM sodium phosphate, pH 7.4, I mM MgC12, 1 mM EGTA, 1 mM dithiothreitol (DTT), 10-20% glycerol, 1 /xg/ml leupeptin, 1 mM PMSF, and 40 mM octylglucoside, then homogenized with a Dounce homogenizer. The clarified solubilized membranes are stirred on ice for 30 min to allow a complete solubilization. Insoluble material is removed by centrifugation at 200,000g for 15 rain as described earlier. Reconstitution of the soluble proteins into the lipid vesicle can be simply achieved by diluting the solubilized membrane with buffer lacking detergent to reduce the detergent concentration below the critical micelle concentration (CMC). One volume of solubilized membrane is mixed with 4 vol of solubilization buffer lacking octylglucoside which reduces the concentration of octylglucoside to 8 raM, well below the CMC of 25 mM, and is left on ice for 30-60 min. An alternative option is to dialyze the mixture against buffer lacking the detergent.
Purification of Cytochrome b Cytochrome b is purified from the membranes and specific granules of neutrophils as described previously. Briefly, membranes and specific granules are purified from large numbers of neutrophils from normal human blood or by leukapheresis of patients with chronic granulocytic leukemia by scaling up the procedures described earlier. The membranes and granules (approximately 1 vol) are homogenized in (approximately 10-20 vol) buffer A (100 mM Tris-acetate, pH 7.4, 100 mM KC1, 20% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, and protease inhibitors 1 mM PMSF, 1 mg/liter leupeptin, 1 mg/liter TLCK, and 1 mg/ liter pepstatin) containing 0.5% sodium cholate and centrifuged at approximately 100,000g. Cytochrome b remains largely in the pellet which is then washed with buffer without detergent and then extracted with buffer containing 1% Triton N-101 in roughly the same ratio of volumes of pellet to buffer. The Triton extract is then passed through columns containing DEAE-, CM-, and n-aminooctyl-Sepharose, and the cytochrome, which fails to attach to these resins, is trapped on heparin agarose from which it is eluted with a linear gradient of 0-1.0 M NaC1.
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Relipidation of Cytochrome b To obtain functional cytochrome b it is essential to reconstitute purified cytochrome b with lipid. This can be achieved by either preparing artificial lipid vesicles (35% phosphatidylcholine, Sigma) or extracting the endogenous lipid from neutrophil membranes. The lipid is then solubilized and combined with purified solubilized cytochrome b. Cytochrome b and the lipid associate upon removal of the detergent. Prior to relipidation of cytochrome b, the detergent is exchanged from Triton N-101 to solubilization buffer containing 40 mM octylglucoside. Cytochrome b is bound to the heparin agarose column in the Triton buffer, is washed with solubilization buffer containing octylglucoside, and is then eluted in break buffer containing 40 mM octylglucoside with a gradient of 0-1.0 M NaC1. To extracted lipids from neutrophil membranes, 200 ml of the membrane (protein concentration 2 mg/ml) is extracted with 1 ml of chloroform/ methanol (2:1, v/v), vortexed vigorously, and then centrifuged for 5 min at 400g in an Eppendorf centrifuge. The denatured proteins are removed from the interface, and the lower organic layer is evaporated under nitrogen. The dried extract of phospholipids is then solubilized in solubilization buffer. To optimize phospholipid/cytochrome b, 100 tzl of various concentrations of the solubilized phospholipids (100-400 p.g phospholipid) is mixed with 40 pmol of cytochrome b (40 tzl) and the mixture is diluted with 800 tzl solubilization buffer lacking detergent and is left on ice for 3060 min. The relipidated cytochrome is now ready to be used in the cellfree reconstitution assay. Preparation of p67 p~, p47Ph% and p21 Racl Isolation of large amounts of p67 ph°~ and p47ph°x from phagocytes is a laborious process since they are expressed at low abundance and are susceptible to proteolysis, particularly p67eh°~. However, it is possible to prepare small amounts of partially purified fractions enriched with these proteins as a good source for reconstitution in the oxidase. Purification of p21 Rac from phagocytes is described in [5] in this volume. Expression of these proteins in E. coli or in insect cells provides a good source of large amounts of pure proteins.
Preparation of Partially Purified p47ph°x and p67ph°xfrom Phagocyte Cytosol Ammonium sulfate precipitation provides a good method for the preparation of a fraction of cytosol that is enriched in p47ph°x and p67ph°x and depleted of p21 Rac. Ammonium sulfate is added over a period of 30 min
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to 10-20 ml of neutrophil cytosol (prepared as described earlier) to obtain a final concentration of 37% saturation and the mixture is stirred for 30 min on ice. The mixture is then centrifuged for 15 min at 200,000g in a TLX Beckman ultracentrifuge. The pellet is resuspended to its initial volume in 100 mM sodium phosphate buffer, pH 7.2, containing 1 mM MgC12, 1 mM DTT, 1 mM PMSF, and 1 tzg/ml leupeptin. The ammonium sulfate is removed from the supernatant by dialysis or by exchanging the buffer on a desalting column such as Sephadex G-25M PD10 (Pharmacia LKB Biotechnology Inc.). The presence of these proteins should be confirmed by Western blot analysis with antibodies which can be obtained from several laboratories active in the field. For reconstitution of the oxidase activity, it is also important to check that this fraction can reconstitute the oxidase activity only when supplemented with membrane fraction and p21 Rac protein as discussed below. Partially purified p47ph°x and p67ph°x can also be prepared by gelfiltration chromatography. Neutrophil cytosol is fractionated on a Superose 12 gel filtration column (Pharmacia) and fractions are collected and analyzed by Western blotting with antibodies against p47ph°x, p67ph°x, and p21 Rac. Typically, p47ph°x and p67ph°x coelute as a 260-kDa complex whereas p21 Rac elutes with R h o - G D I as a 45-kDa complex. It is also possible to prepare p47ph°x and p67ph°x by the absorption of neutrophil cytosol on 2',5'-ADP agarose as described. 13
Preparation of Recombinant p47ph°x, p6T 'h°x, and p21 Racl in E. coli Large quantities of p47ph°x, p67ph°x, and p21 Racl can be rapidly prepared in E. coli using the pGEX expression vector. Expression of tagged proteins in Sf9 cells is also a good expression system. Human p47ph°x,p67ph°x,and p21 Racl cDNAs are cloned into a glutathione S-transferase (GST) expression vector pGEX-2T as described previously. Transformed clones with pGEX containing p47ph°x, p67ph°x, or p21 Racl cDNAs were handled as following: 1. Terrific broth (50 ml) supplemented with 100 mg/ml ampicillin is inoculated with E. coli containing the desired clone rotated overnight at 37° in a shaking incubator. 2. The overnight culture is diluted 1 : 10 into fresh Terrific broth containing ampicillin and is grown for 1 hr. 3. Expression of the fusion protein is induced with 200 tzM IPTG and the culture is grown for an additional 4 hr. 13D. Sha'ag and E. Pick, Biochim. Biophys.Acta 1037, 405 (1990).
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OXIDASE ACTIVITY
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4. The cells are harvested by centrifugation at 300g for 15 min, and the obtained cell pellet is resuspended in 5-7 ml of lysis buffer containing 50 mM Tris, pH 7.5, 5 mM MgCI2, 1 mM DTF, and 1 mM PMSF. 5. The cell suspension is sonicated thoroughly (4 × 15 sec) on ice, and the cell debris is removed by centrifugation at 48,000g for 15 min. 6. The supernatant is incubated for 1 hr at 4° with 0.5-1 ml of glutathione-Sepharose beads (Pharmacia or Sigma) prewashed with lysis buffer. 7. The glutathione beads are washed three times with 10 ml cold lysis buffer by centrifuging the beads at 400g for 5 min and resuspending them in fresh buffer. 8. The bound proteins are eluted by resuspending the beads in 24 ml of lysis buffer containing 5 mM reduced glutathione (Sigma). The suspension is incubated for 2 rain, and the eluted proteins are recovered in the supernatant obtained by spinning down the beads. 9. To release the desired protein from the GST protein, thrombin cleavage is required. The buffer solution containing the fusion protein is exchanged into thrombin buffer containing 50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 5 mM MgCI2, 2.5 mM CaC12, and 1 mM Dq-T. The buffer exchange can be achieved either by dialysis or by desalting on a PD10 column (Pharmacia). 10. The activity of thrombin obtained from different sources (most commonly human or bovine) varies substantially, making it essential to optimize the cleavage conditions. A small-scale reaction is set by incubating 20 t~g of fusion protein with various amounts of thrombin (1-20 units depending on the source of thrombin) for 1 hr at room temperature. The efficiency of the cleavage is analyzed by S D S - P A G E visualized by Coomassie blue staining. 11. Once the optimal ratio of thrombin to fusion protein is obtained, the reaction is scaled up and the rest of the fusion protein is cleaved. 12. The activity of thrombin is inhibited by the addition of 10 /zM APMSF (Sigma Cat. No. A6664), and the efficiency of the cleavage is again assessed by SDS-PAGE. 13. A further purification is required to separate the fusion protein from GST. This can be achieved either by reabsorbing the GST on glutathioneagarose or by separation by anion-exchange chromatography on a Mono Q column (HR 5/5 Pharmacia) under standard conditions. For instance, p21 Racl and p47ph°xwere separated from GST by fractionation on a Mono Q column; the GST bound to the Mono Q (and can be eluted by 250300 mM NaC1) and p21 Racl and p47ph°xremained in the unbound fractions. Typically, 1 mg of pure recombinant protein is produced using this method from 1 liter of broth culture. The proteins were concentrated by centrifuga-
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tion on Centricon 10 membrane concentrators (Amicon) to a concentration of 1 mg/ml, aliquoted to 50/xl/vial, snap frozen in liquid nitrogen, and stored at - 7 0 °. R e c o n s t i t u t i o n of Cell-Free NADPH Oxidase
Assay for Superoxide Production The most widely used assay for measuring superoxide production is the reduction of cytochrome c inhibitable by superoxide dismutase (SOD). The assay has been described in detail by Pick. 14 The superoxide ions generated by the activated oxidase reduce cytochrome c, which has an absorption maximum at 550 nm. The rate of superoxide generation is calculated from the slope of the linear range of the absorbance at 550 nm, using e550 = 2.1 × 104 M -1 cm -1 Since cytochrome c can be reduced by reducing sources other then superoxide, it is essential to demonstrate that the reaction is inhibitable by SOD, indicating that the reduction of cytochrome c is specific.
Cell-Free Assay a. Using Cytosol and Membranes. It is advisable to first optimize the reconstitution of the oxidase in the cell-free assay with the solubilized membrane and cytosol. This helps in identifying an inactive recombinant protein. This system can also be used to test cytosol and membranes from patients with C G D to identify the impaired component as described in this series. Solubilized membrane (30-50 ml) (diluted 1:8 to dilute the detergent, 5-20 mg protein) and 10-40/xl cytosol (50-150 mg) are incubated for 90 sec in 0.9 ml assay buffer consisting of 65 m M sodium phosphate buffer, p H 7.0, 1 m M E G T A , 1 m M MgCI2, 1 0 / z M FAD, and 1 0 0 / z M cytochrome c. To determine the optimal concentrations of the SDS activator, various concentrations of SDS are added to the incubation mixture. SDS (6-15 ml) from a 10 m M stock solution is added to the incubation mixture to achieve optimal oxidase activation. A final concentration of SDS is 80-120 mM. The reaction is then initiated by the addition of 200 tzM N A D P H (20 ml from 10 m M stock), and the rate of superoxide generation is measured for 2 min in a double beam spectrophotometer (Uvikon 860, Kontron). The reference sample contains SOD (Sigma; we use at 50/xg/ ml) in addition to reagents mentioned previously. The same procedure can be applied with partially purified components. The solubilized membrane 14E. Pick, in "Methods in Enzymology" (G. Di Sabato and J. Everse, eds.), Vol. 132, p. 407. Academic Press, San Diego, 1986. 15F. B. Wientjes, J. J. Hsuan, N. F. Totty, and A. W. Segal, Biochem. J. 296, 557 (1993).
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(as mentioned earlier) is incubated with a fraction enriched in p47ph°x and p67ph°xobtained by the methods described earlier, supplemented with either partially purified or recombinant p21 Racl and an optimal concentration of SDS, incubated for 2 min, and electron transport initiated with NADPH. Note: Figure 2 shows that it is possible to demonstrate that reconstituted activity is dependent on the different components described earlier. b. Reconstitution of Cell-Free NADPH Oxidase from Purified Components. Since the recombinant p21 Racl purified from E. coli is predominantly in the GDP-bound form, to obtain a functional GTPase protein it is essential to exchange the nucleotide to either GTP or GTPyS. This is done by incubating the protein (100/xg/ml) in the presence of 100 mM guanine nucleotide in 5 mM EDTA for 10-15 min at room temperature followed by the addition of 10 mM MgC12 to chelate the EDTA and lock the nucleotide into the protein. Once all the reagents are prepared and the cell-free assay is optimized, the system is ready for reconstitution with purified components. Higher enzymatic activities can be obtained by separating the activation process from the catalysis reaction. Thus, the purified components are first preincubated in an optimal SDS concentration in a small volume (100 txl) of assay buffer containing but lacking cytochrome c and containing FAD
~ l 80O
0
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i
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i
20
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+
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.
.
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.
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+
+
+
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p67-phox p47-phox p21rac1 SDS
+ + +
+
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+ + +
+ + +
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.
+ +
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+
FIG. 2. Reconstitution of NADPH oxidase activity in the cell-free system by recombinant
p67ph°x, p47ph°x, p21 Racl, and pure cytochrome b. Solubilized neutrophil membrane (6/zg protein, 4 pmol cytochrome b) or pure relipidated cytochrome b (4 pmol) was incubated either with 30 /zl neutrophil cytosol (100 p~g protein) or with p67ph°x (4.5 nM), p47ph°x (43.5 nM), and p21 Racl (48.5 nM) preloaded with GTP and the optimal concentration of SDS (where indicated).
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and then transferring this to 0.9 ml of catalysis buffer to initiate the reaction with NADPH. Ten microliters of relipidated cytochrome b (0.1-0.5/zg, 1-5 pmol) is preincubated for 2 min at room temperature with 2-40/.d (0.1-4/zg protein) p47ph°x, p67ph°x, and p21 Racl in the presence of an optimal concentration of SDS (100-200/zM, 10-20/zl from 1 mM stock) and 10 t~M FAD, and the volume is complete to 100/xl with assay buffer lacking cytochrome c. The mixture is then transferred to a 1-ml cuvette containing 0.9 ml assay buffer with 100/zM cytochrome c and the reaction is initiated by the addition of 20/xl of NADPH (from 10 mM stock). The superoxide production is monitored for 120 sec in a double-beam spectrophotometer as described earlier. The assay can be performed in a microplate as described in a previous volume of this series.
Calculations Since the reduction of cytochrome c represents the amount of superoxide generated (1 mol of superoxide will reduce 1 tool of cytochrome c), the rate of superoxide generation is calculated from the slope of the linear range of the absorbance at 550 nm, using e550 = 2.1 × 104 M -1 cm 1 of cytochrome c: Absorbance change of 1 unit at 550 n m × 47.6 = nmol 02-. The specific activity is expressed as either nmol O2/min/mg of membrane protein or as mol O2-/mol cytochrome b-245/sec. Typical values should be in the range of 1500-3000 nmol O2-/min/mg protein and 60-120 mol O2-/mol cytochrome b-245/sec, respectively. Acknowledgment We thank the Wellcome Trust for support.
CELLEXPRESSION
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[29] R e c o n s t i t u t i o n o f C e l l - F r e e N A D P H O x i d a s e A c t i v i t y by Purified Components
By
ARIE ABO and ANTHONY W. SEGAL
Introduction The assembly of the N A D P H oxidase of phagocytes on activation is essential for the efficient killing of microorganisms. The components of this system are primarily expressed in professional phagocytes which include neutrophils, macrophages, monocytes, and eosinophils. The activated oxidase utilizes N A D P H as an electron donor to catalaze the reduction of oxygen to superoxide (O2- -) which serves as a precursor for the formation of reduced oxygen species, such as H20:, • OH, and HOC1.1 The electron-transporting component of the N A D P H oxidase is an unusual membrane-bound flavocytochrome b composed of two subunits: a 21-kDa ot subunit (also known as p21ph°x) and a 76- to 92-kDa heavily glycosylated/3 subunit (also known as gp91Ph°x). The /3 subunit contains the binding sites for N A D P H and FAD, the location of the heme(s) is as yet uncertain. The activation of the electron transport oxidase is dependent on the assembly of the cytosolic components p47ph°x and p67ph°x and the Rasrelated small GTP-binding protein p21 Rac on the membrane (Fig. 1). Impairment in several components of the oxidase complex is a direct cause of chronic granulomatous disease (CGD), a rare syndrome associated with chronic infections and impaired microbicidal activity. 2 A key to our understanding and identifying the N A D P H oxidase components emerged with the initial discovery of the cell-free system in which in vitro reconstitution was obtained by the combination of cytosol and membranes prepared from resting phagocytes together with optimal concentrations of amphiphiles like arachidonic acid or SDS. 3 Furthermore, by mixing normal membranes and cytosol with those prepared from CGD neutrophils, it was shown that most patients with autosomal recessive CGD exhibit deficiencies in the cytosolic fraction, whereas the membrane fraction is impaired in X-linked patients. 4 t A. W. Segal and A. Abo, Trends Biochem. Sci. 18, 43 (1993). 2 R. M. Smith and J. T. Curnutte, Blood 77, 673 (1991). 3 y . Bromberg and E. Pick, J. Biol. Chem. 260, 13539 (1985). 4 j. T. Curnutte, R. L. Berkow, R. L. Roberts, S. B. Shurin, and P. J. J. Scott, Clin. Invest.
81, 606 (1988). METHODSIN ENZYMOLOGY,VOL.256
Copyright © 1995 by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[291
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269
Various groups have shown that fractions containing p47 ph°x and p67 ph°x could not alone replace the cytosol in the cell-free assay. We and others then identified an additional cytosolic factor essential for oxidase activity which, on purification, resolved into a heterodimeric complex of proteins subsequently identified as Ras-related small GTP-binding proteins p21 Rac and G D P dissociation inhibitor for R h o ( R h o - G D I ) . 5 R e c o m b i n a n t p21 R a c l or p21 Rac2 loaded with G T P could replace the complex of Rac and G D I . 6-8 Subsequently, it was shown that the oxidase activity was fully reconstituted by purified recombinant p67 ph°x, p47 ph°x, and p21 Rac synthesized in Escherichia coli or Sf9 (Spodoptera frugiperda fall a r m y w o r m ovary) cells together with purified cytochrome b from h u m a n neutrophil or recombinant cytochrome b p r e p a r e d in Sf9 cells. 9-u This chapter describes the preparation of recombinant N A D P H oxidase cytosolic c o m p o n e n t s in E. coli and reconstitution of oxidase activity in the cell-free system with purified cytochrome b or solubilized neutrophil m e m branes.
P r e p a r a t i o n of N e u t r o p h i l M e m b r a n e s a n d Cytosol N o r m a l h u m a n blood is a good source of neutrophils (typical yield is 2 - 3 × 10 s neutrophils/100 ml blood) which are readily purified from the blood and can serve as a source of cytosol and m e m b r a n e fractions for optimization of the cell-free assay. However, this source does not generally provide enough neutrophils for purification of cytochrome b or the cytosolic components. Blood from patients with chronic myeloid leukemia contains up to approximately 100 times the normal neutrophil count and these patients are occasionally leukaphoresed to r e m o v e large n u m b e r of granulocytes. Additional sources of these ceils and purification methods are described in m o r e detail in Segal et al.12 5 A. Abo and E. Pick, J. BioL Chem. 266, 23577 (1991). 6 A. Abo, E. Pick, A. Hall, N. Totty, C. G. Teahan, and A. W. Segal, Nature 353, 668 (1991). 7 U. G. Knaus, P. G. Heyworth, T. Evans, J. T. Curnutte, and G. M. Bokoch, Science 254, 1512 (1991). 8 U. G. Knaus, P. G. Heyworth, B. T. Kinsella, J. T. Curnutte, and G. M. Bokoch, J. Biol. Chem. 267, 23575 (1992). 9 A. Abo, A. Boyhan, I. West, J. Adrian, Thrasher, and A. W. Segal, J. Biol. Chem. 24, 16767 (1992). l0 D. Rotrosen, C. L. Yeung, T. L. Leto, H. L. Malech, and C. H. Kwong, Science 256, 1512 (1992). 11D. Rotrosen, C. L. Yeung, and J. P. Katkin, J. Biol. Chem. 268, 14256 (1993). 12A. W. Segal, A. M. Harper, A. R. Cross, and O. T. G. Jones, in "Methods in Enzymology" (G. Di Sabato and J. Everse, eds.), Vol. 132, p. 378. Academic Press, San Diego, 1986.
270
CELL EXPRESSION
[291
+
0.,)
r,,)
z -O
z;
oa~
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[29]
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Methods Isolation of Neutrophils from Human Peripheral Blood. This is a brief description of neutrophil isolation. More detailed descriptions can be found in Segal et aL t2 Human blood (100 ml) is drawn from a healthy volunteer into preservative-free heparin (5 U/ml) mixed with 0.15 M NaC1 and sedimented with 10% volume of 10% dextran (w/v, average molecular weight 242,000) for approximately 30 min until erythrocytes have sedimented. The supernatant plasma containing the leukocytes is layered on Ficoll/Hypaque (Lymphoprep, Nycomed, Pharma, 5 vol of plasma on 1 vol of gradient) and is centrifuged at 400g for 15 min. The pellet contains the neutrophils and residual erythrocytes. The erythrocytes are lysed by hypotonic lysis. The pellet, up to 0.2 ml in volume, is resuspended in 20 ml of water and is vigorously mixed on a vortex mixer or pipetted up and down for 10 sec. Osmolarity is restored by the addition of 20 ml 0.31 M NaCI (1.8%) containing 5 IU/ml heparin. Preparation of Membranes and Cytosol. The neutrophils are resuspended at a concentration of 2 × 108 cells/ml in a break buffer consisting of 6 mM PIPES, pH 7.3, 6% (w/w) sucrose, 60 mM KC1, 1.8 mM NaC1, 2.3 mM MgCI2, IIzM diisopropyl fluorophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 /xg/ml TLCK. The cells are sonicated three times at 4° for 15 sec, overlaid on a discontinuous sucrose gradient (in 10 mM Tris, pH 7.4) of 15% (w/w) on top of 34% (w/w) on top of 45% (w/w), and centrifuged at 200,000g at 4°C for 15 rain in a TLX Beckman ultracentrifuge using a TLS55 swing-out rotor. The cytosol is collected above the 15% sucrose, the plasma membranes at the 15/34% interface, and the specific granules at the 34/45% interface. The specific granules and plasma membrane are diluted 1 : 4 with cold break buffer lacking sucrose and are pelleted by centrifugation as described earlier.
F~G. 1. Model of the role p21 Rac might play in the activation complex of the NADPH oxidase. The specialized components of the NADPH oxidase include the a and/3 subunits of flavocytochrome b and the two cytosolic proteins, p47ph°x and p67ph°x. p21 Rac is present in the GDP-bound form in a complex with GDI (GDP dissociation inhibition factor), an association that requires its modification by isoprenylation near its C terminus that could also be required for attachment to the membrane. Activation is associated with separation of p21 Rac from GDI, the exchange of GTP for GDP, and its movement with p47ph°x and p67ph°X in an activation complex into association with the flavocytochrome in the membranes. The bound GTP is then hydrolyzed to GDP as a consequence of endogenous GTPase activity of the rac or through modulation by GAPs that accelerate GTPase activity. The function of a third specialized cytosolic protein, p40ph°x, which is associated with p67ph°x but is not required in the cell-free system,15 remains to be determined.
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M e m b r a n e Solubilization and Reconstitution. The membrane and specific granules serve as a good source of cytochrome b for cell-free activation; however, better activity can be obtained if the membrane is solubilized with a detergent such as octylglucoside or deoxycholate which is then removed by dialysis or diluted below the critical micellar concentration to allow reconstitution of the proteins with the lipid vesicles. The pelleted membranes are resuspended to a concentration of 1-2 × 108 cells equivalents/ml in a cold solubilization buffer consisting of 120 mM sodium phosphate, pH 7.4, I mM MgC12, 1 mM EGTA, 1 mM dithiothreitol (DTT), 10-20% glycerol, 1 /xg/ml leupeptin, 1 mM PMSF, and 40 mM octylglucoside, then homogenized with a Dounce homogenizer. The clarified solubilized membranes are stirred on ice for 30 min to allow a complete solubilization. Insoluble material is removed by centrifugation at 200,000g for 15 rain as described earlier. Reconstitution of the soluble proteins into the lipid vesicle can be simply achieved by diluting the solubilized membrane with buffer lacking detergent to reduce the detergent concentration below the critical micelle concentration (CMC). One volume of solubilized membrane is mixed with 4 vol of solubilization buffer lacking octylglucoside which reduces the concentration of octylglucoside to 8 raM, well below the CMC of 25 mM, and is left on ice for 30-60 min. An alternative option is to dialyze the mixture against buffer lacking the detergent.
Purification of Cytochrome b Cytochrome b is purified from the membranes and specific granules of neutrophils as described previously. Briefly, membranes and specific granules are purified from large numbers of neutrophils from normal human blood or by leukapheresis of patients with chronic granulocytic leukemia by scaling up the procedures described earlier. The membranes and granules (approximately 1 vol) are homogenized in (approximately 10-20 vol) buffer A (100 mM Tris-acetate, pH 7.4, 100 mM KC1, 20% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, and protease inhibitors 1 mM PMSF, 1 mg/liter leupeptin, 1 mg/liter TLCK, and 1 mg/ liter pepstatin) containing 0.5% sodium cholate and centrifuged at approximately 100,000g. Cytochrome b remains largely in the pellet which is then washed with buffer without detergent and then extracted with buffer containing 1% Triton N-101 in roughly the same ratio of volumes of pellet to buffer. The Triton extract is then passed through columns containing DEAE-, CM-, and n-aminooctyl-Sepharose, and the cytochrome, which fails to attach to these resins, is trapped on heparin agarose from which it is eluted with a linear gradient of 0-1.0 M NaC1.
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Relipidation of Cytochrome b To obtain functional cytochrome b it is essential to reconstitute purified cytochrome b with lipid. This can be achieved by either preparing artificial lipid vesicles (35% phosphatidylcholine, Sigma) or extracting the endogenous lipid from neutrophil membranes. The lipid is then solubilized and combined with purified solubilized cytochrome b. Cytochrome b and the lipid associate upon removal of the detergent. Prior to relipidation of cytochrome b, the detergent is exchanged from Triton N-101 to solubilization buffer containing 40 mM octylglucoside. Cytochrome b is bound to the heparin agarose column in the Triton buffer, is washed with solubilization buffer containing octylglucoside, and is then eluted in break buffer containing 40 mM octylglucoside with a gradient of 0-1.0 M NaC1. To extracted lipids from neutrophil membranes, 200 ml of the membrane (protein concentration 2 mg/ml) is extracted with 1 ml of chloroform/ methanol (2:1, v/v), vortexed vigorously, and then centrifuged for 5 min at 400g in an Eppendorf centrifuge. The denatured proteins are removed from the interface, and the lower organic layer is evaporated under nitrogen. The dried extract of phospholipids is then solubilized in solubilization buffer. To optimize phospholipid/cytochrome b, 100 tzl of various concentrations of the solubilized phospholipids (100-400 p.g phospholipid) is mixed with 40 pmol of cytochrome b (40 tzl) and the mixture is diluted with 800 tzl solubilization buffer lacking detergent and is left on ice for 3060 min. The relipidated cytochrome is now ready to be used in the cellfree reconstitution assay. Preparation of p67 p~, p47Ph% and p21 Racl Isolation of large amounts of p67 ph°~ and p47ph°x from phagocytes is a laborious process since they are expressed at low abundance and are susceptible to proteolysis, particularly p67eh°~. However, it is possible to prepare small amounts of partially purified fractions enriched with these proteins as a good source for reconstitution in the oxidase. Purification of p21 Rac from phagocytes is described in [5] in this volume. Expression of these proteins in E. coli or in insect cells provides a good source of large amounts of pure proteins.
Preparation of Partially Purified p47ph°x and p67ph°xfrom Phagocyte Cytosol Ammonium sulfate precipitation provides a good method for the preparation of a fraction of cytosol that is enriched in p47ph°x and p67ph°x and depleted of p21 Rac. Ammonium sulfate is added over a period of 30 min
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to 10-20 ml of neutrophil cytosol (prepared as described earlier) to obtain a final concentration of 37% saturation and the mixture is stirred for 30 min on ice. The mixture is then centrifuged for 15 min at 200,000g in a TLX Beckman ultracentrifuge. The pellet is resuspended to its initial volume in 100 mM sodium phosphate buffer, pH 7.2, containing 1 mM MgC12, 1 mM DTT, 1 mM PMSF, and 1 tzg/ml leupeptin. The ammonium sulfate is removed from the supernatant by dialysis or by exchanging the buffer on a desalting column such as Sephadex G-25M PD10 (Pharmacia LKB Biotechnology Inc.). The presence of these proteins should be confirmed by Western blot analysis with antibodies which can be obtained from several laboratories active in the field. For reconstitution of the oxidase activity, it is also important to check that this fraction can reconstitute the oxidase activity only when supplemented with membrane fraction and p21 Rac protein as discussed below. Partially purified p47ph°x and p67ph°x can also be prepared by gelfiltration chromatography. Neutrophil cytosol is fractionated on a Superose 12 gel filtration column (Pharmacia) and fractions are collected and analyzed by Western blotting with antibodies against p47ph°x, p67ph°x, and p21 Rac. Typically, p47ph°x and p67ph°x coelute as a 260-kDa complex whereas p21 Rac elutes with R h o - G D I as a 45-kDa complex. It is also possible to prepare p47ph°x and p67ph°x by the absorption of neutrophil cytosol on 2',5'-ADP agarose as described. 13
Preparation of Recombinant p47ph°x, p6T 'h°x, and p21 Racl in E. coli Large quantities of p47ph°x, p67ph°x, and p21 Racl can be rapidly prepared in E. coli using the pGEX expression vector. Expression of tagged proteins in Sf9 cells is also a good expression system. Human p47ph°x,p67ph°x,and p21 Racl cDNAs are cloned into a glutathione S-transferase (GST) expression vector pGEX-2T as described previously. Transformed clones with pGEX containing p47ph°x, p67ph°x, or p21 Racl cDNAs were handled as following: 1. Terrific broth (50 ml) supplemented with 100 mg/ml ampicillin is inoculated with E. coli containing the desired clone rotated overnight at 37° in a shaking incubator. 2. The overnight culture is diluted 1 : 10 into fresh Terrific broth containing ampicillin and is grown for 1 hr. 3. Expression of the fusion protein is induced with 200 tzM IPTG and the culture is grown for an additional 4 hr. 13D. Sha'ag and E. Pick, Biochim. Biophys.Acta 1037, 405 (1990).
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OXIDASE ACTIVITY
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4. The cells are harvested by centrifugation at 300g for 15 min, and the obtained cell pellet is resuspended in 5-7 ml of lysis buffer containing 50 mM Tris, pH 7.5, 5 mM MgCI2, 1 mM DTF, and 1 mM PMSF. 5. The cell suspension is sonicated thoroughly (4 × 15 sec) on ice, and the cell debris is removed by centrifugation at 48,000g for 15 min. 6. The supernatant is incubated for 1 hr at 4° with 0.5-1 ml of glutathione-Sepharose beads (Pharmacia or Sigma) prewashed with lysis buffer. 7. The glutathione beads are washed three times with 10 ml cold lysis buffer by centrifuging the beads at 400g for 5 min and resuspending them in fresh buffer. 8. The bound proteins are eluted by resuspending the beads in 24 ml of lysis buffer containing 5 mM reduced glutathione (Sigma). The suspension is incubated for 2 rain, and the eluted proteins are recovered in the supernatant obtained by spinning down the beads. 9. To release the desired protein from the GST protein, thrombin cleavage is required. The buffer solution containing the fusion protein is exchanged into thrombin buffer containing 50 mM Tris-HC1, pH 8.0, 150 mM NaC1, 5 mM MgCI2, 2.5 mM CaC12, and 1 mM Dq-T. The buffer exchange can be achieved either by dialysis or by desalting on a PD10 column (Pharmacia). 10. The activity of thrombin obtained from different sources (most commonly human or bovine) varies substantially, making it essential to optimize the cleavage conditions. A small-scale reaction is set by incubating 20 t~g of fusion protein with various amounts of thrombin (1-20 units depending on the source of thrombin) for 1 hr at room temperature. The efficiency of the cleavage is analyzed by S D S - P A G E visualized by Coomassie blue staining. 11. Once the optimal ratio of thrombin to fusion protein is obtained, the reaction is scaled up and the rest of the fusion protein is cleaved. 12. The activity of thrombin is inhibited by the addition of 10 /zM APMSF (Sigma Cat. No. A6664), and the efficiency of the cleavage is again assessed by SDS-PAGE. 13. A further purification is required to separate the fusion protein from GST. This can be achieved either by reabsorbing the GST on glutathioneagarose or by separation by anion-exchange chromatography on a Mono Q column (HR 5/5 Pharmacia) under standard conditions. For instance, p21 Racl and p47ph°xwere separated from GST by fractionation on a Mono Q column; the GST bound to the Mono Q (and can be eluted by 250300 mM NaC1) and p21 Racl and p47ph°xremained in the unbound fractions. Typically, 1 mg of pure recombinant protein is produced using this method from 1 liter of broth culture. The proteins were concentrated by centrifuga-
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tion on Centricon 10 membrane concentrators (Amicon) to a concentration of 1 mg/ml, aliquoted to 50/xl/vial, snap frozen in liquid nitrogen, and stored at - 7 0 °. R e c o n s t i t u t i o n of Cell-Free NADPH Oxidase
Assay for Superoxide Production The most widely used assay for measuring superoxide production is the reduction of cytochrome c inhibitable by superoxide dismutase (SOD). The assay has been described in detail by Pick. 14 The superoxide ions generated by the activated oxidase reduce cytochrome c, which has an absorption maximum at 550 nm. The rate of superoxide generation is calculated from the slope of the linear range of the absorbance at 550 nm, using e550 = 2.1 × 104 M -1 cm -1 Since cytochrome c can be reduced by reducing sources other then superoxide, it is essential to demonstrate that the reaction is inhibitable by SOD, indicating that the reduction of cytochrome c is specific.
Cell-Free Assay a. Using Cytosol and Membranes. It is advisable to first optimize the reconstitution of the oxidase in the cell-free assay with the solubilized membrane and cytosol. This helps in identifying an inactive recombinant protein. This system can also be used to test cytosol and membranes from patients with C G D to identify the impaired component as described in this series. Solubilized membrane (30-50 ml) (diluted 1:8 to dilute the detergent, 5-20 mg protein) and 10-40/xl cytosol (50-150 mg) are incubated for 90 sec in 0.9 ml assay buffer consisting of 65 m M sodium phosphate buffer, p H 7.0, 1 m M E G T A , 1 m M MgCI2, 1 0 / z M FAD, and 1 0 0 / z M cytochrome c. To determine the optimal concentrations of the SDS activator, various concentrations of SDS are added to the incubation mixture. SDS (6-15 ml) from a 10 m M stock solution is added to the incubation mixture to achieve optimal oxidase activation. A final concentration of SDS is 80-120 mM. The reaction is then initiated by the addition of 200 tzM N A D P H (20 ml from 10 m M stock), and the rate of superoxide generation is measured for 2 min in a double beam spectrophotometer (Uvikon 860, Kontron). The reference sample contains SOD (Sigma; we use at 50/xg/ ml) in addition to reagents mentioned previously. The same procedure can be applied with partially purified components. The solubilized membrane 14E. Pick, in "Methods in Enzymology" (G. Di Sabato and J. Everse, eds.), Vol. 132, p. 407. Academic Press, San Diego, 1986. 15F. B. Wientjes, J. J. Hsuan, N. F. Totty, and A. W. Segal, Biochem. J. 296, 557 (1993).
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(as mentioned earlier) is incubated with a fraction enriched in p47ph°x and p67ph°xobtained by the methods described earlier, supplemented with either partially purified or recombinant p21 Racl and an optimal concentration of SDS, incubated for 2 min, and electron transport initiated with NADPH. Note: Figure 2 shows that it is possible to demonstrate that reconstituted activity is dependent on the different components described earlier. b. Reconstitution of Cell-Free NADPH Oxidase from Purified Components. Since the recombinant p21 Racl purified from E. coli is predominantly in the GDP-bound form, to obtain a functional GTPase protein it is essential to exchange the nucleotide to either GTP or GTPyS. This is done by incubating the protein (100/xg/ml) in the presence of 100 mM guanine nucleotide in 5 mM EDTA for 10-15 min at room temperature followed by the addition of 10 mM MgC12 to chelate the EDTA and lock the nucleotide into the protein. Once all the reagents are prepared and the cell-free assay is optimized, the system is ready for reconstitution with purified components. Higher enzymatic activities can be obtained by separating the activation process from the catalysis reaction. Thus, the purified components are first preincubated in an optimal SDS concentration in a small volume (100 txl) of assay buffer containing but lacking cytochrome c and containing FAD
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p67-phox p47-phox p21rac1 SDS
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FIG. 2. Reconstitution of NADPH oxidase activity in the cell-free system by recombinant
p67ph°x, p47ph°x, p21 Racl, and pure cytochrome b. Solubilized neutrophil membrane (6/zg protein, 4 pmol cytochrome b) or pure relipidated cytochrome b (4 pmol) was incubated either with 30 /zl neutrophil cytosol (100 p~g protein) or with p67ph°x (4.5 nM), p47ph°x (43.5 nM), and p21 Racl (48.5 nM) preloaded with GTP and the optimal concentration of SDS (where indicated).
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and then transferring this to 0.9 ml of catalysis buffer to initiate the reaction with NADPH. Ten microliters of relipidated cytochrome b (0.1-0.5/zg, 1-5 pmol) is preincubated for 2 min at room temperature with 2-40/.d (0.1-4/zg protein) p47ph°x, p67ph°x, and p21 Racl in the presence of an optimal concentration of SDS (100-200/zM, 10-20/zl from 1 mM stock) and 10 t~M FAD, and the volume is complete to 100/xl with assay buffer lacking cytochrome c. The mixture is then transferred to a 1-ml cuvette containing 0.9 ml assay buffer with 100/zM cytochrome c and the reaction is initiated by the addition of 20/xl of NADPH (from 10 mM stock). The superoxide production is monitored for 120 sec in a double-beam spectrophotometer as described earlier. The assay can be performed in a microplate as described in a previous volume of this series.
Calculations Since the reduction of cytochrome c represents the amount of superoxide generated (1 mol of superoxide will reduce 1 tool of cytochrome c), the rate of superoxide generation is calculated from the slope of the linear range of the absorbance at 550 nm, using e550 = 2.1 × 104 M -1 cm 1 of cytochrome c: Absorbance change of 1 unit at 550 n m × 47.6 = nmol 02-. The specific activity is expressed as either nmol O2/min/mg of membrane protein or as mol O2-/mol cytochrome b-245/sec. Typical values should be in the range of 1500-3000 nmol O2-/min/mg protein and 60-120 mol O2-/mol cytochrome b-245/sec, respectively. Acknowledgment We thank the Wellcome Trust for support.