Biochem. J. (1984) 223, 639-648Printed in Great Britain

Composition of partially purified NADPH oxidase from pig neutrophils

Paolo BELLAVITE,* Owen T. G. JONES,t Andrew R. CROSS,f Emanuele PAPINI*and Filippo ROSSI*

*Istituto di Patologia Generale, Universitai di Verona, Strada le Grazie, 37134 Verona, Italy, andtDepartment ofBiochemistry, Medical School, University of Bristol, Bristol BS8 ITD, U.K.

(Received 16 April 1984/Accepted 13 June 1984)

The superoxide (02-)-forming enzyme NADPH oxidase from pig neutrophils wassolubilized and partially purified by gel-filtration chromatography. The purificationprocedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductaseactivities. 02'--forming activity was co-purified with cytochrome b-245 and wasassociated with phospholipids. However, active fractions endowed with cytochromeb were devoid of ubiquinone and contained only little FAD. The cytochrome b/FADratio was 1.13:1 in the crude solubilized extract and increased to 18.95: 1 in the par-tially purified preparations. Most of FAD was associated with fractions containingNADH-dependent oxidoreductases. These results are consistent with the postulatedrole of cytochrome b in O2- formation by neutrophil NADPH oxidase, but raisedoubts about the participation of flavoproteins in this enzyme activity.

The production of superoxide (02--) radicalions from neutrophils appears to be due to thespecific activation of a membrane-bound NADPHoxidase, which is inactive in resting cells (Rossi &Zatti, 1964; Patriarca et al., 1971; Hohn & Lehrer,1975; Babior et al., 1976). Much information onthe nature of this enzyme has been accumulated inrecent years, as a consequence of studies onisolated membranes (Cross et al., 1981; Babior etal., 1981; Sloan et al., 1981; Gabig et al., 1982), onsolubilized and partially purified preparations ofoxidase (Tauber & Goetzl, 1979; Babior & Peters,1981; Light et al., 1981; Wakeyama et al., 1982;Bellavite et al., 1983a; Gabig & Lefker, 1984) andon patients affected by chronic granulomatousdisease, a hereditary condition where the 02'-formation by phagocytes is lacking (Segal et al.,1978; Tauber et al., 1983; Gabig, 1983). There is asubstantial agreement that NADPH oxidase con-tains FAD (Babior & Peters, 1981; Light et al.,1981; Cross et al., 1982b; Michell, 1983) and a cyto-chrome b-245 (Cross et al., 1981; Gabig et al.,1982; Borregaard et al., 1982; Bellavite et al.,1983a; Morel & Vignais, 1984) and that it needsphospholipids for optimal activity (Gabig &Babior, 1979). Other authors indicated also ubi-

Abbreviation used: DCIP, 2,6-dichlorophenol-indophenol.

quinone as a possible oxidoreduction component(Crawford & Schneider, 1982, 1983), but this wasnot confirmed by others (Cross et al., 1983).However, true evidence of the participation ofindividual components in the 02--generatingsystem is lacking, because attempts to isolate andpurify the oxidase in an active state have beenunsuccessful. The main reason for this is themarked instability of the enzyme activity ondetergent extraction (Patriarca et al., 1974; Tauber& Goetzl, 1979; Babior& Peters, 1981 ; Light et al.,1981; Bellavite et al., 1983a; Gabig, 1983). More-over, the respiratory burst of neutrophils isreversible when the stimulatory agent is removed(Rossi et al., 1983), indicating that the return of theNADPH oxidase to the resting state can easilyoccur.

In spite of many attempts made in recentyears in our laboratory (Bellavite et al., 1983a,b;Serra et al., 1983), we could not achieve completepurification. However, in the course of thesestudies, we were able to solubilize the 02'_-forming enzyme from pig neutrophils with mini-mum loss of activity and to obtain a partiallypurified preparation by gel-filtration chromato-graphy. The analysis of such a preparation, withspecial regard to cytochrome b, FAD, quinone andphospholipid content, is reported in the presentpaper.

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ExperimentalMaterials

Ficoll 400 was purchased from Pharmacia FineChemicals, Uppsala, Sweden. Dextran (Mr300000) was purchased from Serva Feinbio-chemica, Heidelberg, West Germany. Phorbol-12-myristate-13-acetate, phenylmethanesulphonylfluoride, di-isopropyl phosphorofluoridate, cyto-chrome c (type VI), DCIP, NADPH (type III),NADH (type III), Lubrol PX and sodium deoxy-cholate were purchased from Sigma Chemical Co.,St. Louis, MO, U.S.A. Sodium deoxycholate wasrecrystallized from ethanol (MacLennan, 1971).Ultrogel AcA-22 was purchased from LKB Pro-dukter, Bromma, Sweden. Human CuZn super-oxide dismutase was a gift from Dr. J. V. Bannister(Department of Inorganic Chemistry, Universityof Oxford, Oxford, U.K.). Ubiquinone-10 was agift from Dr. W. T. Griffiths (Department of Bio-chemistry, University of Bristol, Bristol, U.K.). Allother reagents were the highest grade available.

Isolation of neutrophilsFresh pig blood, made incoagulable with 0.8%

sodium citrate and 20mM-EDTA, was transferredto plastic cylinders, and dextran was added at thefinal concentration of 2.5% (w/v). After sedimenta-tion of erythrocytes, the supernatant was centri-fuged at 500g for 10min. Contaminating erythro-cytes were lysed by suspending the cell pellet in0.2% NaCl for min. Hypo-osmotic and hyper-osmotic (1.2% NaCl) solutions employed for thehaemolytic treatment contained 2mM-EDTA and0.5 i.u. of heparin/ml. Usually two to four cycles ofhypo-osmotic treatment were necessary in order toremove all erythrocytes. Leucocytes were suspend-ed in Krebs-Ringer phosphate buffer (122mM-NaCl/4.8 mM-KCl/ 1.2mM-MgCl2/ 17mM-sodiumphosphate buffer, pH 7.4) containing 5 mM-glucose,2mM-NaN3, 2mM-EDTA and 0.5i.u. of hepar-in/ml (KRP medium), at the concentration ofabout 109 cells/ml. Any macroscopic aggregatesand fibrin clusters were removed by filtration ongauze. Leucocyte suspension was layered on aFicoll solution (5.7% Ficoll 400, 9% sodiumdiatrizoate, 0.2mM-EDTA) and centrifuged at500g for 20min at room temperature. The pellet,containing more than 95% pure neutrophils, wassuspended in ice-cold KRP medium at a concen-tration of about 109 cells/ml and treated with3mM-di-isopropyl phosphorofluoridate for 5min.Neutrophils were then washed twice and finallysuspended in KRP medium.

Cell activation and preparation of subcellularparticles

Neutrophils were activated by exposing the cellsuspension (5 x 107 cells/ml) to 1 jug of phorbol-12-

myristate-13-acetate/ml for 5min at 370C withstirring. Preliminary experiments demonstratedthat this treatment caused the maximum possibleactivation of the respiratory burst of neutrophils.The incubation was stopped by addition of anexcess of ice-cold KRP medium and immediatelycentrifuged. After this point, all experimental stepswere performed at 0-40C. Non-activated (resting)neutrophils were centrifuged without previousexposure to phorbol-12-myristate-13-acetate. Thepellet was resuspended in 10mM-Tris/HCl buffer,pH 7.0, containing 0.34M-sucrose, 2mM-NaN3,2mM-EDTA and 1 mM-phenylmethanesulphonylfluoride, and sonicated with two to four bursts of10s at 100W, until 90% of cells were disrupted.The homogenate was centrifuged at 400g for10min in order to sediment nuclei and unbrokencells. Then 2 vol. of the supernatant was layered on1 vol. of lOmM-Tris/HCl buffer, pH 7.0, containing0.68M-sucrose, 2mM-NaN3, 2mM-EDTA and1 mM-phenylmethanesulphonyl fluoride and cen-trifuged at IOOOOg for 1 h. The pellet, containingthe subcellular particles, was suspended in 10mM-sodium phosphate buffer, pH 8.0, containing 20%(v/v) glycerol, 1 mM-phenylmethanesulphonyl fluo-ride, 2mM-NaN3, 1 mM-EGTA and 1 mM-MgSO4(glycerol/phosphate buffer) at a concentration of10mg of protein/ml.

Solubilization of subcellular particles and gelfiltration

To the subcellular particles 0.4% Lubrol PX and0.4% sodium deoxycholate were added. The mix-ture was kept at 0°C for 10min under magneticstirring, then sonicated with three bursts of 20sat 100 W. The detergent-treated particles werecentrifuged at 100000g for 1 h. Then 0ml of the100000g supernatant was immediately applied toan Ultrogel AcA-22 column (3.8cm x 16cm) equili-brated with glycerol/phosphate buffer. Fractionsof volume 4ml were collected at a flow rate of1 ml/min.

AssaysAssays of enzyme activity were carried out with

a Perkin-Elmer 576 double-beam spectrophoto-meter at 220C. Reference and sample cuvettescontained 50mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid]/NaOH buffer,pH 7.0, 1 mM-diethyltriaminepenta-acetic acid,2mM-NaN3,0.15mM-NADPH or 0.15mM-NADHand the appropriate electron acceptor. The finalvolume was 1 ml. NAD(P)H-cytochrome c reduc-tase activities were measured in the presence of80 M-cytochrome c and of 60Mg of superoxidedismutase. The reaction was started by adding theenzyme to the sample cuvette and an equal volumeof glycerol/phosphate buffer to the reference

1984

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Composition of partially purified NADPH oxidase

cuvette, and the absorption increase at 550nmwas monitored. Cytochrome c reduction was calcu-lated by using the absorption coefficient21.1 mm-1Icm-1 (Van Gelder & Slater, 1962).NAD(P)H oxidase activity was measured as O2*-production by the rate of cytochrome c reductionmeasured in the absence of superoxide dismutaseminus the rate of NAD(P)H-cytochrome c reduc-tase activity (Babior et al., 1976). NADH-DCIPreductase activity was measured in the presence of60.uM-DCIP and of 60ig of superoxide dismutase,and the reaction was started by adding the enzymeto the reference cuvette and an equal volume ofglycerol/phosphate buffer to the sample cuvette.Since DCIP is decolorized on reduction, theenzyme-catalysed reaction in the reference cuvettecaused a net increase of the differential absor-bance, which was recorded at 600nm. DCIPreduction was calculated by using the absorptioncoefficient 16.1 mm-'1 cm-' (Dawson et al., 1969)and is expressed in terms of e /min, on the basisthat 1 mol of DCIP is reduced by 2equiv. of e-.Cytochrome b was measured by reduced-

minus-oxidized difference spectroscopy byusing the absorption coefficient (E559 -E540)21.6mM- 1 cm-1 (Cross et al., 1982a).

Flavins were extracted by heating the sample at100°C for 5min before precipitating the proteinwith ice-cold 0.6M-HC104. After centrifugation at1000OOg for 2h, the supernatant was adjusted topH 7.5 with 5M-KOH and with addition ofNa2HPO4 to the final concentration of 0.1 M. Thewhite precipitate (KC104) was removed by centri-fugation at lOOOg for 10min. FAD and FMN wereassayed fluorimetrically by the method ofFaeder &Siegel (1976).

Ubiquinone-10 was extracted with light petro-leum (b.p. 50-60C) as described by Redfearn

(1967) and was measured by u.v. absorption at275nm after purification by reverse-phase high-pressure liquid chromatography on a 200mmApex-ODS silica (pore size 5Mm) column elutedwith acetonitrile/diethyl ether (4: 1, v/v), withubiquinone-10 as standard. Standard ubiquinone-10 was determined by oxidized-minus-reduceddifference spectroscopy by using the absorptioncoefficient (82 7 5,ox. -627 5,red.) 12.25 mM*cm-1 (Red-fearn, 1967).

Proteins were measured, after trichloroaceticacid precipitation, by the method of Lowry et al.(1951), with albumin as standard.

Phospholipid determination was carried outafter extraction with chloroform/methanol (2:1,v/v). Phosphate was determined by the method ofBartlett (1959).

ResultsSolubilization ofNADPH oxidase, cytochrome b andFAD

Solubilization of membrane-bound enzymes isoften complicated by loss of catalytic activity, andNADPH oxidase in particular is known to beunstable on detergent extraction. Preliminaryexperiments were therefore performed in order tooptimize the solubilization procedure. The bestresults (i.e. maximum solubilization with mini-mum enzyme inactivation) were obtained bytreating the subcellular particles, suspended inglycerol-containing buffer, with a mixture of 0.4%Lubrol PX and 0.4% sodium deoxycholate. Asshown in Table 1, in these conditions the treatmentof subcellular particles with detergent caused aslight increase in the specific activity of the °2 --forming enzyme, due probably to a better accessi-bility of substrates and of cytochrome c to the

Table 1. Solubilization ofNADPH oxidase, oj cytochrome b, ofFAD and ofproteins from subcellular particles ofphorbol-12-myristate-13-acetate-activated neutrophils

Subcellular particles were treated with 0.4% Lubrol PX and 0.4% deoxycholate as described in the Experimentalsection and were centrifuged at lOOOOOg for 1 h. NADPH oxidase activity and cytochrome b, FAD and proteincontents of various fractions were assayed as described in the Experimental section. 'Specific' refers to enzymeactivity or cytochrome b, FAD or protein content/mg of protein. 'Total' refers to total activity or content obtained inthe fraction from 109 neutrophils. The '%' values concern the total activity or content of the fraction with respect tothe total activity or content of subcellular particles (treated). The means+ S.E.M. for three separate experiments arereported.

SubcellularparticlesSpecific

Subcellular particles(treated)

e A ToaSpecific Total

NADPH oxidase 41.3+7.2 58.6+15.0(nmol of O2'-/min)

Cytochrome b (pmol) 112.4±8.3 125.3+20.1FAD (pmol) 79.2+11.7 80.5+10.5Protein (mg) - -

621.2

1328.2853.2

10.6+ 0.7

100000g supernatantr A- I

Specific Total %54.9+14.9 323.9 52.1

1000OOg pelletr

Specific29.9+12.1

121.9+ 19.9 719.2 54.1 85.4+6.0107.1 + 15.6 631.9 74.0 54.8+6.6

- 5.9+0.8 55.7 -

Total %170.4 27.4

468.8 36.6312.3 36.6

5.7+0.8 53.8

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solubilized enzyme. No modifications of cyto-chrome b and of FAD content were caused bydetergents. After ultracentrifugation, the super-natant contained about 50% of protein, ofNADPH oxidase and of cytochrome b. The totalrecovery of enzyme activity (supernatant+ pellet)was 79.5%, indicating a small loss of activity. Thesupernatant contained 74% of the FAD present insubcellular particles. Solubilized FAD was pro-tein-bound, i.e. in the form of flavoprotein, sinceafter chromatography of the 1000OOg supernatanton Sephadex G-25 at least 85% of FAD was co-eluted together with the protein peak in the voidvolume (results not shown).

Gel filtration of the solubilized extractThe solubilized preparation was filtered through

an Ultrogel AcA-22 column, and the chromato-graphic profile shown in Fig. 1 was obtained.Proteins (Fig. la) were separated into two regions:a smaller peak was eluted in the void volume,whereas most of the proteins appeared as a largepeak in the included volume. When the absorb-ance at 280nm of the fractions was measured(Fig. lb), the peak eluted in the void volumeappeared to be relatively greater than the proteinpeak measured with the Lowry method (Fig. la).This indicates the presence of other non-proteincompounds absorbing in the u.v. These compo-nents were identified as phospholipids by measur-ing the phosphorus content. The first peak of gelfiltration contained 1.34mg of phospholipid/mg ofprotein, a value much higher than that of thesecond peak (0.10mg of phospholipid/mg ofprotein) and that of the 10OOOOg supematant(0.15mg-of phospholipid/mg of protein).

Fig. l(c) shows that NADPH oxidase activitywas eluted as a major peak in the void volume andthat a smaller peak of activity was retained by theresin. A similar chromatographic behaviour wasexhibited by cytochrome b (Fig. le), with the onlydifference that the second peak was relativelygreater than the second peak of oxidase activity.

Superoxide dismutase-insensitive NADPH-dependent cytochrome c-reducing activity(NADPH-cytochrome c reductase, Fig. ld) wasvery low in all fractions, and it was mostlyrepresented in the second peak. The activities ofsuperoxide dismutase-insensitive NADH-depen-dent cytochrome c reductase and DCIP reductasewere much higher and easier to measure than thecorresponding activities measured with NADPHas substrate. The elution profile of one of thesediaphorase enzymes is reported in Fig. 1(1): thebulk of NADH-DCIP reductase activity wasfound in the included volume of the chromato-graphic fractionation, but a small contaminationby this enzyme was still present in the first peak. A

similar profile was obtained by measuringNADH-cytochrome c reductase activity (resultsnot shown).

Enzyme activities, cytochrome b and FAD content ofthe two peaks separated by gel filtration

In the subsequent chromatographic experimentsafter gel filtration all the fractions were dividedinto two groups, according to the two main peaks'measured as absorbance at 280nm, and the varioussamples were pooled. For example, in the experi-ment shown in Fig. 1 the pool of the first peakwould have been composed of fractions 11-16, thepool of the second peak of fractions 17-40. Table 2reports the enzyme specific activities and the cyto-chrome b and FAD specific content of the twopeaks compared with those of the 100lOOg super-natant loaded on the column. NAD(P)H-depen-dent O2---forming activity present in the first peakwas purified 4-5-fold with respect to the 1000OOgsupernatant. As expected, the activity was muchhigher with NADPH than with NADH as sub-strate. A very similar purification was observed asregards the cytochrome b, so that the oxidase/cytochrome b ratio in the partially purified fractionremained very similar to the ratio in the unfrac-tionated supernatant. NADPH-cytochrome c re-ductase activity of the first peak increased only 1.5-fold, whereas NADH-cytochrome c reductaseand NADH-DCIP reductase activities markedlydecreased. Since the exclusion limit of UltrogelAcA-22 resin is of about 1.2 x 106 daltons, theseresults indicate that the chromatography allowedthe separation of high-molecular-mass com-plexes, enriched in phospholipids, NADPH oxi-dase activity and cytochrome b, from oxidoreduc-tase enzymes that are in a more soluble form andare retained by the resin. Unlike the 02- -formingenzyme, these oxidoreductases use NADH aspreferential electron donor.

Unexpected results were obtained on measure-ment of the flavin content. The first peakcontained little FAD, so that its specific contentdecreased from 117.5pmol/mg of protein in the10OOOOg supernatant to 35.7 pmol/mg of protein inthe fractions where NADPH oxidase activity andcytochrome b concentration were found to beincreased. As a consequence, the cytochrome blFAD ratio increased from 1.13:1 to 18.95:1 andthe NADPH oxidase/FAD ratio increased from0.31:1 to 4.20:1. In contrast, the NADH-cyto-chrome c reductase/FAD ratio remained almostconstant, indicating a similar distribution in thechromatographic separation. Tables 1 and 2 do notreport the values of FMN determination becausethis flavin cofactor was present only in traceamounts (less than 10-20% of total flavins) andthen not in all preparations tested, so that its

1984

642

Composition of partially purified NADPH oxidase

.* 601E

500C...

_ 40

30a)av*~ 200

-

10

Z 0

240

.. 200

0

X160

U)

° 80

2 40

U 0

(c) a) 01U

150

e_

0 v 40E .=O e-C 300 10

I- 20

10

z

(d)

I10

Fraction no.

Fig. 1. Gel filtration of the solubilized extract of subcellular particles from phorbol-12-myristate-13-acetate-activated pigneutrophils

A lOml portion of the IOOOOOg supernatant was chromatographed on an Ultrogel AcA-22 column, and 4mlfractions were collected and assayed as described in the Experimental section. The iOOOOOg supernatant contained6mg of protein/ml and 750pmol of cytochrome b/ml, and the enzyme activities were: NADPH oxidase, 220nmol of02' /min per ml; NADPH-cytochrome c reductase, 64nmol of e-/min per ml; NADH-DCIP reductase, 280nmolof e /min per ml.

presence could be attributed to breakdown ofFADduring the extraction procedure. In order to checkif the finding of a small amount ofFAD in the firstpeak was due to incomplete extraction, in someexperiments 0.5% deoxycholate was added duringthe heat-extraction of flavins. No increase ofFADmeasured in the first peak was caused by thedetergent, indicating that the presence of phospho-

lipids does not interfere with the extractionprocedure.

In some experiments the proteinase inhibitorphenylmethanesulphonyl fluoride was omittedfrom the glycerol/phosphate buffer and the flavinextraction was carried out after the treatment ofthe samples with 5mg/ml of trypsin at 37°C for 1 h.Trypsin treatment did not significantly change the

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Table 2. NAD(P)H oxidase, NAD(P)H-cytochrome c reductase and NADH-DCIP reductase activities and cytochrome b,flavin and protein contents in various fractions obtained from phorbol-12-myristate 13-acetate-activated neutrophilsThe lOOOOOg supernatant from phorbol-12-myristate-13-acetate-activated neutrophils was chromatographed on anUltrogel AcA-22 column, and the fractions of the two main protein peaks were pooled as described in the text.Assays were carried out as described in the Experimental section. Specific activities and contents are expressed asfollows: NADPH oxidase and NADH oxidase, nmol of O2'-/min per mg of protein; NADPH-cytochrome creductase, NADH-cytochrome c reductase and NADH-DCIP reductase, nmol of e-/min per mg of protein;cytochrome b and FAD, pmol/mg of protein; protein, mg of protein recovered in fraction from 109 neutrophils.Recovery values concern the sum of total activity or content of the first and second peaks with respect to totalactivity or content of lOOOOOg supernatant. The means+S.E.M. for four separate experiments are reported.

Specific activity or content

NADPH oxidaseNADH oxidaseNADPH-cytochrome c reductaseNADH-cytochrome c reductaseNADH-DCIP reductaseCytochrome bFADProteinNADPH oxidase/cytochrome b ratioCytochrome b/FAD ratioNADPH oxidase/FAD ratioNADH-cytochrome c reductase/FAD ratio

lOOOOOgsupernatant36.6+6.110.5+ 2.39.8+5.196.8+28.576.5 +9.5133.1+18.4117.5+ 12.25.9+0.80.271.130.310.82

First peakfrom gel filtration

150.0+ 30.258.6+ 18.314.4+ 5.729.2+7.749.9+ 16.2

676.8 +4.635.7+5.90.5+0.1

0.2218.954.200.82

Second peakfrom gel filtration

9.5+1.91.0+0.93.6+0.4

128.9+30.458.3 +9.259.8 +9.8122.3+3.55.2+0.30.160.490.081.05

Table 3. NAD(P)H oxidase, NAD(P)H-cytochrome c reductase and NADH-DCIP reductase activities and cytochrome b,flavin and protein contents in various fractions obtained from resting neutrophils

The 1000OOg supernatant from resting neutrophils was chromatographed on an Ultrogel AcA-22 column, and thefractions of the two main protein peaks were pooled as described in the text. Assays were carried out as described inthe Experimental section. Specific activities and contents are expressed in the units indicated in Table 2 legend. Thevalues of two separate experiments are reported.

Specific activity or content

NADPH oxidaseNADH oxidaseNADPH-cytochrome c reductaseNADH-cytochrome c reductaseNADH-DCIP reductaseCytochrome bFADProtein

lOOOOOgsupernatant0.9-1.70.7-0.74.4-9.1

71.6-119.555.2-65.597.3-127.089.1-94.48.3-9.4

First peakfrom gel filtration

0.8-3.95.4-1.710.4-8.431.7-38.921.1-26.9

698.8-934.531.5-51.70.6-0.5

Second peakfrom gel filtration

1.1-0.90.8-0.84.3-5.7

98.8-117.839.3-55.151.7-65.382.8-88.09.3-9.7

amount of extracted FAD, indicating that flavin isnon-covalently bound to the proteins.

Table 3 reports the results of two chromato-graphic fractionations carried out on preparationsobtained from non-stimulated neutrophils.NAD(P)H oxidase activity was practically un-detectable, whereas the rates of superoxide dis-mutase-insensitive cytochrome c reduction andDCIP reduction were in the same range as thosemeasured in fractions obtained from phorbol- 12-myristate-13-acetate-activated neutrophils. Cyto-chrome b and FAD distribution in the two peaks

obtained from gel filtration did not change withrespect to the distribution observed in Table 2.

Ubiquinone content

Fig. 2 shows the high-pressure-liquid-chromato-graphic profiles of extracts of subcellular particlesand of the first peak obtained from Ultrogel AcA-22 chromatography. Ubiquinone content was verylow, even in the subcellular particles (about20pmol/mg of protein). For this reason in thisexperiment the highest possible sensitivity had tobe used, causing the 'noise' present in the reported

1984

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57.655.744.8119.972.782.694.396.6

644

Composition of partially purified NADPH oxidase

(d)

Fig. 2. High-pressure-liquid-chromatography recording traces of ubiquinone analysisLight-petroleum extracts of ubiquinone-10 standard (140pmol) (a), of subcellular particles from activatedneutrophils (4.4mg of protein, 460pmol of cytochrome b) (b), of the first peak from Ultrogel AcA-22chromatography of resting preparations (1.0mg of protein, 643 pmol of cytochrome b) (c) and of the first peak fromUltrogel AcA-22 chromatography of activated preparations (0.8mg of protein, 514pmol of cytochrome b) (d) werefractionated by high-pressure liquid chromatography as described in the Experimental section, and the absorbanceat 275nm of the eluate was monitored.

traces. Ubiquinone in the first peak was undetect-able. Considering the sensitivity limits of themethod and the amount of sample extracted, it canbe calculated that the ubiquinone/cytochrome bratio in this fraction is certainly less than 1:20.

DiscussionIn spite of the central role played by NADPH

oxidase in the generation of toxic radicals byphagocytes, the structure and the components ofthis enzyme (or enzymic system) are not com-pletely defined. The main reason for this is the lackof complete purification of the enzyme. At present,information on the nature ofNADPH oxidase canbe obtained by functional studies or by analysingpartially purified enzyme as compared with non-purified preparations. This latter procedure wasadopted in the present work.Sound evidence of the participation of some

component in the O2--forming system can beobtained only by studying enzyme preparationsthat are endowed with their full 02'--forming

activity. Care was therefore taken in order tosolubilize and isolate the enzyme with minimuminactivation. Satisfactory results were obtained bysubjecting the solubilized extract to gel-filtrationchromatography on Ultrogel AcA-22 (Tables 1 and2). The method allowed the isolation of an high-molecular-mass complex where the radical-gener-ating oxidase was purified 4-5-fold with respect tothe non-fractionated preparation (Table 2). Prob-ably the high catalytic activity of this complex waspreserved by the presence of phospholipids, whichare necessary for the activity of NADPH oxidase(Gabig & Babior, 1979).The peak containing the O2 --forming activity

was separated by gel filtration from a second peakcontaining most of NAD(P)H-cytochrome c re-ductase and NADH-DCIP reductase activity(Fig. 1). This separation indicates that superoxidedismutase-insensitive reduction of electron accep-tors such as cytochrome c and DCIP is catalysed byenzymes not involved in the 02 - formation. Twoother observations support this conclusion: (a) thesuperoxide dismutase-insensitive oxidoreductase

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activities use NADH as preferential substrate and(b) they are not activated in fractions obtainedfrom phorbol-12-myristate-13-acetate-treated cells(compare Tables 2 and 3). The physiological role ofthese diaphorase enzymes in the neutrophil is notknown.

Oxidase activity co-purified with cytochrome b,and this is a further indication that this cyto-chrome, whose particular feature is low midpointpotential (Em,7.0 =-245mV) (Cross et al., 1981),is strictly related to the O2 --generating electron-transport system.

In addition to cytochrome b, it has beensuggested that a quinone and FAD may beinvolved in the radical-generating oxidase. Wetherefore measured the amount ofthese cofactors inthe partially purified preparations. The quinoneanalysis was not able to demonstrate the presenceof quinone in the oxidase-cytochrome b-phospho-lipid complex. Traces of ubiquinone were found inthe subcellular particles. The discrepancy with theresults of other authors (Crawford & Schneider,1982, 1983) may be explained in terms of purity ofthe preparation. As reported by Cross et al. (1983),the quantity of ubiquinone in neutrophils is verylow (25.5 pmol/mg of cell protein) and it isassociated with the mitochondrial fraction. In thepresent investigation the isolation of neutrophilsby a method that allowed us to obtain cellpreparations at least 95% pure, the solubilizationof cell-free particles and the gel filtration gave anactive enzyme fraction practically free of quin-ones. This excludes the participation of thesecofactors from the 02- -forming activity of neutro-phils and agrees with the results obtained by Crosset al. (1983) with pure membrane fractions.

Unexpectedly, the FAD content of the purifiedoxidase preparation was very low as comparedwith the cytochrome b content. Since no doubts onthe presence of a flavoprotein in the system havebeen raised until now, this point deserves a specialcomment. First of all, we should rule out possibletrivial explanations such as inadequate extractionand loss of flavin during the experiment. In-adequate extraction of FAD can be excluded forthe following reasons. (a) The amount of FADextracted from non-purified fractions was about100pmol/mg of protein and the cytochrome blFAD ratio was about 1:1. These-values are in thesame range as those previously reported by us andby others for crude cell fractions (Cross et al.,1982b; Gabig, 1983; Bellavite et al., 1983b). (b)Addition of deoxycholate during flavin extractiondid not increase the amount of measured FAD. (c)Total recovery of flavins from gel-filtration chro-matography was very good (Table 2). Loss ofFADfrom the enzyme during solubilization and duringthe chromatography can be excluded, because it

should have been followed by loss of activity. Acertain inactivation during the experimental stepswas unavoidable, as indicated by the incompleterecovery of 02 --forming activity after gel filtra-tion (Table 2). However, the decay of activitycannot be compared with the decrease of FADconcentration during purification. In fact, assum-ing that the cytochrome b is a structural componentof the enzyme, the NADPH oxidase/cytochrome bratio could be taken as a parameter of thefunctional integrity of the enzyme. As indicated byTable 2, this ratio only slightly decreased in thepartially purified preparation with respect to the100OOOg supernatant (0.22 and 0.26 respectively),whereas the cytochrome b/FAD and NADPHoxidase/FAD ratios changed by a factor of 15-fold.Moreover, the NADPH oxidase/cytochrome bratio in the first peak of gel filtration was greaterthan in the second peak (Table 2 and Fig. 1),indicating that the inactivation affected the por-tion of the oxidase that was retained by the resinmore than the portion that was excluded.The most logical explanation of the distribution

of FAD after gel filtration could be found bylooking at the distribution of cytochrome c oxido-reductase and DCIP oxidoreductase. These en-zymes underwent a chromatographic separationthat was practically superimposable on that ofFAD, and the NADH-cytochrome c reductase/FAD ratio remained constant in all the fractions.Since it is known that diaphorase enzymes areflavoproteins (Mahler & Cordes, 1969), it isconceivable that most of the FAD present in thevarious fractions is associated with these enzymes.What about the small amount of FAD remain-

ing in the first peak after gel filtration? It could beattributed either to a bimodal distribution ofFADin the column fractions, or to spreading ofFAD ofthe included peak. Our data do not allow us todistinguish between these two possibilities, be-cause the sensitivity of the flavin assay did notpermit accurate measurements of the FAD contentof each column fraction. However, since thedistribution of superoxide dismutase-insensitiveoxidoreductases is clearly bimodal (Fig. 1), it isconceivable that at least part ofFAD present in thefirst peak is attributable to the small contamina-tion by these flavoproteins. However, it cannot beexcluded that a small portion ofFAD is associatedwith the O2 --forming system. If this is the case,one should hypothesize that the NADPH oxidaseof neutrophils is formed by a flavoprotein sur-rounded by a number (20 or more) of cytochrome bmolecules. Such a system could work as proposedfor the interaction between cytochrome P-450 andthe cytochrome P-450 reductase, whose ratio wasfound to be 10-25:1 (Yang, 1977; Gut et al., 1983).One could speculate that in neutrophil membrane

1984

646

Composition of partially purified NADPH oxidase 647

only a minimum fraction (5%) of the cytochrome bis bound to the flavoprotein in a functionalcomplex, whereas the bulk of cytochrome b is anon-active (not bound) form. However, this doesnot mean that the activation process consists in theaggregation between a flavoprotein and a cyto-chrome b molecule, because our data (Tables 2 and3) indicate that the FAD and cytochrome bcontents in complexes isolated from resting cellsand from activated cells are very similar.Of course, the results presented in this paper do

not exclude the alternative explanation, i.e. thatNADPH oxidase does not contain FAD asprosthetic group. This conclusion contrasts withmany reports suggesting the participation of aflavoprotein in the radical-generating oxidase(Babior & Peters, 1981; Light et al., 1981; Cross etal., 1982b; Michell, 1983). However, these reportsare based, not on the analysis of purified enzyme,but on the effect of stimulatory (FAD) or inhibi-tory (FAD analogues, quinacrine) compounds(Babior & Peters, 1981; Light et al., 1981; Bellaviteet al., 1983b), and the absolute specificity of theseeffects remains to be demonstrated. Cross et al.(1982b) reported a cytochrome b/FAD ratio of 1: 1or even of 0.5:1, but the data refer to wholemembranes or phagocytic vesicles respectively. Ina previous report from this laboratory (Bellavite etal., 1983b) we indicated a cytochrome b/FAD ratioof 2.4: 1, but on a much-less-purified preparation.More recently Gabig & Lefker (1984) 'resolved' the'flavoprotein and the cytochrome b components ofthe NADPH-dependent O2---generating oxidase',but, since they analysed non-purified preparationssolubilized from membranes, it is impossible to saywhat portion of the solubilized FAD was due to thepresence of other flavoproteins, different from theNADPH oxidase.

In recent work (Bellavite et al., 1984) weobtained evidence that NADPH oxidase does notreduce DCIP, thus confirming data from anotherlaboratory (Babior & Peters, 1981), and this is avery anomalous behaviour for a flavoprotein. Inconclusion, the present study, based on partiallypurified preparation of oxidase, excluded theparticipation of quinones and raised many doubtson the flavoprotein nature of the oxidase. In anycase, the cytochrome b/FAD ratio in the O2 --forming system should be much different from thatpreviously reported. Probably the final answer willbe given when completely pure and fully activeoxidase will be available.

This work was supported by grant from MinisteroPubblica Istruzione (Fondi 40%) and from the WellcomeTrust. P. B. was the recipient of a travel grant from theWellcome Trust.

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