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Electron transfer reactions in the N A D P H oxidase system of neutrophils - involvement of an N A D P H - c y t o c h r o m e c reductase in the oxidase system H i r o t a d a Fujii a n d K a t s u k o K a k i n u m a ~t)parttll('tlt o]" l i t l l t l t t l . l a
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(Received 2SJune It)t)ll Keywords: NADPH,,xidase:NADPIIc~l,~hmme, *cdtlcta~c:'~tlDert,xidt"gun.rat;on; Fla.uen/~'me: Cyt(Jchr(mtch-55~:(NculnH~hil) Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with beptylthioglucoside in a high yield. The sogubilized preparation from myristate-stimulated ceils tsample S/ showed iligh O z- generating acl~ ity, and the preparation from resting cells (sample R) had no activity, hut the two samples had equal amounts of fiavins and cytochrome b-558 ( ~ t b-558). The electron transfer reactions to exogenous cytochrome c {eyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent eyt c rednctase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reduetase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Phomreduction of the NADPH oxldase system wan examined in the absence of NADPH under anaerobic conditions by monitoring the reduction cat-s of exogenous cyt c using a flashlight with cztt-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcltation at about 450 urn. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt e by sample R was independent of the tight wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Unde- aerobic conditions, no direct reduction of either eyt c or cyt b-558 was observed. These results suggest that an NADPX-cyt c reductase (a membrane-bound fiavoproteln) is involved in the NADPH oxidase system of stimulated neutrophils.
Neutrophils contain a membrane-associated NADPH oxidase system that is dormant in resting cells but becomes activated in stimulated cells, catalyzing one electron reduction of molecular oxygen to superoxide anion (O~) (for reviews see Reg. I-3). This oxidasc system has been thought to be an electron transferring chain consisting of a flavoprotein [4-7], a b-type cytoehrome (eyl b-558) [8,91 and cyttlseHc components [10,11]. However. the roles of these components in the electron transport system arc uncertain. Since the dis-
Abbreviations: cyh cyw~hrome; (:GO. chronic granulomatous disease: ttTG. heptylthioglucoside;SOD, superoxide disrnutase. Correspondence: K. K.kinuma. Departmem of Inflammatory Research, The Tokyo MetropolitanInstitute of MedicalSciences.S-IS Honkomagome, Bunkyo-ku, Tokyo 113,Japan.
covery of the absence of cyt b-558 in ncutrophils of patients with X-linked chronic granulomatous disease {CGD) [8], detailed information on the eytochrome has been obtained by cloning the mutant gene responsible for this type of CGD and by immunological and biochemical studies on the encoded gene [12,13]. Ojt b-558 located in the membrane is a hcterodimcr consisting of a large glycosylated subunit (91 kDa) and a small suhunit (22 kDa) [12,13]. Cyt b-558 has been proposed to be the terminal site of the electron transport system because of its low redox potential (Em = - 2 4 0 mV)[14], but its functional role in the electron transfer reaction has no1 been determined. In addition, two cytosolic proteins, p-47 phox and p-67 phox, have recently been shown to be essential components of the NADPH oxidase system with autosomal recessive CGD [It]. In contrast to the large number of studies on cyt b-558, there have been few on the participation of a
202 flavoprotein in the NADPH oxidase system. However. the following findings suggest that a flavoprotein is a component of the osidase: (1) the enzyme achvity is inhibited by micromolar concentrations of a flavin analog, 5-carba-5-deaza-FAD [5]. (2) The velocity of O~ generation by a reconstituted solubilized oxidase with several gavin analogues increases with increase in the redox potentials of these FAD analogues, whereas the apparent K m values of the oxidam for all these analogues are similar to that for FAD [15]. (3) A flavoprotein purified from neutrophil membranes shows highly NADPH-specific cyt c reductase or oxidoreductase activity [7,16]. (4) Neutrophil plasma membranes contain an FAD enzyme with a low redox potential that reacts with NADPH to form a flavin semiquinone only in the stimulated state of the oxidase [6]. However, it is controversial whether the O~ forming NADPH oxidase system of neutrophils has NADPH diaphorase activity [17 22]. The experimental conditions and samples used in previous studies difte~d. No NADPH diaphorase activity was found using a solubilized oxidase fraction under aerobic conditions [17] or a partially purified NADPH oxidase fraction under anaerobic conditions [lg]. These preparations all contained eyt b-558. On the other hand, NADPH-eyt c reductase activity was found in eyt b-558-free, purified osidase preparations under aerobic conditions [16,1920]. In addition, a conversion from NADPH-cyt c rcductase activity to O 2 forming activity was observed by two laboratories [16,19]. In the present study, we further studied the electron transfer reactions from an NADPH-reduced and photoreducible enzyme to eyt b-558 or exogenous cyt c in solubilized oxidase fractions from stimulated and resting cells under aerobic and anaerobic conditions. The present results suggest that an NADPH-eyt c reduetase (a flavin enzyme) located in neutrophll membranes is involved in the NADPH oxidase system. Materials and Methods
M2tterials Fatty acids such as myfistic acid and araehidonie acid from Wako Pure Chemical, Tokyo, were dissolved in ethanol as described previously [23]. Heptylthioglueoside (HTG) was purchased from Dojindo Laboratories, Kumamoto. NADPH and NADH were from Oriental Yeast, Tokyo. Superoxlde dismutase (SOD), cytochrome c (type VI, from horse heart), FAD, FMN and menadione were purchased from Sigma. Other reagents were of analytical grade. Preparation of neutrophils Neutrophils were obtained from pig blood by reported methods [6,24].
Preparation of membrane vesicles from stimule:~.! , rnd restbzg neutropbils Pairs of stimulated and resting cell samples were prepared with and without myristate as reported previously [6,16]. The resulting ceil pellets were mixed with ice-cold 0.34 M sucrose containing KRP arid promptly frozen and stored at -80°C. The stored cell suspensions were th~'..~'d at room temperature, mixed with phenylmcthylsullo,,yl fluoride at a final concentration of I raM, and was subjected to sonic irradiation at 0°C in a Branson Sonifier, model 185 equipped with a controller to set the sonication time at 6 -< 1.5 s at 20 W with intervals of i s. The post-nuclear supernatants were separated from the cell sonicates by centrifugation at 4 0 0 × g for 20 rain, and used for collection of membrane vesicles by centrifugation at 100000 × g for 60 min at 4°C. Solubilization of the NADPH oxidase The membrane vesicles of stimulated and resting cells were suspended at a final concentration of 5 mg protein per ml in a solubilizing mixture consisting of 1% (w/v) heptylthioglucoside (HTG), 30% glycerol and 50 mM phosphate buffer (pH 7.4). The mixtures were gently stirred in an ice-bath for 30 min, and centrifuged at 1 0 0 0 0 0 × g for 60 rain to obtain the supernatants of the solubilized membranes from resting cells (sample R) and stimulated cells (sample S). The solubilizing yield (%) of NADPH oxidase was determined as the ratio of the activity of the solubilized sample to that of membrane vesieles-HTG mixture. Spectrophotometric analysis of exogenous cyt c and cyt b-558 under anaerobic conditions Time-dependent spectral changes of eyt c in the solubilized oxidase were measured in the range of 500-600 nm under anaerobic conditions. The reaction mixture, consisting of an aliquot (10 g.l) of the solubilized oxidase (3-4 mg ot protein/ml) and 730 ~tl of 0.1 M phosphate buffer (pH 7.0) containing 30 p~M cyt c and 1.5 mM MgCI2, was placed in an air-tight microcuvette (10 mm light path and 3 mm width). Time-dependent spectral changes of eft b-558 were measured in the range of 400-600 nm under anaerobic conditions in a euvette containing reaction mixture consisted of 60 /xl of the same solubilized sample and 69 #1 of 0.I M phosphate buffer (pH 7.0). Strictly anaerobic conditions for the reaction mixtures were achieved using pure argon and a system of glucose and glucose oxidase as follows: the cuvette containing one of the above reaction mixtures was hermetically sealed with an air-tight silicone-rubber stopper with two in~erted needles (input and output) for flushing with oxygen-free argon, which was obtained by trapping traces of oxygen in commercial 99.99~5% pure argon in an Oxisorb (Messer Griesheim
GMBR, Dusseldorf) [6]. Subsequently, I1) mM glucose and glucose oxidase (40 units/ml) were addcd into thc cuvette through a gas-tight syringe. All additional solutions, such as NADPH and NADH, were also kept under strictly anaerobic conditions obtained with a glucose-glucose oxidase system and pure argon, before their additions to the reaction mixture with a gas-tight syringe. The cu'":ttc was thermostatically regulated at 25~C, the reaction mixture was stirred and spectral changes were measured in a Unisoku Biospectropbotometer US-401 (Unisoku Co., Osaka), attached to a personal computer (NEC PC 9~01). The extent of reduction of cyt b-558 was calculated taking the extinction coefficient at 558-540 nm as 21,6" 10~ I reel i cm-I [9]. Values are shown as percentages of that of eyt b-558 fully reduced with dithionite. Phororeduction of exogenous cyt c and cyt b-558 A 50 W Moritex halogen lamp was used as a light source, and light was introduced into the abo'~c microcuvette through a light guide (5 mm diameter and 15 em length of optical hbers) ur, d,:r anaerobic conditions. Photoirradiation was first carried oilt with eight cut-off filters (Y41, 43, 45, 46, 48, 49, 50 and 51, Toshiba Co., Tokyo) in the range of 402-505 nm to determine the optimum wavelength for photoexcitation. The photoreduction rates of cyt c and cyt b-558 were then obtained by corrections of the light energy in the wavelength ranges to the same intensity of photoirradiation. Photoreduction was performed by irradiation at a selective wavelength of 450 nm with a sharp band-path filter (450 nm at the maximum transmittance and 11.5 om band pass, specially order-made by Hitachi Co., Hitachi). Assay o f enzyme activity NADPH-depeudant O 2 generating activity was measured as the rate of cyt c reduction in zhe absence of superoxide dismutase (SOD) minus the rate with SOD in assay medium consisting of 1.5 mM MgCI z and 0.1 M phosphate buffer (pH 7.0) [25]. The reaction was started by adding 0.1 mM NADPH, and the increase in the absorption at 550-540 nm was followed at 25°C in a Hitachi 556 spcctrophotometer. NADPH-cyt c reduetase activity was measured in the same reaction mixture as for the assay of O~- generation by tracing the rate of cyt c reduction at 550-540 nm under the same anaerobic eonditions as those described above. Anaerobiosis was confirmed by the absence of difference in the NADPH-depgndent cyt c reduction of the solubilized oxidase in the presence and absence of excess SOD (300 units/ml). Myeloperoxidase activity was measured by the guaiacol method cf Chance and Maehly [26]. Tetraguaiacol formation was examined at 25"C, and recorded at 470 nm.
Deiermmations of FAD aml FMN The contents of FAD and FMN were determined by the method of Faedcr and Siegel [27] with some modifications [6]
Stimulunon-dependent reduction of c)'t c i:l the pre~ence and absence of o~ygen Pairs of stimulated and resting ceils were prepared with and without myristate, respectively, which were then fraetionated to obtain membrane vesicles. Treatment of membrane vesicles with heptyltbioglucoside (HTG) resulted in a high yield (100%) of solubdized oxidase. The solubilized fractions from stimulated and resting cells {samples S and R, respectively) contained almost equal amounts of proteins (S, 3LI-L-_6.9 m s / 1 0 lu cells: R, 34.2 + 4.5 mg/10 I'~ cells, n = 6), cvt b-558 (S, 323 + 12 pmol/mg of protein; R, 349 + 32 pmol/mg of protein, n = 6) and noneovalently bound FAD (S, 125_+7 pmoi/mg of protein; R; 130_+11 pmol/mg of protein, n - 6) (negligible amounts of FMN) For characterization of the electron transfer reactions in the NADPH oxidase system, NADPH-dependent reduction of exogenous cyt c was studied using the preparation of HTG-solubilized oxidase under aerobic and anaerobic conditions. Fig. l shows the NADPH-depcndcnt cyt c reductions by samples S and R under aerobic (Fig. IA) and anaerobic (Fig. IB) conditions. Under aerobic conditions, the cyt c reducing activity in sample S was markedly higher than that iu sample R and was completely inhibited by SOD, showing that sample S had high NADPH oxidase activity, generating 280 nmol O ~ / m i n per mg of protein. Sample R had no O~ generating activity. No SOD-insensitive reduction of cyt c was detected in sample S under aerobic conditions. On the contrary, direct reductions of cyt c by both .samples were observed under anaerobic conditions, as shown in Fig. IB, and the rate catalyzed by sample S was more than that by sample R. Since the rates of cyt c reduction by sample S in the presence and absence of excess SOD (300 units/ml)were similar, the anaerobiosis in the present system was sufficient to prevent the formation of O~ by the NADPH oxidase. Therefore. these results suggest that the reduction of eyt c is due to direct electron transfer from the oxidase itself, not to O~. Under anaerobic conditions, cyt c is reduced by NADPH itself in the absence of added samples, and thus its reduction rate (blank) was subtracted from those obtained with samples S and R, respectively: the initial reduction rate catalyzed by sample S (58 nmol/rmin per mg of protein) was about t.5-fold that by sample R (42 nmol/min per mg of protein). When NADH was used in place of NADPH, both sample S and R showed low levels of reduction of
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Fig. I. Spectrophotometric tracings of o;-dependent (A} and -independent cyl c reduction (B) I~."HTG-s~lubilized samples S and R. prepared from stimulated BIId resting cells, respectively. The reactlon mixtures(A and B) contained sample S or R 130 pg prntein/ml) and 30 #M cyt c in 73n ~tl of 0,1 M pll~phale buffer (oH 7.at. Under ae~bic eondaions (A). 0 2 dependenl ~vtoehlome c rpd)~elion was started by adding 0.1 mM NADPH. Before u~ay of NADPH-cyt t rcductase activity (BL ana,~robiosis in the reaction mixture in an air-tight cuveue was achieved by hushing w,h pure argon and additit,n of glu~se (10 raM) and glucose oxidase 140 units/ml). The reaction was started by adding 0.1 mM NADPH in the presence or absence of SOD 4300 units/ml) to confirm ana~mbiosis, The rates of cyt c reduction were traced by measuring the absorption difference at 550 540 nm at ZS° C. The blank value of Cyl c reduction was measured withuut solubilizcd samples under identical cunditiuns.
cyt c a n d t h e r e was n o m a r k e d difference in their (eduction rates (14 a n d 18 nmol cyt c / r a i n p e r m g of protein by sample R a n d S, respectively). T h e s e results suggest that the activity for direct reduction of exogenous cyt c is e n h a n c e d by stimulation of cells a n d is NADPH-spccific.
Effects o f exogenous F A D an~l F M N on N A D P H - d e p e n dent reduction o f cyt c T h e reductions of cyt c by samples S a n d R were studied in the p r e s e n c e of various concentrations (0.110 u.M) of F A D or F M N u n d e r a n a e r o b i c conditions (Fig. 2). T h e cyt c-reducing activity of s a m p l e S inc r e a s e d with increases in the concentrations of F A D a n d F M N in t h e assay mtxture, a n d F M N was more effective titan F A D . T h e results in Fig. 2 show that 1 ,~M F A D or F M N resulted in maximal activity In contrast to sample S, sample R showed no increase in cyt c-reducing activity u n d e r the s a m e conditions in the p r e s e n c e of 10 taM F A D or F M N (Fig. 2 a n d T a b l e D. Addition of N A D H in place of N A D P H to the same system u n d e r a n a e r o b i c conditions did not e n h a n c e cyt c reduction by exogenous F A D a n d F M N with e i t h e r sample S or R (Fig. 2). Next, the effects of m e n a d i o n e
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'FRN or e ~ (~M) Fig. 2. Effects of exogenous FMN and FAD on NADPI[- or NADH-dependenl cyt (,-reducing activity by s~lubilized samples S and R under anaerobic conditions. NADPH-dependcnt cyt c reduc0ons by sample~ S and R ~ r e measured in the presence of various concentralions of FMN (S (*) and R to)) and of FAD (S ( • ) and R ([])1. NADH-dependent cyt t reducing activity was also measured under similar conditions in the presence of FMN (S t a t and R (a)), Experimental conditions were as for Fig. IB except for the presence of FAD or PMN. The reaction w:,s started by adding 0.1 mM NADPH (;r NADH. on t h e cyt c-reduclng activity were e x a m i n e d in a n a e r o bic conditions with samples S a n d R (Table I). M e n a dione (10 ,u.M) e n h a n c e d t h e cyt c-reducing activity by sample S, but not by s a m p l e R, consisten ~- with the results with F M N a n d F A D (Fig. 2). T h e e n h a n c e m e n t of activity of sample S by m e n a d i o n e was c o n c e n t r a TABLE I
NADPH-del~,ndent ~ductions of cyt c and cyl b-558 hi HTG-solubllized fracture of stimulated (S) and resting (R) cell~un&', aerobic and anaerobic conditions m tile presence of FMN, FAD and menadion¢ Experimenlal conditions for eyt c reduelion were as for Pigs. I and 2. Reduction of cyt h-558 in samples S and R w~s measured as described in Fig. 3. Values are means for duplicate determinations. n.d., non-detectable L~tochrome c reduction tnmol/rainperrag ofprotein)
Cytoehromeb-SSB reduction (nmol/min per ms ofprotein)
Anaerobic = YMN t5 ~M) FAD ',s ~zM) Menadione (1O ~M)
S8 104 ",~ 75
42 45 44 40
0.21 I).20 0.21) 0.08
0.20 0.19 [).20 0.1O
Aerobic FMN (l(g) ~tM) FAD(1D0 ~M) Menadione (sop/aM)
280 278 281 281
n.d. n,d. n.d. n.d.
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Time (rain) Fig ]. Reductitms of cyt b-558 in samples S and R with 0.1 mM NADPH or NADH under anaerobic canditkms. Tbe initial reducllon of cyt h-558 in sample S to) with NADPH was compared with Ihat in sample R (o) under anaerobic conanions. Similar expertments were carried out with NADH in sample S ( • ) and R I ~ ). The HTG-solubilized sample (6-7 mg protein/roD in 0.1 M phospbale buffer IpH 7.it). w~,s mlxed with OA mM NADPH or NADH anaerobically and the reduced minus oxidized spectrum at 250C was recorded. The rate of NADPH-dependent reduction was calculated as the percentage of fully reduced eyt b-55g with dithinnite. The concentration of cyt b-558 in the cuvettc wos 2 to 3/xM.
t i o n - d e p e n d e n t up to 80O p,M. T h e s e results showed that stimulation of t h e N A D P H oxidase system resuited in e n h a n c e m e n t of N A D P H - c y t c r e d u c t a s c activity in the m e m b r a n e s . In the a b s e n c e of a d d e d
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Reduction of cyt b-558 bl samples S and R witb NADPH under anaerobic conditions Next. we e x a m i n e d the rates of reduction of cyt b-558 with N A D P H in samples S a n d R. Addition of 0.1 m M N A D P H u n d e r anaerobic conditions resulted in slow reduction of cyt b-558 in both sample S a n d R, as shown in Fig. 3 a n d T a b l e I. T h e initial rates of reduction of cyt h-558 with 0.1 m M N A D P H in samvies S a n d R were virtually the s a m e even though the former h a d high O~ g e n e r a t i n g activity a n d the latter h a d none as described above. T h e reduction rate of cyt b-558 with 0.1 m M N A D P H was higher t h a n that with 0.1 m M N A D H , but no a p p r e c i a b l e specificity for the nucleotide was observed (Fig. 3). T h e reduction rate of
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flavins, this e n h a n c e m e n t was slight because m;leromolecular c.~t c had little access to the active site of the oxidase system, but the m a r k e d e n h a n c e m e n t of N A D P H - c y t c reduct . . . . . . . . . . in the p . . . . . . . f small molecules of flavins which could shuttle one electron from the active site af the N A D P H oxidz:~e to exogenous cyt c [28]. In sample S, F M N was more effective than F A D for the reduction of cyt c. probably due to its higher rate constant for the single-electron transfer reaction as seen in the effects of FMN and F A D on N A D ( P ) H - d e p e n d e n t flavin enzymes [28,29], In contrast, exogenous flavins a n d m e n a d i o n e had no effect on 0 2 prtMuction (Table 1), suggesting that oxygen is accessible to an active site of the N A D P H oxidase without any help of these electron-shuqting c o m p o u n d s a n d accepts one electron directly from the
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a rain Time (rain) Fig. 4. Phutoreductlons of exogenous cyt c IA) and cyt b-558 (B) in samples S and R wilh time under anaerobic condillons. (A} Photoreduction~ of exogenouscyt ¢ by sample S (Q) and R (o }were measured under anaerobic conditions. "lhe ~nditions for the assayof cyt c reductase aClivay were as for Fig, I except for the absence of NADPI-I.The blank ( a ) for phxdoreduelion of cyt c was measured in the same manner without samples in the assay medium. Photoreduction was carried out hy flashlight (hv) with a 50 W halogen lamp. (B) Photoreducdons of cyt h-558 in samples S tl} and R to} plotted as functions of time at 25*(:= The rate of reduction is shown as a pe~entage of fully reduced cyt h-558 with dithionite. Anaerobic conditions were obtained as described for Figs. 1-3.
cyt b-558 in samples R a n d S were c o m p a r e d using pairs of samples from eight different preparations of pig blood cells. No appreciable differences were f o u n d in t h e reduction rates in samples R a n d S of these preparations. The effects of F A D , F M N , a n d m e n a dione on the N A D P H - d e p e n d e n t reduction of eyl b-558 in the S a n d R samples were e x a m i n e d u n d e r anaerobic conditions (Table 1). Results showed that m e n a dione (lt} ,~M) inhibited the reduction of cyt b-558 but did not inhibit O 2 production by sample S (Table I).
Photoreductions of ~ogetloz~s cyt sample A and R in anaerobiosis
c
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and cyt b-538 in I~oth
Unexpectedly, the results obtained h e r e gave evidence for the involvement of an N A D P H - ~ ' t c reductase in the N A D P H oxidase system in stimulated cells. Thus, we next focused attention on t h e N A D P H - c y t c reductase with rcspccl to the redox center. Flavoproteins can be reduced by light u n d e r anaerobic conditions, so we examined the photoreductions of exogenous cyt c a n d cyt b-558 in samples S a n d R by flashlight u n d e r anaerobic conditions. Typical results of the reductions of cyt c a n d cyt b-558 are s h o w n in Fig. 4A a n d B, respectively. T h e p h o t o r e d u c t i o n rate of cyt c in sample S was m u c h h i g h e r t h a n t h a t in sample R (Fig. 4A). T h e e n h a n c e m e n t of t h e activity in sample S was consistent with that obtained with N A D P H (Figs. 1 a n d 2), suggesting that the N A D P H - c y t c reductase is possibly a flavin enzyme. In contrast to t h e rate of reduction of cyt c, t h e rate of p h o t o r e d u e t i o n of cyt b-558 in sample S was almost the same as t h a t in sample R (Fig. 4B), consistent with the results o b t a i n e d with N A D P H (Fig. 3). F r o m the results in Fig. 4A a n d B, the photoreduction rates of eyt c a n d cyt b-558 in sample S were 8.3 a n d 0.084 n m o l / m i n p e r m g of protein, respectively; that is, most of t h e electrons (99%) produced by p h o t o r e d u c t i o n w e r e trausferred to cyt c a n d only 1% to cyt b-558. W h e n a small a m o u n t of oxygen was a d d e d to the p h o t o i r r a d i a t e d samples, the spectrum of the p h o t o r e d u c e d cyt c immediately disappeared, suggesting that the p h o t o r e d u c e d flavoprotein is highly oxygen-sensitive.
~ o
O400
~
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500
Wavelength (nn~) Fig. 5. Wavelcnglh dependence oa
photorcducaons of L~t c in samples S (o) and R (0) under anaerobic condilions, Photoreductions of exogenous cyt c in samples S and R were carried oul by irradiation by flashlighl passed through each of eight cutoff optical filters. Experimental conditions were as fnr Pic. 4A. Photoreduction of cyt c in Ihe assay medium withoul sample was used as the blank. The rules of eyl c reduction were corrected for the energy level of Iho light cul-off at different wa~lcngths to normalize the light intensily and arc pinned against the wavelength. lower than t h a t in sample S. T h e spectroscopic studies suggested that photoexeitation of a flavin e n z y m e occurs in sample S, resulting in electron transfer from t h e p h o t o r e d n c e d flavin to e x o g e n o u s cyt c. T h e r e f o r e , f o r f u r t h e r examination of the electron transfer reactions in samples S a n d R, we studied the 'effect' of photoirradiation o n samples containing cyt c at t h e optimal
Wat~elength for photoreduction of cytochromes W e analyzed the wavelength for p h o t o r e d u c t l o n of cyt c a n d cyt b-55g in samples S a n d R using eight cut-oft filters (the r a n g e of 402 to 505 n m ) a n d a hand-pass filter (450 n m at the m a x i m u m transmittance a n d 11.5 a m b a n d pass). T h e p h o t o r e d u c t i o n rates of exogenous cyt c in samples S a n d R were m e a s u r e d at 550 n m , a n d values were corrected for t h e energy levels of the light passed t h r o u g h the cut-off filters. T h e photorednction of eyt c in sample S d e p e n d e d o n t h e wavelength, a n d was maximal at about 45O n m , as s h o w n in Fig. 5. In contrast, photoreduction of eyt c in sample R showed a b r o a d p e a k a n d the activity was far
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Wavelength ( ~ ) Fig. 6. Phmoreduced minus OXidizeddifference spectra of ~ g e n o u s Cyl c in saml~les s and R by pholoirradiation with a band-pass optical filter at 450 nm under anaerobic conditions. (Right) Photoreduction of cyl c in sample $ or R was carried out by photoirradiafinn with a band pass oplical filter for selection of 450 nm light (left). The blank was recorded in the same manner by photoirradiation of reaction mixture in the absence of the ~lubdized samples. (Left) Optical property of tile band-pass filter used in this experiment.
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O400 4an 5on 'W'JaveIertath (r,_m) Fig. 7. Wavelengthdependence of phott)reductions of LVt h-55~;in ~mples S and U under unaerobic conditions Experimental~ndi lions were as for Fig. 4B with the same cut-oft filte~ :is for Fig 5 The photoreducfiun rates of cyt b-558 in samples S (o) 4nd R (c,) were delerrnined with ~rrection as for Fig. 5 Io I~ormalize th," intensityof the lighl passed through the cut-off filters. light-wavelength of 450 nm by light passed through the band-path filter (Fig. 6, left). As shown in Fig. 6, photoreduction of cyt c by selective irradiation at 450 nm was observed only in sample S, the rate of photoreduction in sample R being the same as that of the blank and independent of the wavelength. In Fig. 7, the photoreduction rates of cyt b-558 in samples S and R are plotted as functions of the cut-off wavelength of irradiated light in a similar way to that in Fig. 5. The reduction rates of cyt b-558 in samples S and R showed a similar wavelength-dependency to that in Fig. 5, the maximum reduction occurring at about 450 nm in both the S and R sample. These results suggest that cyt b-558 in both samples accepts electrons from a photoredueed flavin localized in the membranes. Discussion
In the present study, we showed that under strict anaerobic conditions HTG-solubilized preparations from stimulated and resting neutropbils exhibited NADPH-dependent cyt c reducing activity and that the activity of stimulated neutrophils was much higher than that of resting ones in the presence of FMN and FAD (Table I). This finding suggests that an NADPHeyt c reduetase located in the membranes becomes active during stimulation of the ceils. We obtained similar results on the enhancement of NADPH-dependent cyt c reduetase activity in solubilized oxidase fractions from pig blood neutrophils activated with
other stimulators, such as arachidonic acid and phorbol my-tstatc ~cctate. However, the saturated fatty acid seems preferable lot studies on free radicals as described !ater. We exan~ined membrane fractions of resting and stimulated cells, but found that the NADPH-dcpeadenl reductien of exogenous cyt e was mot,, niarkcd in solubilized oxidase preparations than in membrane preparations, probably due to more ready accessibility of the exogenous electron aeceptor to the site of an intrinsic flavin enzyme. Of several detergents tested, heplyhbioglueoside was the best for demonstrating the diaphorase activity under anaerobic conditions. The present results obtained by photorcduction gave evidence for the involvement of flavin in the NADPHcyt c reductase as its active site. Under anaerobic conditions, photoirradiation of the oxidase system at a selective wavelength of 450 nm, which is known to cause excitation of flavoproteins [3fl]. resulted in marked cyt c-reductase activity in the sample of stimulated cells but little in the sample of resting ones (Fig. 6). In the absence of the substrate tNADPH), photoreduction of flavoproteins involves the uptake of two electron equivalents by the flavin from intramolecular and intermolecular donors at the N(5), (3(8) or C(4) positions of the isoallonazine ring after transition to the flavin triplet (singlet) excitable state by absorption of light [31]. These results suggest that stimulation of neutrophilg causes modification of the flavoprJtein to active and photon-sensitive state that catalyzes marked reduction of exogenous cyt c. We found that the cyt c-redoctase activity induced by photorcduction is also very unstable in air. An NADPH-~--yt c reductase separated from other redox components is relatively oxygen-stable, but becomes unstable in the presence of cyt b-558 [16,32], suggesting that it may form flavin semiquinoae in the stimulated state, transferring a single electron to an oxidizable terminal component in the NADPH oxidase system. The photoreduced sample S failed to generate O.; even though the preparation could be reduced by light in anaerobic:sis. Therefore, NADPH seems to work not only as a specific reduetam, but also as a cofactor for a conformatiorial modification of the NADPH oxidase system to an 0 2 generating state. Previous EPR studies in our laboratory [6] showed that upon addition of NADPH in anaerobiosis, membranes from stimulated ceils gave an EPR signal of flavin semiquinone as a neutral (blue) semiquiaone. The saturated fatty acid, myristate, seems preferential as a stimulator for demonstrating flavin free radicals in ESR spectra because peroxide radicals formed in unsaturated fatty acids exhibit ESR signals at g = 2, which interfere with the ESR signals of flavin free radicals (Ref. 6 and unpublished data). The idea of involvement of a flavin semiquinone in the NADPH
oxidasc syslem in sfimulaled ceils is supportcd by the present findings wilh exogenous F A D , F M N a n d menadione, all of which can transfer a single electron by their shuttling between flavin serniquinonc a n d exogenous cyt c. leading to the e n h a n c e m e n t of N A D P H d e p e n d e n t cyl c-reductase activity in preparations fronl stimulated ceils (Fig, 2 a n d Table 1). In contrast, none of these tl~rec reagents h a d any effect on O;- production in the N A D P H oxidase s~slenL even at the high concentrations used: m e n a d i o n e at up to 0.8 raM. a n d F M N a n d F A D at 0.I m M . These results suggest that oxygen is 0 more specific electron accepter than these artificial compound:, in the oxidase ,~ystem. In contrast to the m a r k e d difference in N A D P H - c y t c rednctase activities in resting a n d stimulated samples, there was no difference in lheir reductions of cyt b-55g by either N A D P H or light, as s h o w n in Figs. 3 a n d 4. The present results failed to support the hypothesis that cyt b-558 in the oxidase system is the terminal electron d o n o r to oxygen. This heine p r o l e i n is an essential c o m p o n e n t of the oxidase as j u d g e d by the fact that neutrophils of patients with X-link inherited C G D lack this protein. Since the redox potential of eyt h-558 is unusually low, -2d-5 mY, this ferrous h e m e has been t h o u g h t to be highly autoxidizable, resulting in O ~ formation [14]. However, cyt b-558 in sample R was reduced to the same extent as that in sample S by N A D P H (Fig. 3), but did not form any O ~ . This finding indicates that O~- generation should be interpreted nol only by t h e r m o d y n a m i c possibilities, b u t also by phagocyte-specific control mechanisms. In a cell-free systenl, the O 2 f o r m i n g activity of the N A D P M oxidase d e p e n d s o n the presence of cyt b-558, F A D a n d cb'tosolic c o m p o n e n t s such as p47-phox, p67phox [11] and a small nueleotide-binding c o m p o n e n t ( s ) [33]. O u r recent sludies on cytosolic c o m p o n e n t s suggest thai neither p47-phox n o r p67-phox is a flavia enzyme [34]. These cytosolie c o m p o n e n t s a p p e a r to be activating factors modifying redox c o m p o n e n t s such as cyt b-558 a n d / o r the florin enzyrae r a t h e r than electron transport c o m p o n e n t s . It is very likely that the e n h a n c e d activity of N A D P H - d e p e n d e n t cyt c reduction m a y be catalyzed by an F A D enzyme, an N A D P H cyt c reductase, reported from this laboratory [16]. Since plasma m e m b r a n e s of pig blood neutrophils contained very similar a m o u n t s of F A D (and negligible a m o u n t s of F M N ) in both resting and stimulated states [6,16], the F A D enzyme may be an intrinsic m e m b r a n e protein like cyt b-558 r a t h e r t h a n a cytosolic protein. Vignais' g r o u p reported similar reductase activity in purified preparations from m e m b r a n e s of bovine neutrophils [19] a n d recently proposed a model for the diaphorase-o×idase transition based o n their findings that in rabbit peritoneal neutrophils, a m e m b r a n e h o u n d N A D P H - c y t c reductase was converted to a n
O , - g c n e t a t i n g oxidase d u r i n g slimulation [35]. T h e i r model is in accord with ours. Acknowledgements We are very grateful to Prof. Takashi Yonetani, D e p a r l m e n t of Biochemistry a n d Biophysics, Univt.,'sity of Pennsylvania and Dr. Takeshi Nishino, Departm e n t of Biochemistry, Y o k o h a m a City University School of Medicine, for t h e i r helpful discussions a n d critical r e a d i n g of o u r manuscript. This work was supported in part by grants from the Ministry of Education, Science a n d Culture, Japan. References
i Bahior. B.M. (1984) alo~d 64, 959-9fi6. 2 Rossi, F. (19861 Biochim. Biophys. Acts 852, 65-89. ~ Segal. A.W. (1089} J. Clin. Invesl. 83. 1785 1703. Parkinson, J.F. and Gahig, T.G. (10881 J. Binenerg. Bionlernb. 20, 653 677. 5 Light. D.R. Walsh. C., O'Canaghan, AM., G ~ z l . E.J. and Tauher. A.I. (10511 Biochemistry 20, 1468-147b. 6 Kakinuma. K, Kanedu. M.. Chiha, T. and Ohnishi, T. (19561 J, Biol. Chem. 261, 9426 9432. 7 Markert. M.. Gla-~.s, GA. and Babior, aM. (19851 pro¢. Nan. Acad. gel. USA 82, 3114-3145, 8 Segah A.W. and Jones, O.T.G. t 19781Nature 276, 515-517. 9 Gabig. T.G., Sche~ish. E.W. and Sanlinga. J T. 0952) J. Biol. Chefs. 257, 4114-4119. 10 grombers, "~. and Pick, E. (19541 CeLl. lmmunol. 58, 213-221. 11 Clark, R.A.. Malech. H.L., Ganin, J.l, Nunoi. H., Volpp, B.D.. Pearson. D.W., Nauseef, WM. and Curnutte. J.T. (19591 New EngL J, Mcd. 32h 647-652. 12 Royel-Pokora, El., Kunkel, L.M.. Monaco, A.P.. Golf, 5.C., Newburger, P.E., aaehner, R.L., Cole. F.S., Curnune, J.T. and Orkins, S.H. (19861 Nature 322, 82-38. 13 Teahan, C. R ~ e , P., Parker, P., ToUy. N. and Segal, A.W. (19871 Nalure 32% 720-72L 14 Cross, A.R., Jones~ O.T.G. Harper. A.M. and Segal. A.W. (19811 Biechem. J. 194, 599 606. 15 Parkin~n, J.F. and Gabig, T.G. (19881 J. Biol. Chem. 263, 8669-8863 16 Kak[numa. K. Kaneda, M. and Fukuhara, Y. (19871 J. Biol. Chem. 262, 12316-12322. 17 Babior. B.M. and Peters. W.A. (10811 J. Biol. Chem. 256. 23212323. 18 aellavite. P., Bianca, V., Serra, M.C.. PapinL E. and Rossi, F. (1984) FEnS Lell. 170,157-161. 19 Doussiere, J. and Vignais. P.V. {19851 Biochemistry 24. 72317239. 20 Kogma, tl., Takahashi, K., Sakane. F. and Koyama, J. t19871 L Biochem (T,ikyo) ID2, 1083-113gg. 21 Green, T.R. and Wu, D.E. 0955) FEllS Lett. 179. 82-86. 22 Green. T.R. and Pratt, K.L. (1990~ J. Biol. Chem. 7~5. 1932410320. 23 Kakinuma, K (19741 Biochim. Biophys ~cla 348, 76=85, 24 Wakeyama, H.. Takeshige, K.. Takayan~:g~..' and M[nakami, S. (1982) Bioehem. J. 205, 593 601 25 nobler. B.M. Cumune. J.T. and McMurric,. n.J. (19761L Clln. Invest. 58, 9[59-990. 26 Chance, B. and Maeld~, A.C. (1'1551 Methods Enzyraul. 2. 764775.
2O') 27 Fueder, E J and Siegul. L M 11';73) Atoll. Bio~llcnl ,~3, 332 33(, 2£ Kishore. G . M u,ld S,,,cI1. E tZ (1')791 13k,chenl. Bi,~pily,. C(~mmun. 87. 518-523. 29 Tu. S.-C.. Romero. F.A an0 W;irlg. L.-I I. 11q311 Arch. Hi,,chem Biophys. 2O9. a23-z32 311 Chance. B. and Schoener. B 11966)in Fhvins ;rod Fl:l%,,rlr,,teh,, (Shller, E.C.. ed.). pp. 4Y7, Elsevier. ,"-.rrl:lerdurn. 31 ;lcelis. P.F. (lI)'Jll ill (l~erlliS[~ ~lnd Uiochcmlsl D ol F]~l,OUll zymcs (MiJller. F.. ed~. V,,t L pp. 171 194. ( R ( " Pre,s. B,~ca Rillt,n.
32 K;~,il;UmLi, K. (Inb~,, I'. tllld Fujii. H (1989~ ill Medical r~i,,chemical :lied ( hcl~li¢:d A-pqCls 0f Fr~c ~tdlc;Ll~ (Ihy;llshi, ( ) Niki, E . K.rl&~. M. and Yoshikawa. I . Cds. I. p p 1311 13]-1. Elsevier Amsterdam ~ Atl;iMui. I lind Pick. I (iq,,lll J. [ euko;)le Bml. 48. Ill7 115. C h l h . T Kaneda M Fmll }1. ('k*rk. R A ,~lauseef. W ~] .nO Kttkirlllnl;i K liL)t2t}) Biochcm I]inphys Res ('l~mmun. 173. ~v Lap,,rl,. F [),,u,,i,'re I und Vigmlis. P.",. (1,1,,i)) Eur J []hlchcm. /w4.31H ~11'~
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Electron transfer reactions in the N A D P H oxidase system of neutrophils - involvement of an N A D P H - c y t o c h r o m e c reductase in the oxidase system H i r o t a d a Fujii a n d K a t s u k o K a k i n u m a ~t)parttll('tlt o]" l i t l l t l t t l . l a
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~t'xottch, Jill" Tokyo ~lt'fgf;l)tstift*n ]$l([dtltll' ¢,[].h'tlt( al Scit'ttccx "F,~k)o lJ,tpan~
(Received 2SJune It)t)ll Keywords: NADPH,,xidase:NADPIIc~l,~hmme, *cdtlcta~c:'~tlDert,xidt"gun.rat;on; Fla.uen/~'me: Cyt(Jchr(mtch-55~:(NculnH~hil) Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with beptylthioglucoside in a high yield. The sogubilized preparation from myristate-stimulated ceils tsample S/ showed iligh O z- generating acl~ ity, and the preparation from resting cells (sample R) had no activity, hut the two samples had equal amounts of fiavins and cytochrome b-558 ( ~ t b-558). The electron transfer reactions to exogenous cytochrome c {eyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent eyt c rednctase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reduetase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Phomreduction of the NADPH oxldase system wan examined in the absence of NADPH under anaerobic conditions by monitoring the reduction cat-s of exogenous cyt c using a flashlight with cztt-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcltation at about 450 urn. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt e by sample R was independent of the tight wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Unde- aerobic conditions, no direct reduction of either eyt c or cyt b-558 was observed. These results suggest that an NADPX-cyt c reductase (a membrane-bound fiavoproteln) is involved in the NADPH oxidase system of stimulated neutrophils.
Neutrophils contain a membrane-associated NADPH oxidase system that is dormant in resting cells but becomes activated in stimulated cells, catalyzing one electron reduction of molecular oxygen to superoxide anion (O~) (for reviews see Reg. I-3). This oxidasc system has been thought to be an electron transferring chain consisting of a flavoprotein [4-7], a b-type cytoehrome (eyl b-558) [8,91 and cyttlseHc components [10,11]. However. the roles of these components in the electron transport system arc uncertain. Since the dis-
Abbreviations: cyh cyw~hrome; (:GO. chronic granulomatous disease: ttTG. heptylthioglucoside;SOD, superoxide disrnutase. Correspondence: K. K.kinuma. Departmem of Inflammatory Research, The Tokyo MetropolitanInstitute of MedicalSciences.S-IS Honkomagome, Bunkyo-ku, Tokyo 113,Japan.
covery of the absence of cyt b-558 in ncutrophils of patients with X-linked chronic granulomatous disease {CGD) [8], detailed information on the eytochrome has been obtained by cloning the mutant gene responsible for this type of CGD and by immunological and biochemical studies on the encoded gene [12,13]. Ojt b-558 located in the membrane is a hcterodimcr consisting of a large glycosylated subunit (91 kDa) and a small suhunit (22 kDa) [12,13]. Cyt b-558 has been proposed to be the terminal site of the electron transport system because of its low redox potential (Em = - 2 4 0 mV)[14], but its functional role in the electron transfer reaction has no1 been determined. In addition, two cytosolic proteins, p-47 phox and p-67 phox, have recently been shown to be essential components of the NADPH oxidase system with autosomal recessive CGD [It]. In contrast to the large number of studies on cyt b-558, there have been few on the participation of a
202 flavoprotein in the NADPH oxidase system. However. the following findings suggest that a flavoprotein is a component of the osidase: (1) the enzyme achvity is inhibited by micromolar concentrations of a flavin analog, 5-carba-5-deaza-FAD [5]. (2) The velocity of O~ generation by a reconstituted solubilized oxidase with several gavin analogues increases with increase in the redox potentials of these FAD analogues, whereas the apparent K m values of the oxidam for all these analogues are similar to that for FAD [15]. (3) A flavoprotein purified from neutrophil membranes shows highly NADPH-specific cyt c reductase or oxidoreductase activity [7,16]. (4) Neutrophil plasma membranes contain an FAD enzyme with a low redox potential that reacts with NADPH to form a flavin semiquinone only in the stimulated state of the oxidase [6]. However, it is controversial whether the O~ forming NADPH oxidase system of neutrophils has NADPH diaphorase activity [17 22]. The experimental conditions and samples used in previous studies difte~d. No NADPH diaphorase activity was found using a solubilized oxidase fraction under aerobic conditions [17] or a partially purified NADPH oxidase fraction under anaerobic conditions [lg]. These preparations all contained eyt b-558. On the other hand, NADPH-eyt c reductase activity was found in eyt b-558-free, purified osidase preparations under aerobic conditions [16,1920]. In addition, a conversion from NADPH-cyt c rcductase activity to O 2 forming activity was observed by two laboratories [16,19]. In the present study, we further studied the electron transfer reactions from an NADPH-reduced and photoreducible enzyme to eyt b-558 or exogenous cyt c in solubilized oxidase fractions from stimulated and resting cells under aerobic and anaerobic conditions. The present results suggest that an NADPH-eyt c reduetase (a flavin enzyme) located in neutrophll membranes is involved in the NADPH oxidase system. Materials and Methods
M2tterials Fatty acids such as myfistic acid and araehidonie acid from Wako Pure Chemical, Tokyo, were dissolved in ethanol as described previously [23]. Heptylthioglueoside (HTG) was purchased from Dojindo Laboratories, Kumamoto. NADPH and NADH were from Oriental Yeast, Tokyo. Superoxlde dismutase (SOD), cytochrome c (type VI, from horse heart), FAD, FMN and menadione were purchased from Sigma. Other reagents were of analytical grade. Preparation of neutrophils Neutrophils were obtained from pig blood by reported methods [6,24].
Preparation of membrane vesicles from stimule:~.! , rnd restbzg neutropbils Pairs of stimulated and resting cell samples were prepared with and without myristate as reported previously [6,16]. The resulting ceil pellets were mixed with ice-cold 0.34 M sucrose containing KRP arid promptly frozen and stored at -80°C. The stored cell suspensions were th~'..~'d at room temperature, mixed with phenylmcthylsullo,,yl fluoride at a final concentration of I raM, and was subjected to sonic irradiation at 0°C in a Branson Sonifier, model 185 equipped with a controller to set the sonication time at 6 -< 1.5 s at 20 W with intervals of i s. The post-nuclear supernatants were separated from the cell sonicates by centrifugation at 4 0 0 × g for 20 rain, and used for collection of membrane vesicles by centrifugation at 100000 × g for 60 min at 4°C. Solubilization of the NADPH oxidase The membrane vesicles of stimulated and resting cells were suspended at a final concentration of 5 mg protein per ml in a solubilizing mixture consisting of 1% (w/v) heptylthioglucoside (HTG), 30% glycerol and 50 mM phosphate buffer (pH 7.4). The mixtures were gently stirred in an ice-bath for 30 min, and centrifuged at 1 0 0 0 0 0 × g for 60 rain to obtain the supernatants of the solubilized membranes from resting cells (sample R) and stimulated cells (sample S). The solubilizing yield (%) of NADPH oxidase was determined as the ratio of the activity of the solubilized sample to that of membrane vesieles-HTG mixture. Spectrophotometric analysis of exogenous cyt c and cyt b-558 under anaerobic conditions Time-dependent spectral changes of eyt c in the solubilized oxidase were measured in the range of 500-600 nm under anaerobic conditions. The reaction mixture, consisting of an aliquot (10 g.l) of the solubilized oxidase (3-4 mg ot protein/ml) and 730 ~tl of 0.1 M phosphate buffer (pH 7.0) containing 30 p~M cyt c and 1.5 mM MgCI2, was placed in an air-tight microcuvette (10 mm light path and 3 mm width). Time-dependent spectral changes of eft b-558 were measured in the range of 400-600 nm under anaerobic conditions in a euvette containing reaction mixture consisted of 60 /xl of the same solubilized sample and 69 #1 of 0.I M phosphate buffer (pH 7.0). Strictly anaerobic conditions for the reaction mixtures were achieved using pure argon and a system of glucose and glucose oxidase as follows: the cuvette containing one of the above reaction mixtures was hermetically sealed with an air-tight silicone-rubber stopper with two in~erted needles (input and output) for flushing with oxygen-free argon, which was obtained by trapping traces of oxygen in commercial 99.99~5% pure argon in an Oxisorb (Messer Griesheim
GMBR, Dusseldorf) [6]. Subsequently, I1) mM glucose and glucose oxidase (40 units/ml) were addcd into thc cuvette through a gas-tight syringe. All additional solutions, such as NADPH and NADH, were also kept under strictly anaerobic conditions obtained with a glucose-glucose oxidase system and pure argon, before their additions to the reaction mixture with a gas-tight syringe. The cu'":ttc was thermostatically regulated at 25~C, the reaction mixture was stirred and spectral changes were measured in a Unisoku Biospectropbotometer US-401 (Unisoku Co., Osaka), attached to a personal computer (NEC PC 9~01). The extent of reduction of cyt b-558 was calculated taking the extinction coefficient at 558-540 nm as 21,6" 10~ I reel i cm-I [9]. Values are shown as percentages of that of eyt b-558 fully reduced with dithionite. Phororeduction of exogenous cyt c and cyt b-558 A 50 W Moritex halogen lamp was used as a light source, and light was introduced into the abo'~c microcuvette through a light guide (5 mm diameter and 15 em length of optical hbers) ur, d,:r anaerobic conditions. Photoirradiation was first carried oilt with eight cut-off filters (Y41, 43, 45, 46, 48, 49, 50 and 51, Toshiba Co., Tokyo) in the range of 402-505 nm to determine the optimum wavelength for photoexcitation. The photoreduction rates of cyt c and cyt b-558 were then obtained by corrections of the light energy in the wavelength ranges to the same intensity of photoirradiation. Photoreduction was performed by irradiation at a selective wavelength of 450 nm with a sharp band-path filter (450 nm at the maximum transmittance and 11.5 om band pass, specially order-made by Hitachi Co., Hitachi). Assay o f enzyme activity NADPH-depeudant O 2 generating activity was measured as the rate of cyt c reduction in zhe absence of superoxide dismutase (SOD) minus the rate with SOD in assay medium consisting of 1.5 mM MgCI z and 0.1 M phosphate buffer (pH 7.0) [25]. The reaction was started by adding 0.1 mM NADPH, and the increase in the absorption at 550-540 nm was followed at 25°C in a Hitachi 556 spcctrophotometer. NADPH-cyt c reduetase activity was measured in the same reaction mixture as for the assay of O~- generation by tracing the rate of cyt c reduction at 550-540 nm under the same anaerobic eonditions as those described above. Anaerobiosis was confirmed by the absence of difference in the NADPH-depgndent cyt c reduction of the solubilized oxidase in the presence and absence of excess SOD (300 units/ml). Myeloperoxidase activity was measured by the guaiacol method cf Chance and Maehly [26]. Tetraguaiacol formation was examined at 25"C, and recorded at 470 nm.
Deiermmations of FAD aml FMN The contents of FAD and FMN were determined by the method of Faedcr and Siegel [27] with some modifications [6]
Stimulunon-dependent reduction of c)'t c i:l the pre~ence and absence of o~ygen Pairs of stimulated and resting ceils were prepared with and without myristate, respectively, which were then fraetionated to obtain membrane vesicles. Treatment of membrane vesicles with heptyltbioglucoside (HTG) resulted in a high yield (100%) of solubdized oxidase. The solubilized fractions from stimulated and resting cells {samples S and R, respectively) contained almost equal amounts of proteins (S, 3LI-L-_6.9 m s / 1 0 lu cells: R, 34.2 + 4.5 mg/10 I'~ cells, n = 6), cvt b-558 (S, 323 + 12 pmol/mg of protein; R, 349 + 32 pmol/mg of protein, n = 6) and noneovalently bound FAD (S, 125_+7 pmoi/mg of protein; R; 130_+11 pmol/mg of protein, n - 6) (negligible amounts of FMN) For characterization of the electron transfer reactions in the NADPH oxidase system, NADPH-dependent reduction of exogenous cyt c was studied using the preparation of HTG-solubilized oxidase under aerobic and anaerobic conditions. Fig. l shows the NADPH-depcndcnt cyt c reductions by samples S and R under aerobic (Fig. IA) and anaerobic (Fig. IB) conditions. Under aerobic conditions, the cyt c reducing activity in sample S was markedly higher than that iu sample R and was completely inhibited by SOD, showing that sample S had high NADPH oxidase activity, generating 280 nmol O ~ / m i n per mg of protein. Sample R had no O~ generating activity. No SOD-insensitive reduction of cyt c was detected in sample S under aerobic conditions. On the contrary, direct reductions of cyt c by both .samples were observed under anaerobic conditions, as shown in Fig. IB, and the rate catalyzed by sample S was more than that by sample R. Since the rates of cyt c reduction by sample S in the presence and absence of excess SOD (300 units/ml)were similar, the anaerobiosis in the present system was sufficient to prevent the formation of O~ by the NADPH oxidase. Therefore. these results suggest that the reduction of eyt c is due to direct electron transfer from the oxidase itself, not to O~. Under anaerobic conditions, cyt c is reduced by NADPH itself in the absence of added samples, and thus its reduction rate (blank) was subtracted from those obtained with samples S and R, respectively: the initial reduction rate catalyzed by sample S (58 nmol/rmin per mg of protein) was about t.5-fold that by sample R (42 nmol/min per mg of protein). When NADH was used in place of NADPH, both sample S and R showed low levels of reduction of
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Fig. I. Spectrophotometric tracings of o;-dependent (A} and -independent cyl c reduction (B) I~."HTG-s~lubilized samples S and R. prepared from stimulated BIId resting cells, respectively. The reactlon mixtures(A and B) contained sample S or R 130 pg prntein/ml) and 30 #M cyt c in 73n ~tl of 0,1 M pll~phale buffer (oH 7.at. Under ae~bic eondaions (A). 0 2 dependenl ~vtoehlome c rpd)~elion was started by adding 0.1 mM NADPH. Before u~ay of NADPH-cyt t rcductase activity (BL ana,~robiosis in the reaction mixture in an air-tight cuveue was achieved by hushing w,h pure argon and additit,n of glu~se (10 raM) and glucose oxidase 140 units/ml). The reaction was started by adding 0.1 mM NADPH in the presence or absence of SOD 4300 units/ml) to confirm ana~mbiosis, The rates of cyt c reduction were traced by measuring the absorption difference at 550 540 nm at ZS° C. The blank value of Cyl c reduction was measured withuut solubilizcd samples under identical cunditiuns.
cyt c a n d t h e r e was n o m a r k e d difference in their (eduction rates (14 a n d 18 nmol cyt c / r a i n p e r m g of protein by sample R a n d S, respectively). T h e s e results suggest that the activity for direct reduction of exogenous cyt c is e n h a n c e d by stimulation of cells a n d is NADPH-spccific.
Effects o f exogenous F A D an~l F M N on N A D P H - d e p e n dent reduction o f cyt c T h e reductions of cyt c by samples S a n d R were studied in the p r e s e n c e of various concentrations (0.110 u.M) of F A D or F M N u n d e r a n a e r o b i c conditions (Fig. 2). T h e cyt c-reducing activity of s a m p l e S inc r e a s e d with increases in the concentrations of F A D a n d F M N in t h e assay mtxture, a n d F M N was more effective titan F A D . T h e results in Fig. 2 show that 1 ,~M F A D or F M N resulted in maximal activity In contrast to sample S, sample R showed no increase in cyt c-reducing activity u n d e r the s a m e conditions in the p r e s e n c e of 10 taM F A D or F M N (Fig. 2 a n d T a b l e D. Addition of N A D H in place of N A D P H to the same system u n d e r a n a e r o b i c conditions did not e n h a n c e cyt c reduction by exogenous F A D a n d F M N with e i t h e r sample S or R (Fig. 2). Next, the effects of m e n a d i o n e
t
o
1o
'FRN or e ~ (~M) Fig. 2. Effects of exogenous FMN and FAD on NADPI[- or NADH-dependenl cyt (,-reducing activity by s~lubilized samples S and R under anaerobic conditions. NADPH-dependcnt cyt c reduc0ons by sample~ S and R ~ r e measured in the presence of various concentralions of FMN (S (*) and R to)) and of FAD (S ( • ) and R ([])1. NADH-dependent cyt t reducing activity was also measured under similar conditions in the presence of FMN (S t a t and R (a)), Experimental conditions were as for Fig. IB except for the presence of FAD or PMN. The reaction w:,s started by adding 0.1 mM NADPH (;r NADH. on t h e cyt c-reduclng activity were e x a m i n e d in a n a e r o bic conditions with samples S a n d R (Table I). M e n a dione (10 ,u.M) e n h a n c e d t h e cyt c-reducing activity by sample S, but not by s a m p l e R, consisten ~- with the results with F M N a n d F A D (Fig. 2). T h e e n h a n c e m e n t of activity of sample S by m e n a d i o n e was c o n c e n t r a TABLE I
NADPH-del~,ndent ~ductions of cyt c and cyl b-558 hi HTG-solubllized fracture of stimulated (S) and resting (R) cell~un&', aerobic and anaerobic conditions m tile presence of FMN, FAD and menadion¢ Experimenlal conditions for eyt c reduelion were as for Pigs. I and 2. Reduction of cyt h-558 in samples S and R w~s measured as described in Fig. 3. Values are means for duplicate determinations. n.d., non-detectable L~tochrome c reduction tnmol/rainperrag ofprotein)
Cytoehromeb-SSB reduction (nmol/min per ms ofprotein)
Anaerobic = YMN t5 ~M) FAD ',s ~zM) Menadione (1O ~M)
S8 104 ",~ 75
42 45 44 40
0.21 I).20 0.21) 0.08
0.20 0.19 [).20 0.1O
Aerobic FMN (l(g) ~tM) FAD(1D0 ~M) Menadione (sop/aM)
280 278 281 281
n.d. n,d. n.d. n.d.
n.d. n,d. n.d. ILd.
n.d. n.d. n.d. n.d.
205
/
.... tij~O
2n
I i
"g.
/ $ //
O
,0 i
¢
t0
moa ,/tt/~j,/A
J
~--~ 0.o
0.5
1.0
t.5
20
l0
Time (rain) Fig ]. Reductitms of cyt b-558 in samples S and R with 0.1 mM NADPH or NADH under anaerobic canditkms. Tbe initial reducllon of cyt h-558 in sample S to) with NADPH was compared with Ihat in sample R (o) under anaerobic conanions. Similar expertments were carried out with NADH in sample S ( • ) and R I ~ ). The HTG-solubilized sample (6-7 mg protein/roD in 0.1 M phospbale buffer IpH 7.it). w~,s mlxed with OA mM NADPH or NADH anaerobically and the reduced minus oxidized spectrum at 250C was recorded. The rate of NADPH-dependent reduction was calculated as the percentage of fully reduced eyt b-55g with dithinnite. The concentration of cyt b-558 in the cuvettc wos 2 to 3/xM.
t i o n - d e p e n d e n t up to 80O p,M. T h e s e results showed that stimulation of t h e N A D P H oxidase system resuited in e n h a n c e m e n t of N A D P H - c y t c r e d u c t a s c activity in the m e m b r a n e s . In the a b s e n c e of a d d e d
o
SOD
1
I-i-i,~
.
terminal site with a high affinity.
Reduction of cyt b-558 bl samples S and R witb NADPH under anaerobic conditions Next. we e x a m i n e d the rates of reduction of cyt b-558 with N A D P H in samples S a n d R. Addition of 0.1 m M N A D P H u n d e r anaerobic conditions resulted in slow reduction of cyt b-558 in both sample S a n d R, as shown in Fig. 3 a n d T a b l e I. T h e initial rates of reduction of cyt h-558 with 0.1 m M N A D P H in samvies S a n d R were virtually the s a m e even though the former h a d high O~ g e n e r a t i n g activity a n d the latter h a d none as described above. T h e reduction rate of cyt b-558 with 0.1 m M N A D P H was higher t h a n that with 0.1 m M N A D H , but no a p p r e c i a b l e specificity for the nucleotide was observed (Fig. 3). T h e reduction rate of
++i+lt//
/ ~.
flavins, this e n h a n c e m e n t was slight because m;leromolecular c.~t c had little access to the active site of the oxidase system, but the m a r k e d e n h a n c e m e n t of N A D P H - c y t c reduct . . . . . . . . . . in the p . . . . . . . f small molecules of flavins which could shuttle one electron from the active site af the N A D P H oxidz:~e to exogenous cyt c [28]. In sample S, F M N was more effective than F A D for the reduction of cyt c. probably due to its higher rate constant for the single-electron transfer reaction as seen in the effects of FMN and F A D on N A D ( P ) H - d e p e n d e n t flavin enzymes [28,29], In contrast, exogenous flavins a n d m e n a d i o n e had no effect on 0 2 prtMuction (Table 1), suggesting that oxygen is accessible to an active site of the N A D P H oxidase without any help of these electron-shuqting c o m p o u n d s a n d accepts one electron directly from the
/~o
I o..O
i
I
R
,o+
~a~'A" V--q
~/~/~
bl ~
0 0
. . . . . . . . . . lO
20
a rain Time (rain) Fig. 4. Phutoreductlons of exogenous cyt c IA) and cyt b-558 (B) in samples S and R wilh time under anaerobic condillons. (A} Photoreduction~ of exogenouscyt ¢ by sample S (Q) and R (o }were measured under anaerobic conditions. "lhe ~nditions for the assayof cyt c reductase aClivay were as for Fig, I except for the absence of NADPI-I.The blank ( a ) for phxdoreduelion of cyt c was measured in the same manner without samples in the assay medium. Photoreduction was carried out hy flashlight (hv) with a 50 W halogen lamp. (B) Photoreducdons of cyt h-558 in samples S tl} and R to} plotted as functions of time at 25*(:= The rate of reduction is shown as a pe~entage of fully reduced cyt h-558 with dithionite. Anaerobic conditions were obtained as described for Figs. 1-3.
cyt b-558 in samples R a n d S were c o m p a r e d using pairs of samples from eight different preparations of pig blood cells. No appreciable differences were f o u n d in t h e reduction rates in samples R a n d S of these preparations. The effects of F A D , F M N , a n d m e n a dione on the N A D P H - d e p e n d e n t reduction of eyl b-558 in the S a n d R samples were e x a m i n e d u n d e r anaerobic conditions (Table 1). Results showed that m e n a dione (lt} ,~M) inhibited the reduction of cyt b-558 but did not inhibit O 2 production by sample S (Table I).
Photoreductions of ~ogetloz~s cyt sample A and R in anaerobiosis
c
~ t z-'.
and cyt b-538 in I~oth
Unexpectedly, the results obtained h e r e gave evidence for the involvement of an N A D P H - ~ ' t c reductase in the N A D P H oxidase system in stimulated cells. Thus, we next focused attention on t h e N A D P H - c y t c reductase with rcspccl to the redox center. Flavoproteins can be reduced by light u n d e r anaerobic conditions, so we examined the photoreductions of exogenous cyt c a n d cyt b-558 in samples S a n d R by flashlight u n d e r anaerobic conditions. Typical results of the reductions of cyt c a n d cyt b-558 are s h o w n in Fig. 4A a n d B, respectively. T h e p h o t o r e d u c t i o n rate of cyt c in sample S was m u c h h i g h e r t h a n t h a t in sample R (Fig. 4A). T h e e n h a n c e m e n t of t h e activity in sample S was consistent with that obtained with N A D P H (Figs. 1 a n d 2), suggesting that the N A D P H - c y t c reductase is possibly a flavin enzyme. In contrast to t h e rate of reduction of cyt c, t h e rate of p h o t o r e d u e t i o n of cyt b-558 in sample S was almost the same as t h a t in sample R (Fig. 4B), consistent with the results o b t a i n e d with N A D P H (Fig. 3). F r o m the results in Fig. 4A a n d B, the photoreduction rates of eyt c a n d cyt b-558 in sample S were 8.3 a n d 0.084 n m o l / m i n p e r m g of protein, respectively; that is, most of t h e electrons (99%) produced by p h o t o r e d u c t i o n w e r e trausferred to cyt c a n d only 1% to cyt b-558. W h e n a small a m o u n t of oxygen was a d d e d to the p h o t o i r r a d i a t e d samples, the spectrum of the p h o t o r e d u c e d cyt c immediately disappeared, suggesting that the p h o t o r e d u c e d flavoprotein is highly oxygen-sensitive.
~ o
O400
~
~
--
4SO
500
Wavelength (nn~) Fig. 5. Wavelcnglh dependence oa
photorcducaons of L~t c in samples S (o) and R (0) under anaerobic condilions, Photoreductions of exogenous cyt c in samples S and R were carried oul by irradiation by flashlighl passed through each of eight cutoff optical filters. Experimental conditions were as fnr Pic. 4A. Photoreduction of cyt c in Ihe assay medium withoul sample was used as the blank. The rules of eyl c reduction were corrected for the energy level of Iho light cul-off at different wa~lcngths to normalize the light intensily and arc pinned against the wavelength. lower than t h a t in sample S. T h e spectroscopic studies suggested that photoexeitation of a flavin e n z y m e occurs in sample S, resulting in electron transfer from t h e p h o t o r e d n c e d flavin to e x o g e n o u s cyt c. T h e r e f o r e , f o r f u r t h e r examination of the electron transfer reactions in samples S a n d R, we studied the 'effect' of photoirradiation o n samples containing cyt c at t h e optimal
Wat~elength for photoreduction of cytochromes W e analyzed the wavelength for p h o t o r e d u c t l o n of cyt c a n d cyt b-55g in samples S a n d R using eight cut-oft filters (the r a n g e of 402 to 505 n m ) a n d a hand-pass filter (450 n m at the m a x i m u m transmittance a n d 11.5 a m b a n d pass). T h e p h o t o r e d u c t i o n rates of exogenous cyt c in samples S a n d R were m e a s u r e d at 550 n m , a n d values were corrected for t h e energy levels of the light passed t h r o u g h the cut-off filters. T h e photorednction of eyt c in sample S d e p e n d e d o n t h e wavelength, a n d was maximal at about 45O n m , as s h o w n in Fig. 5. In contrast, photoreduction of eyt c in sample R showed a b r o a d p e a k a n d the activity was far
4ZO
4~0
4~a
nO0
5ZO
n,m
Beo
nBo
60o
Wavelength ( ~ ) Fig. 6. Phmoreduced minus OXidizeddifference spectra of ~ g e n o u s Cyl c in saml~les s and R by pholoirradiation with a band-pass optical filter at 450 nm under anaerobic conditions. (Right) Photoreduction of cyl c in sample $ or R was carried out by photoirradiafinn with a band pass oplical filter for selection of 450 nm light (left). The blank was recorded in the same manner by photoirradiation of reaction mixture in the absence of the ~lubdized samples. (Left) Optical property of tile band-pass filter used in this experiment.
?s
°a
z~
O400 4an 5on 'W'JaveIertath (r,_m) Fig. 7. Wavelengthdependence of phott)reductions of LVt h-55~;in ~mples S and U under unaerobic conditions Experimental~ndi lions were as for Fig. 4B with the same cut-oft filte~ :is for Fig 5 The photoreducfiun rates of cyt b-558 in samples S (o) 4nd R (c,) were delerrnined with ~rrection as for Fig. 5 Io I~ormalize th," intensityof the lighl passed through the cut-off filters. light-wavelength of 450 nm by light passed through the band-path filter (Fig. 6, left). As shown in Fig. 6, photoreduction of cyt c by selective irradiation at 450 nm was observed only in sample S, the rate of photoreduction in sample R being the same as that of the blank and independent of the wavelength. In Fig. 7, the photoreduction rates of cyt b-558 in samples S and R are plotted as functions of the cut-off wavelength of irradiated light in a similar way to that in Fig. 5. The reduction rates of cyt b-558 in samples S and R showed a similar wavelength-dependency to that in Fig. 5, the maximum reduction occurring at about 450 nm in both the S and R sample. These results suggest that cyt b-558 in both samples accepts electrons from a photoredueed flavin localized in the membranes. Discussion
In the present study, we showed that under strict anaerobic conditions HTG-solubilized preparations from stimulated and resting neutropbils exhibited NADPH-dependent cyt c reducing activity and that the activity of stimulated neutrophils was much higher than that of resting ones in the presence of FMN and FAD (Table I). This finding suggests that an NADPHeyt c reduetase located in the membranes becomes active during stimulation of the ceils. We obtained similar results on the enhancement of NADPH-dependent cyt c reduetase activity in solubilized oxidase fractions from pig blood neutrophils activated with
other stimulators, such as arachidonic acid and phorbol my-tstatc ~cctate. However, the saturated fatty acid seems preferable lot studies on free radicals as described !ater. We exan~ined membrane fractions of resting and stimulated cells, but found that the NADPH-dcpeadenl reductien of exogenous cyt e was mot,, niarkcd in solubilized oxidase preparations than in membrane preparations, probably due to more ready accessibility of the exogenous electron aeceptor to the site of an intrinsic flavin enzyme. Of several detergents tested, heplyhbioglueoside was the best for demonstrating the diaphorase activity under anaerobic conditions. The present results obtained by photorcduction gave evidence for the involvement of flavin in the NADPHcyt c reductase as its active site. Under anaerobic conditions, photoirradiation of the oxidase system at a selective wavelength of 450 nm, which is known to cause excitation of flavoproteins [3fl]. resulted in marked cyt c-reductase activity in the sample of stimulated cells but little in the sample of resting ones (Fig. 6). In the absence of the substrate tNADPH), photoreduction of flavoproteins involves the uptake of two electron equivalents by the flavin from intramolecular and intermolecular donors at the N(5), (3(8) or C(4) positions of the isoallonazine ring after transition to the flavin triplet (singlet) excitable state by absorption of light [31]. These results suggest that stimulation of neutrophilg causes modification of the flavoprJtein to active and photon-sensitive state that catalyzes marked reduction of exogenous cyt c. We found that the cyt c-redoctase activity induced by photorcduction is also very unstable in air. An NADPH-~--yt c reductase separated from other redox components is relatively oxygen-stable, but becomes unstable in the presence of cyt b-558 [16,32], suggesting that it may form flavin semiquinoae in the stimulated state, transferring a single electron to an oxidizable terminal component in the NADPH oxidase system. The photoreduced sample S failed to generate O.; even though the preparation could be reduced by light in anaerobic:sis. Therefore, NADPH seems to work not only as a specific reduetam, but also as a cofactor for a conformatiorial modification of the NADPH oxidase system to an 0 2 generating state. Previous EPR studies in our laboratory [6] showed that upon addition of NADPH in anaerobiosis, membranes from stimulated ceils gave an EPR signal of flavin semiquinone as a neutral (blue) semiquiaone. The saturated fatty acid, myristate, seems preferential as a stimulator for demonstrating flavin free radicals in ESR spectra because peroxide radicals formed in unsaturated fatty acids exhibit ESR signals at g = 2, which interfere with the ESR signals of flavin free radicals (Ref. 6 and unpublished data). The idea of involvement of a flavin semiquinone in the NADPH
oxidasc syslem in sfimulaled ceils is supportcd by the present findings wilh exogenous F A D , F M N a n d menadione, all of which can transfer a single electron by their shuttling between flavin serniquinonc a n d exogenous cyt c. leading to the e n h a n c e m e n t of N A D P H d e p e n d e n t cyl c-reductase activity in preparations fronl stimulated ceils (Fig, 2 a n d Table 1). In contrast, none of these tl~rec reagents h a d any effect on O;- production in the N A D P H oxidase s~slenL even at the high concentrations used: m e n a d i o n e at up to 0.8 raM. a n d F M N a n d F A D at 0.I m M . These results suggest that oxygen is 0 more specific electron accepter than these artificial compound:, in the oxidase ,~ystem. In contrast to the m a r k e d difference in N A D P H - c y t c rednctase activities in resting a n d stimulated samples, there was no difference in lheir reductions of cyt b-55g by either N A D P H or light, as s h o w n in Figs. 3 a n d 4. The present results failed to support the hypothesis that cyt b-558 in the oxidase system is the terminal electron d o n o r to oxygen. This heine p r o l e i n is an essential c o m p o n e n t of the oxidase as j u d g e d by the fact that neutrophils of patients with X-link inherited C G D lack this protein. Since the redox potential of eyt h-558 is unusually low, -2d-5 mY, this ferrous h e m e has been t h o u g h t to be highly autoxidizable, resulting in O ~ formation [14]. However, cyt b-558 in sample R was reduced to the same extent as that in sample S by N A D P H (Fig. 3), but did not form any O ~ . This finding indicates that O~- generation should be interpreted nol only by t h e r m o d y n a m i c possibilities, b u t also by phagocyte-specific control mechanisms. In a cell-free systenl, the O 2 f o r m i n g activity of the N A D P M oxidase d e p e n d s o n the presence of cyt b-558, F A D a n d cb'tosolic c o m p o n e n t s such as p47-phox, p67phox [11] and a small nueleotide-binding c o m p o n e n t ( s ) [33]. O u r recent sludies on cytosolic c o m p o n e n t s suggest thai neither p47-phox n o r p67-phox is a flavia enzyme [34]. These cytosolie c o m p o n e n t s a p p e a r to be activating factors modifying redox c o m p o n e n t s such as cyt b-558 a n d / o r the florin enzyrae r a t h e r than electron transport c o m p o n e n t s . It is very likely that the e n h a n c e d activity of N A D P H - d e p e n d e n t cyt c reduction m a y be catalyzed by an F A D enzyme, an N A D P H cyt c reductase, reported from this laboratory [16]. Since plasma m e m b r a n e s of pig blood neutrophils contained very similar a m o u n t s of F A D (and negligible a m o u n t s of F M N ) in both resting and stimulated states [6,16], the F A D enzyme may be an intrinsic m e m b r a n e protein like cyt b-558 r a t h e r t h a n a cytosolic protein. Vignais' g r o u p reported similar reductase activity in purified preparations from m e m b r a n e s of bovine neutrophils [19] a n d recently proposed a model for the diaphorase-o×idase transition based o n their findings that in rabbit peritoneal neutrophils, a m e m b r a n e h o u n d N A D P H - c y t c reductase was converted to a n
O , - g c n e t a t i n g oxidase d u r i n g slimulation [35]. T h e i r model is in accord with ours. Acknowledgements We are very grateful to Prof. Takashi Yonetani, D e p a r l m e n t of Biochemistry a n d Biophysics, Univt.,'sity of Pennsylvania and Dr. Takeshi Nishino, Departm e n t of Biochemistry, Y o k o h a m a City University School of Medicine, for t h e i r helpful discussions a n d critical r e a d i n g of o u r manuscript. This work was supported in part by grants from the Ministry of Education, Science a n d Culture, Japan. References
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32 K;~,il;UmLi, K. (Inb~,, I'. tllld Fujii. H (1989~ ill Medical r~i,,chemical :lied ( hcl~li¢:d A-pqCls 0f Fr~c ~tdlc;Ll~ (Ihy;llshi, ( ) Niki, E . K.rl&~. M. and Yoshikawa. I . Cds. I. p p 1311 13]-1. Elsevier Amsterdam ~ Atl;iMui. I lind Pick. I (iq,,lll J. [ euko;)le Bml. 48. Ill7 115. C h l h . T Kaneda M Fmll }1. ('k*rk. R A ,~lauseef. W ~] .nO Kttkirlllnl;i K liL)t2t}) Biochcm I]inphys Res ('l~mmun. 173. ~v Lap,,rl,. F [),,u,,i,'re I und Vigmlis. P.",. (1,1,,i)) Eur J []hlchcm. /w4.31H ~11'~
