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INTRODUCTION |
NADPH oxidase is a multicomponent electron transport system mainly
expressed in phagocytic cells of the immune system. Normally dormant,
NADPH oxidase is activated after stimulation of these cells resulting
in the vigorous production of superoxide (O
2) at the expense
of NADPH (1). The physiologic role of the enzyme is to contribute to
microbial killing by the generation of reactive oxygen species derived
from O
2 that are toxic to microorganisms. Genetic defects in
components of NADPH oxidase lead to a severe predisposition to
bacterial and fungal infections, a syndrome known as chronic
granulomatous disease (2). The NADPH oxidase enzyme complex consists of
a membrane-bound flavocytochrome, flavocytochrome b
245 that contains two molecules of heme and
one molecule of FAD in a gp91phox/p22phox heterodimer
(3-8). In unstimulated cells, the other components of the complex,
p47phox, p67phox, and Rac2, are present in the cytosol;
upon activation, they associate with flavocytochrome
b
245 on the membrane to form the active enzyme
(9-13). A fourth cytosolic component, p40phox, is also
implicated in NADPH oxidase activity, but the role it plays is poorly
understood at present (14, 15). Understanding of the mechanism and
activation of NADPH oxidase has been greatly facilitated by the
development of a cell-free system. In the simplest form,
NADPH-dependent O
2 can be generated by mixing
neutrophil membranes (containing flavocytochrome
b
245) and neutrophil cytosol (containing
p40phox, p47phox, p67phox, and Rac2) with an
anionic amphiphile and NADPH. In both whole cells and the cell-free
system, there is a characteristic lag period before the oxidase
activity appears after the addition of stimulus (to whole cells) or
amphiphile (to the cell-free system). Previous work has shown that a
decrease in the lag period can only be achieved by simultaneous
incubation of neutrophil membranes and cytosol with the anionic
amphiphile and not by preincubation of one or the other with anionic
amphiphile (16, 17). Recent reports using intrinsic tryptophan
fluorescence and circular dichroism suggest that SDS and
AA1 caused conformational
changes in p47phox at concentrations similar to those required
to induce oxidase activity (18, 19). Furthermore, Sumimoto and
co-workers (20) reported that C-terminally truncated forms of
p47phox and p67phox exhibit a diminished requirement
for SDS, which would be consistent with p47phox (or
p67phox) being a primary target for anionic amphiphiles.
We have previously provided evidence that electron transfer in NADPH
oxidase is regulated at several points. First, at the site where
electrons enter the oxidase, because in the nonactivated enzyme, FAD
does not become reduced in the presence of NADPH (21, 22). Second,
there is a kinetic barrier between the flavin center and the heme,
because they are not in thermodynamic equilibrium during oxidase
turnover (23-26). Finally, rapid electron transfer within the oxidase
only occurs in the presence of oxygen, suggesting regulation at the
level of heme (21, 23, 27, 28). To further understand the events that
occur during oxidase activation, we have taken advantage of a cell-free
system consisting of highly purified flavocytochrome
b
245 and recombinant p47phox,
p67phox, and Rac2 to perform experiments aimed at dissecting
the target(s) of the anionic amphiphile during oxidase activation.
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EXPERIMENTAL PROCEDURES |
Recombinant Proteins--
Oligonucleotide primers corresponding
to each of the human NADPH oxidase subunits were used to amplify
respective cDNA from a human neutrophil cDNA library
(CLONTECH, Palo Alto, CA). Polymerase chain
reaction-amplified products were purified by agarose gel electrophoresis and ligated into the baculovirus transfer vector pVL1393 (Pharmingen, San Diego, CA). Constructs were used to transform Escherichia coli and single colonies were picked from plated
LB broth cultures under selective pressure of 50 µg/ml carbenicillin. Purified vector clones were sequenced to confirm cDNA sequence integrity. Purified pVL1393 plasmid was used for homologous
recombination with BaculoGold baculovirus (Pharmingen) before infection
of Sf9 insect cells. These Sf9 cultures were amplified
twice before plaque assay to identify individual transfectant clones.
Virus from individual plaques was picked and again amplified two or
three times in Sf9 insect cells. Supernatants from these final
Sf9 cultures were aliquoted and stored as master viral stocks.
Large scale production of recombinant oxidase factors was carried out
in Sf9 cells (rp47phox and rpRac2) or Hi5 cells
(rp40phox and rp67phox). Briefly, master viral stocks
were used at a multiplicity of infection of 5-10, infecting 2 × 107 insect cells in 15-20 ml of TMN-FH medium (Pharmingen)
in 150-mm diameter culture dishes; 50 of these plates were used for
each production run. Cells were allowed to incubate for three days at
28 °C before harvest; all subsequent manipulations and purifications were carried out at 4 °C. Insect cells were obtained by scraping the
culture dishes and centrifuging the pooled cultures at 500 × g for 7 min. The cell pellet was resuspended in 200 ml of
phosphate-buffered saline and again centrifuged at 500 × g for 7 min. This cell pellet was then resuspended in 50 ml
of disruption buffer: 10 mM PIPES, pH 7.3, 100 mM KCl, 3 mM NaCl, 3.5 mM
MgCl2, 48 µg/ml [4-(2-aminoethyl)benzene sulfonyl
fluoride, HCl], 1 µg/ml aprotinin, 151 µg/ml benzamidine, 5 mM diisopropylfluorophosphate, 50 µg/ml leupeptin, 0.7 µg/ml pepstatin. Cell suspensions were disrupted by nitrogen
cavitation (450 psi, 20 min) and (for the cytosolic factors
rp40phox, rp47phox, and rp67phox) the cavitate
was subjected to a 10,000 × g centrifugation for 10 min. The resulting supernatant was then collected and centrifuged at
100,000 × g for 1 h; the supernatant from this
high speed spin used as starting material for chromatographic
purifications. Treatment of the cavitate differed slightly for rpRac2:
only the 100,000 × g centrifugation was performed, and
the resulting pellet was then solubilized in 50 ml of extraction
buffer: 20 mM Tris, pH 7.5, 0.6% (w/v) CHAPS. This extract
was centrifuged at 100,000 × g for 1 h, and the
supernatant was collected for subsequent chromatography.
rp40phox was purified at pH 6.0 by anion exchange (HiLoad
Q-Sepharose fast flow) (Amersham Pharmacia Biotech), hydrophobic
interaction chromatography (HiLoad phenyl-Sepharose high performance)
(Amersham Pharmacia Biotech). Pure rp40phox was frozen to
80 °C at 10 µM in 20 mM Tris, pH 6.0, 0.1 mM dithiothreitol, 0.15 mM
phenylmethylsulfonyl fluoride, 5.3 mM ammonium sulfate.
rp47phox was purified at pH 7.5 by cation exchange (HiLoad
SP-Sepharose fast flow), heparin chromatography (1 ml HiTrap heparin) (Amersham Pharmacia Biotech). Pure rp47phox was frozen to
80 °C at 10 µM in 20 mM Tris, pH 7.5, 0.1 mM dithiothreitol, 0.15 mM
phenylmethylsulfonyl fluoride.
rp67phox was purified at pH 7.0 by anion exchange (HiLoad
Q-Sepharose fast flow), hydrophobic interaction chromatography (HiLoad phenyl-Sepharose high performance), and size-exclusion gel
chromatography (HiLoad SuperDex 75) (Amersham Pharmacia Biotech).
rp67phox was frozen to
80 °C at 10 µM in 20 mM Tris, pH 7.0, 0.1 mM dithiothreitol, 0.15 mM phenylmethylsulfonyl fluoride.
rpRac2 detergent extract was purified by anion exchange (HiLoad
Q-Sepharose fast flow). Fractions containing pure rpRac2 were pooled
(~15 ml) and diluted to 100 ml with nucleotide loading buffer: 20 mM Tris, pH 7.5, 1 mM EDTA, 2 mM
dithiothreitol, 100 µM GTP
S. Concentration was
performed in a 200-ml stirred cell through a YM10 membrane (Amicon,
Beverly, MA) to ~6 ml, and volume brought back to 100 ml with fresh
nucleotide loading buffer. The sample was again concentrated to ~6
ml, then brought to 100 µM fresh GTP
S and 5 mM MgCl2. rpRac2 was then frozen to
80 °C
at 2 µM.
Each of the recombinant oxidase factors was analyzed by mass
spectrometry to confirm correct molecular weight; proteins were also
subjected to SDS-polyacrylamide gel electrophoresis and subsequent silver staining to assess homogeneity. Western blotting of
SDS-polyacrylamide gel electrophoresis gels and subsequent incubation
with anti-peptide (all subunits) and monoclonal (p47 only) antibodies
showed that the recombinant proteins were immunoreactive and migrated
identically to their counterparts in purified human neutrophil cytosol.
Activation of NADPH Oxidase in the Cell-free
System--
Cell-free NADPH oxidase assays were performed at 21 °C
in 96-well plates as described previously (29) using a final
concentration of 80 µM SDS or 20 µM AA.
These concentrations were found to elicit the maximum rate of
O
2 production. Reactions were carried out in a final volume of
150 µl, which contained 0.3 pmol of flavocytochrome b
245, 15 pmol of p47phox, 10 pmol of
p67phox, and 4 pmol of Rac2. 10 pmol of p40phox was
included where noted. For preincubation assays, each reaction mixture
was split into three equal parts. The first contained the proteins to
be incubated with anionic amphiphile, the anionic amphiphile (80 µM SDS or 20 µM AA) and cytochrome
c. The second contained NADPH and the proteins not to be
preincubated with amphiphile. The third portion contained relaxation
buffer and either 160 µM SDS or 40 µM AA,
to maintain the final amphiphile concentration in the assay at 80 µM SDS or 20 µM AA. GTP
S was always
included in the portion containing Rac2, and FAD was always included in the portion containing flavocytochrome b
245.
After 5 min of preincubation, the three portions were mixed together in
the microtiter plate, and the reduction of cytochrome c was
monitored at 550 nm. The time taken between mixing and the first
measurement was approximately 25 s. The "complete"
preincubation mixture refers to the reaction mixture that contained all
the assay components (including anionic amphiphile) with the exception
of NADPH.
To check that the rates of reduction of cytochrome c reflect
the true O
2-generating activity of the system and are not a result of a lack of sensitivity at low rates of O
2 production, control assays were carried out at a range of cytochrome c
concentrations using a sensitive spectrophotometer (Perkin-Elmer Lambda
18) and 1-cm path length cuvettes. A 5-fold increase in cytochrome
c concentration gave the same rate as the standard assay
over a wide range of oxidase activities and therefore probably
accurately reflects the true rate of O
2 even at low rates
of O
2 production (data not shown).
INT reductase assays were performed as described above for the
O
2 assay, except 100 µM INT was substituted for
the cytochrome c and 300 units/ml of SOD were included in
the mixture. The rate of reduction of INT was determined from the
absorbance change at 490 nm as described previously (30).
Spectrophotometric Assays--
In cases where it was
advantageous to examine the earliest parts of the time course, assays
were performed in semi-micro cuvettes in a Perkin-Elmer Lambda 18 spectrophotometer. Each reaction mixture contained the same
concentrations of reactants as the microtiter assay in a final volume
of 750 µl.
Measurement of the Lag Time--
Three methods were used to
estimate the lag times; (a) time to maximum reaction
velocity; (b) extending a tangent from the steepest part of
the curve to the x axis, and; (c) measuring the onset time (time taken to produce an arbitrary amount of O
2). Each method gave qualitatively the same results. Of the three, the
onset time was found to give the most reproducible results and was used
to calculate the data presented here. The onset time of each reaction
was taken as the time needed by that mixture to generate the same
amount of O
2 as made by the complete reaction mixture during
the initial 100 s following the addition of NADPH.
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RESULTS |
NADPH Oxidase Activity Is Independent of Both p47phox
and Anionic Amphiphile at High Concentrations of p67phox
and Rac2--
Two groups have recently reported that NADPH oxidase can
be activated in a cell-free system in the absence of p47phox,
under conditions where p67phox and Rac are present at much
higher concentrations than are normally employed (31, 32). In both
cases, they reported that the activity was also dependent upon the
presence of anionic amphiphile (AA or lithium dodecyl sulfate). In our
hands however, we found p47phox-independent oxidase activity
was completely independent of anionic amphiphiles at concentrations of
p67phox and Rac similar to those used by Pick, Lambeth, and
co-workers (31, 32) in these previous reports (Fig.
1). This difference may be due to
differences in the flavocytochrome b
245
preparation (purity, reconstituted lipid composition, FAD content,
detergent, etc.) or that we employed Rac2 in our experiments as opposed
to Rac1. More than 96% of Rac in human neutrophils is the Rac2 form (33). Because the absence of p47phox allowed activation of the
system without addition of amphipathic stimulus, the data strongly
suggest that the primary target for the anionic amphiphiles was
p47phox.

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Fig. 1.
NADPH oxidase activation is independent of
anionic amphiphile at high p67 and Rac2 concentrations in
the absence of p47phox. NADPH oxidase activity was
determined as described under "Experimental Procedures." The
reactions were initiated by the addition of 160 µM NADPH
after 5 min of preincubation with or without SDS. The shaded
bars refer to the standard concentrations of Phox proteins (2.0 nM flavocytochrome b 245, 100 nM p47phox, 67 nM p67phox, 26 nM Rac2). The open bars refer to a system
containing high concentrations of cytosolic phox components (2.0 nM flavocytochrome b 245, 3.1 µM p47phox, 4.0 µM p67phox,
and 175 nM Rac2). For comparison, the high concentration
system employed by Lambeth and colleagues (31) used 10 nM
flavocytochrome b 245, 6.0 µM
p47phox, 6.0 µM p67phox, and 2.0 µM Rac1, and the system used by Pick and colleagues (32)
used 1.75 nM flavocytochrome b 245,
880 nM p47phox, 2.96 µM
p67phox, and 1.0 µM Rac2. The values are the
means ± S.E. of two separate experiments performed in
duplicate.
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Kinetics of O
2 Production after Preincubation of Mixtures
of phox Components with SDS--
As shown previously, preincubation of
all phox components together with SDS for 5 min (the
complete mixture) before the addition of NADPH completely abolished the
lag seen under conditions of no preincubation (Fig.
2, trace b versus trace
a). Preincubation of flavocytochrome b
245
with SDS alone or preincubation of cytosolic phox components
with SDS alone, did not shorten the lag for NADPH oxidase activity
below the control reaction without any SDS preincubation. Similarly,
preincubation of flavocytochrome b
245 with SDS
and preincubation of cytosolic components with SDS concomitantly but in
separate tubes did not significantly reduce the lag time (Fig. 2,
trace c). Preincubation of the recombinant cytosolic
components with SDS in the absence of flavocytochrome b
245 tended to decrease the final maximum
activity, however, consistent with previous studies using neutrophil
cytosol (34) (Fig. 2, trace c). The results of preincubation
of cytosolic components individually and in combination with
flavocytochrome b
245 and SDS are shown in Fig.
3. The lag was only significantly reduced when p47phox and flavocytochrome b
245
were preincubated together with SDS (bars D, H,
and J). Preincubations of p67phox + flavocytochrome
b
245 + SDS (bar E) or Rac2 + flavocytochrome b
245 + SDS (bar F)
did not reduce the lag. Somewhat surprisingly, the simultaneous
presence of p67phox + p47phox + flavocytochrome
b
245 +SDS (bar G) in the
preincubation mixture did not decrease the lag, despite the fact that
preincubation of p47phox + flavocytochrome
b
245 + SDS (bar D) alone did. This
suggests that p67phox can interfere with productive
interactions between p47phox and flavocytochrome
b
245. Interestingly, if Rac2 is also present,
the lag is once again reduced (bar J), suggesting that interactions between p67phox and Rac2 may be important during
the activation process.

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Fig. 2.
Time course of NADPH oxidase activity.
The SOD inhibitable rate of cytochrome c reduction was
followed spectrophotometrically as described under "Experimental
Procedures." Trace a, the reaction was
initiated by the addition of SDS to the complete mixture
(i.e. no preincubation). Trace b, the reaction
mixture containing all the components except NADPH was preincubated for
5 min before the addition of NADPH. Trace c, flavocytochrome
b 245 and the cytosolic phox components were
incubated with SDS in separate tubes for 5 min before mixing and
initiating the reaction with NADPH. The traces are representative of
three separate experiments.
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Fig. 3.
The effect of the composition of the
SDS preincubation mixture on the kinetics of the activation of
O 2 generation by NADPH oxidase. NADPH oxidase
activity and onset time were determined by the SOD-inhibitable rate of
cytochrome c reduction in the microtiter plate assay as
described under "Experimental Procedures." A plus
sign (+) beneath the bar indicates that component(s) were
present in the portion of the reaction mixture preincubated in the
presence of SDS; a minus sign ( ) beneath the bar indicates
that a given component was not pre-exposed to SDS before the reaction
was started. The values are the means ± S.E. of three separate
experiments performed in duplicate.
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We have recently reported that in the absence of Mg2+ there
is a significant SDS-independent spontaneous activation of oxidase activity in this system (29). A series of experiments using Mg2+-free conditions gave qualitatively the same results as
those shown, except in each case there was more
O
2-generating activity evident at the time of the first
reading in the Mg2+-free experiments.
Kinetics of O
2 Production after Preincubation of Mixtures
of phox Components with AA--
A similar series of experiments to
those described above was performed using AA in place of SDS. In both
types of experiment, the maximal velocity is similar regardless of
activator, although the onset time is somewhat shorter with AA. As in
the case of SDS, preincubation of flavocytochrome
b
245 with AA (Fig. 4, bar B) or preincubation of
cytosolic components with AA (bar C) did not shorten the lag
for NADPH oxidase activity below the control reaction without any AA
preincubation (bar A). Similarly, preincubation of
flavocytochrome b
245 with AA or recombinant cytosol with AA concomitantly but separately did not significantly reduce the lag time. As in the case of SDS, preincubation of
p47phox + flavocytochrome b
245 + AA
(Fig. 4, bar D) caused significant reduction in the lag.
Unlike the SDS incubations, however, the combination of p47phox + p67phox + flavocytochrome b
245 + AA
(bar G) reduced the lag maximally, whereas the combination
of p47phox + Rac2 + flavocytochrome
b
245 + AA (bar H) did not reduce the lag at all. This suggests that Rac2 can interfere with productive interactions between p47phox and flavocytochrome
b
245 when AA is the activator. Because this is
not the case for SDS, it indicates there may be significant differences
in the way in which SDS and AA cause oxidase activation.

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Fig. 4.
The effect of the composition of the AA
preincubation mixture on the kinetics of the activation of O 2
generation by NADPH oxidase. NADPH oxidase activity and onset time
were determined by the SOD-inhibitable rate of cytochrome c
reduction in the microtiter plate assay as described under
"Experimental Procedures." A plus sign (+) beneath the
bar indicates that component(s) were present in the portion of the
reaction mixture preincubated in the presence of SDS; a minus sign ( )
beneath the bar indicates that a given component was not pre-exposed to
AA before the reaction was started. The values are the means ± S.E. of three separate experiments performed in duplicate.
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The Effect of p40phox on the Kinetics of NADPH Oxidase
Activation--
The effect of p40phox was also examined in a
series of experiments. In no case did p40phox appear to affect
the lag time or maximal velocity of the reaction (data not shown).
Kinetics of INT Reduction after Preincubation of Mixtures of phox
Components with SDS--
We next examined the kinetics of activation
of INT reductase activity. We have previously shown that INT accepts
electrons from the flavin center of NADPH oxidase and that there is
separate control of electron transfer between NADPH and enzyme FAD and between enzyme FAD and heme (25, 30). As can be seen in Fig. 5, the lag is less pronounced when INT is
the electron acceptor, suggesting there is significant INT reductase
activity before there is appreciable O
2 formed. This does not
appear to be an artifact due to lack of sensitivity of the cytochrome
c assay at low rates of O
2 formation, as
described under "Experimental Procedures." Most
strikingly, there is a very high rate of INT reductase activity in
assays where p47phox + flavocytochrome
b
245 + SDS or p47phox + Rac2 + flavocytochrome b
245 + SDS were preincubated, and these activities had virtually no lag (Fig. 5A,
traces a and b). The rates in both cases were
much higher than the rate obtained when all the components (except
NADPH) were preincubated together (trace e). In
contrast, preincubations containing flavocytochrome b
245 + SDS, p67phox + flavocytochrome
b
245 + SDS, or p67phox + Rac2 + flavocytochrome b
245 + SDS showed the longest lag and the lowest maximal rates (Fig. 5B, traces
d, b, and c). Incubation of the
cytosolic factors with SDS in the absence of flavocytochrome
b
245 also gave rise to rather low linear rates
(Table I.).

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Fig. 5.
The effect of the composition of the SDS
preincubation mixture on the kinetics of INT reductase activity by
NADPH oxidase. INT reduction was followed using a kinetic
microtiter plate assay as described under "Experimental
Procedures." In panel A, the preincubation mixtures
contained SDS and as follows: flavocytochrome
b 245 + p47phox (a);
flavocytochrome b 245 + p47phox + Rac2
(b); flavocytochrome b 245 + p47phox + p67phox (c); no phox proteins (no
preincubation) (d); flavocytochrome
b 245 + p47phox + p67phox + Rac2 (complete preincubation) (e); p47phox+
p67phox + Rac2 (f). In panel
B, the preincubation mixtures contained SDS and as follows:
flavocytochrome b 245 + Rac2
(a); flavocytochrome b 245 + p67phox (b); flavocytochrome
b 245 + p67phox + Rac2 (c);
flavocytochrome b 245 (d); complete
preincubation but no NADPH addition (e). The traces are the
averaged values of two wells of a representative experiment of three
performed in duplicate.
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Kinetics of INT Reduction after Preincubation of phox Components
with AA--
As in the case of SDS activation, the lag was much less
pronounced during AA-dependent activation when INT was the
electron acceptor. Unlike SDS activation, however, all incubations
showed broadly similar initial rates, with the incubation containing all the Phox proteins having the highest rate. Preincubations of
cytosolic factors with AA in the absence of flavocytochrome b
245 again gave somewhat lower rates of INT
reductase activity (Table I).
Kinetics of NADPH Oxidation, INT Reduction, and O
2
Formation in the Presence and Absence of INT--
Further evidence for
an intermediate state of activation of NADPH oxidase was provided by
experiments following NADPH oxidation in the presence or absence of
INT. NADPH oxidation was recorded at 340 nm after the addition of SDS
to the cuvette. The results are shown in Fig.
6. As can be seen from the traces, in the
presence of INT, the onset of NADPH oxidation is faster than in the
absence of the dye, and the rate is fastest during the first 2 min. In the absence of INT, NADPH oxidation follows a similar time course to
O
2 production, with a long lag before reaching the maximum rate. The complementary experiments are shown in Fig.
7 following the reduction of cytochrome
c by O
2 (at 550 nm) or the reduction of INT (at 490 nm). The traces parallel the NADPH oxidation rates, with the reduction
of INT having a rapid onset and reaching the maximum rate within the
first 2 min.

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Fig. 6.
The kinetics of NADPH oxidation by NADPH
oxidase in the presence or absence of the electron acceptor INT.
NADPH oxidation was determined spectrophotometrically as described
under "Experimental Procedures." The reactions were started by the
addition of SDS. The mixing time (before the start of the recording)
was approximately 10 s. Trace a, NADPH oxidation in the
absence of INT; trace b, NADPH oxidation in the presence of
100 µM INT. The traces are representative of three
separate experiments.
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Fig. 7.
The kinetics of INT reduction and O 2
formation after the activation of NADPH oxidase by SDS. INT
reduction and O 2 formation were measured
spectrophotometrically as described under "Experimental
Procedures." The reactions were started by the addition of SDS. The
mixing time (before the start of the recording) was approximately
10 s. Trace a, O 2 production (cytochrome
c reduction); trace b, INT reduction. The traces
are representative of three separate experiments.
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DISCUSSION |
The principal action of both SDS and AA appears to be to promote
an interaction between p47phox and flavocytochrome
b
245. This process requires the simultaneous presence of both flavocytochrome b
245 and
p47phox and results in almost complete elimination of the lag
and subsequent high rates of oxidase activity. The secondary effect of
SDS appears to be to promote interactions between Rac2 and
flavocytochrome b
245 (or the flavocytochrome
b
245·p47phox complex), whereas the
secondary effect of AA appears to promote interactions between
p67phox and flavocytochrome b
245.
Taken together, the results presented above support a model in which
association of the cytosolic factors with flavocytochrome b
245 initially results in an intermediate
state of activation of NADPH oxidase by a relatively rapid process. In
this intermediate state, electron transfer is facilitated from NADPH to
the FAD center of the enzyme, where electrons can be diverted to INT. In the intermediate state, however, electron transfer does not proceed
normally to the heme centers, and thus little O
2 is formed. The intermediate state of activation is converted to the fully active
state in a rate-limiting process that is responsible for the lag.
A model that describes the observed kinetics is shown in Scheme
1. For simplicity, only the reactions
involving flavocytochrome b
245 are shown. In
this model, the anionic amphiphile (SDS or AA) causes a conformational
change in p47phox that causes it to associate with
flavocytochrome b
245. It seems that this
conformational change only occurs in a productive manner when
flavocytochrome b
245 is also present in the incubation mixture, because preincubation of p47phox with SDS
or AA does not decrease the lag if flavocytochrome
b
245 is not present. This complex is
represented by p47*·cytb. The binding of p47phox to
flavocytochrome b
245 allows the high affinity
binding of p67phox and Rac2 as previously proposed by Lambeth
and colleagues (35), resulting in the intermediate state in which
electron transfer can proceed from NADPH to INT. The formation of this
intermediate state is relatively rapid, and consequently, little lag is
evident in the onset of INT reductase activity. A subsequent slow step, possibly involving a conformational change in flavocytochrome b
245 (cytb
cytb* in Scheme 1), results in
the final fully active NADPH oxidase. The relative rapid formation of
the intermediate complex relative to the fully activated form would
explain why INT reductase activity dominates the initial phase of
oxidase activity, whereas O
2 production predominates the
latter phase (Fig. 6).

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Scheme 1.
A kinetic model for the activation of NADPH
oxidase by anionic amphiphiles (see text for details). It should
be noted that in Scheme 1 the rapidly formed complex in the lower left
(p47*.cytb.Rac.p67), which has high reactivity toward INT, has low
reactivity toward O 2, whereas the final slowly formed
complex at the lower right (p47*.cytb*.Rac.p67), which has high
reactivity with O 2, has little or no reactivity toward
INT.
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