From the Department of Molecular and Experimental
Medicine and the
Department of Immunology, The Scripps Research
Institute, La Jolla, California 92037
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The leukocyte NADPH oxidase is an enzyme present
in phagocytes and B lymphocytes that when activated catalyzes the
production of O The NADPH oxidase is a membrane-associated enzyme that catalyzes
the one electron reduction of oxygen to O The phosphorylation of p47PHOX is a well recognized concomitant
of oxidase activation in whole cells, but the mechanism of activation of the oxidase is not fully understood (8-14). One key to
understanding the activation of the oxidase emerged with the discovery
of the cell-free activation system (15-17) in which it was shown that NADPH oxidase activity could be induced in a mixture of membrane and
cytosol by the addition of amphiphiles like arachidonic acid (15, 17)
and SDS (16, 18). Recently, increasing attention has been paid to
cell-free systems in which the oxidase is activated without using
anionic amphiphiles (19-22). Our studies showed that the oxidase can
be activated by p47PHOX phosphorylated by protein kinase C in a
cell-free system containing neutrophil membrane and cytosol (21). In
addition, these studies also revealed a that the phosphorylation of
p47PHOX was not the only ATP-dependent step in the
activation of the oxidase by protein kinase C. A preceding
phosphorylation event occurs in the membranes rendering them capable of
supporting oxidase activation. The target of this event has yet to be
determined. Although these experiments showed a direct relationship
between the phosphorylation of p47PHOX and the activation of
the oxidase, the use of whole cytosol made it difficult to recognize
whether cytosolic factors other than those necessary for activation by
SDS are required for oxidase activation by a kinase. In this paper we
report studies of a recombinant cell-free system containing only
membrane and cytosolic oxidase components (p47PHOX,
p67PHOX, and Rac2). Our findings suggest that the cytosolic
components phosphorylated p47PHOX, p67PHOX, and Rac2
are sufficient for partial activation of the oxidase.
Materials--
Chemicals and enzymes were obtained from the
following sources: dextran and Ficoll-Hypaque from Amersham Pharmacia
Biotech; phosphatidylserine, diacylglycerol,
isopropyl- Preparation of Neutrophil Fractions--
Neutrophil cytosol and
membrane were prepared as described by Borregaard et al.
(23). Neutrophils were prepared from normal subjects by dextran
sedimentation and Ficoll-Hypaque fractionation of freshly drawn
citrate-anticoagulated blood. The neutrophils were suspended at
108 cells/ml in a modified relaxation buffer (0.1 M KCl, 3 mM NaCl, 3.5 mM
MgCl2, 10 mM PIPES buffer, pH 7.3). Plasma
membrane and cytosol were prepared by nitrogen cavitation followed by
centrifugation through a Percoll gradient. Both cytosol and membrane
were divided into aliquots and stored at Production and Purification of Recombinant p67PHOX
from Baculovirus-infected Sf9 Cells--
Purified recombinant
p67PHOX was produced by means of the baculovirus system
described by Leto et al. (1991), using a
p67PHOX-expressing recombinant virus generously provided by
T. L. Leto. Large scale production of pure recombinant
p67PHOX was achieved by infecting monolayer cultures of
Sf9 cells in 150 cm2 flasks at a density of
1-2 × 106 cells/ml (24). Cells were harvested
72 h postinfection, washed twice in phosphate-buffered saline by
centrifugation at 400 × g for 10 min, and then
resuspended to 5 × 107/ml in lysis buffer (50 mM KCl, 3 mM NaCl, 2 mM
MgCl2, 0.1 mM dithiothreitol, 1 mM
EDTA, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin,
2 mM phenylmethylsulfonyl fluoride, 5.4 mM PIPES, pH 7.5). All subsequent work was conducted at 4 °C. Cells were disrupted by sonication (4 × 10 s) and centrifuged at
400 × g for 10 min. The supernatant fraction
containing p67PHOX was brought to 45% saturation with solid
ammonium sulfate. The resulting precipitate was isolated by
centrifugation (1200 × g at 4 °C for 30 min), then
dissolved in 10 ml of buffer A (20 mM Tris, pH 7.5, 0.1 mM dithiothreitol, 1 mM EDTA, 2 mM
EGTA, 0.15 mM phenylmethylsulfonyl fluoride) and dialyzed
overnight against the same buffer. The dialyzed solution was applied to
a Mono Q-Sepharose column (Amersham Pharmacia Biotech) previously
equilibrated with buffer A and washed with 5 volumes of the same
buffer. Proteins were eluted from the column by fast protein liquid
chromatography with a 0.1-0.3 M NaCl gradient in the same
buffer at a flow rate of 0.8 ml/min. The fractions containing purified
p67PHOX were pooled and stored at Preparation of Recombinant GST-p47PHOX and Rac2 Fusion
Proteins--
Recombinant fusion proteins composed of glutathione
S-transferase (GST) linked downstream to p47PHOX or
Rac2 were isolated from Escherichia coli transformed with pGEX-1 Phosphorylation of GST-p47PHOX--
Phosphorylation
of recombinant GST-p47PHOX was carried out using 200 µg of
fusion protein in final volume of 200 µl. The reaction mixture
contained 1 mM ATP, 10 mM magnesium acetate,
1.0 mM CaCl2, 10 µg phosphatidylserine, 1 µg diolein, and 0.5 unit of protein kinase C in 200 µl of
relaxation buffer (0.1 M KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM PIPES buffer, pH
7.3). The lipids were added as mixed liposomes prepared by dissolving
2.5 mg/ml phosphatidylserine and 1 mg/ml diacylglycerol in chloroform,
removing the chloroform under a stream of nitrogen, and then sonicating
the dried lipids for 2 min on ice in 0.8 ml of 20 mM Tris
buffer, pH 7.4. Incubations were carried out for 30 min at 37 °C.
The phosphorylated protein, designated
p47PHOXP6,2
was separated from the reaction mixture as described elsewhere (21).
Cell-free Activation of the NADPH Oxidase with
p47PHOXP6--
Activation of the NADPH oxidase
in the cell-free system was directly measured by following the
superoxide dismutase-inhibitable reduction of cytochrome c
at 550 nm in a dual beam recording spectrophotometer. The complete
reaction mixture contained 5 × 106 cell equivalents
of membrane (12 ± 1.4 pmol of cytochrome
b558) incubated for 10 min at 30 °C with 50 µM GTP Cell-free Activation of the NADPH Oxidase with SDS--
In these
experiments, 5 × 106 cell equivalents of membrane
were incubated for 10 min at 30 °C with 105 pmol of GST-Rac2, 75 pmol of p67PHOX and 70 pmol of GST p47PHOX plus 50 µM GTP Cytochrome b558 Determination--
Neutrophil
membranes (1 × 107 cell eq) were resuspended in 400 µl of Triton buffer (0.1 M potassium phosphate buffer, pH
7.25, containing 2% Triton (v/v). Cytochrome
b558 content was measured as the
dithionite-reduced minus oxidized absorption assuming
Electrophoresis and Immunoblotting--
Protein samples were
subjected to SDS-PAGE using the Laemmli buffer system (28). The gels
were stained with Coomassie Blue. Alternatively, the separated proteins
were electrophoretically transferred onto a nitrocellulose sheet (29)
and probed with partially purified rabbit polyclonal antibodies raised
against full-length Rac2 or the C-terminal decapeptides from
p47PHOX and p67PHOX. These antibodies were used at
1:1000, 1:2000, and 1:5000 dilution for GST-Rac2, GST-p47PHOX,
and p67PHOX, respectively. The proteins were visualized with a
1:2000 dilution of horseradish peroxidase-labeled goat anti-rabbit Ig
antibody (Caltag) and the ECL enzymatic chemoluminescence detection
system (Renaissance, DuPont).
Absence of Cytosolic Factors from the Neutrophil Membrane--
In
the experiments described below, neutrophil membranes were mixed with
recombinant cytosolic oxidase proteins in various combinations.
SDS-PAGE gels of the recombinant proteins used for these experiments
are shown in Fig. 1. Since the
experiments involved the addition of cytosolic components to the assay
mixtures, it was important to determine whether any of these components
was present in the neutrophil membrane that was to be used in the experiments. For this purpose, membrane and cytosol in equal amounts (expressed as cell equivalents) were subjected to SDS-PAGE and immunoblotting, developing with antibodies against the cytosolic components that were to be added in recombinant form. The components were readily visible in the cytosol, but could not be detected in the
membrane.
Activation of the Cell-free Recombinant Leukocyte NADPH Oxidase by
p47PHOXP6--
In the cell-free system, the
leukocyte NADPH oxidase has customarily been activated using certain
anionic amphiphiles including SDS. In an earlier study in which we
supplemented the cell-free system with p47PHOX (added as the
GST fusion protein), we found that the enzyme could be activated
without detergent, provided the p47PHOX was first
phosphorylated by protein kinase C (21). We believe that the activation
of the cell-free oxidase by protein kinase C may represent a more
physiological process than activation by anionic amphiphiles, because
in intact cells, as in the kinase-activated cell-free system, oxidase
activation is associated with the phosphorylation of
p47PHOX.
It has been shown by others that in a cell-free system in which
neutrophil cytosol has been replaced by the two recombinant cytosolic
oxidase subunits p47PHOX and p67PHOX together with the
small GTPase Rac2, O
Experiments with a cell-free system containing membrane and cytosol
indicated that the phosphorylation of p47PHOX was an essential
prerequisite for O
The effect of protein concentration on O Activation of the NADPH Oxidase by SDS Versus Activation by
p47PHOXP6--
p47PHOXP6
produced similar rates of production of O It has been known for many years that p47PHOX becomes
heavily phosphorylated on serine residues when the oxidase is activated (8-14, 33). Here we present evidence that p47PHOX
phosphorylated by protein kinase C is capable of activating the leukocyte NADPH oxidase in a recombinant cell-free system consisting of
neutrophil membrane, p67PHOX and Rac2, therefore identifying
the minimum cytosolic components necessary for
kinase-dependent activation of the oxidase. These findings
strongly suggest that the phosphorylation of p47PHOX that
occurs in whole cells during the activation of the leukocyte oxidase is
functionally significant and that protein kinase C is a kinase capable
of activating p47PHOX by phosphorylation. In addition, our
results with GTP The mechanism of activation of the NADPH oxidase by anionic amphiphiles
is still not clear, but our results confirm previous studies
demonstrating that the activation of the NADPH oxidase by SDS is not
kinase dependent (20). Furthermore, the finding that oxidase activation
is associated with the phosphorylation of p47PHOX both in
intact cells and in the kinase-dependent cell-free system suggests that as compared with amphiphiles, the activation of the
oxidase by protein kinase C may represent a more physiological process.
The fact that p47PHOXS379A is nonfunctional both in whole cells
and in the kinase-dependent cell-free system, yet is
capable of participating in O The foregoing experiments also showed that the addition of
p47PHOXP6 to a cell-free oxidase activating system
is not enough to activate the oxidase to its full extent. A number of
cytosolic components can be postulated as candidate factor(s) that
allow the oxidase to become fully activated. These include lipids
(e.g. arachidonic acid (36)), proteins (e.g.
p40PHOX (37)), possibly other kinases, or perhaps a hitherto
undiscovered oxidase component. Nevertheless, our findings show that
neutrophil membrane, p47PHOX, p67PHOX, and Rac2 are
sufficient for protein kinase C-mediated activation of the oxidase,
albeit at a relatively low level. The participation of other components
in the kinase-dependent activation of NADPH oxidase is the
subject of an ongoing investigation.
2 from oxygen at the expense of NADPH. A
correlation between the activation of the oxidase and the
phosphorylation of p47PHOX, a cytosolic oxidase component, is
well recognized in whole cells, and direct evidence for a relationship
between the phosphorylation of this oxidase component and the
activation of the oxidase has been obtained in a number of cell-free
systems containing neutrophil membrane and cytosol. Using superoxide
dismutase-inhibitable cytochrome c reduction to quantify
O
2 production, we now show that p47PHOX phosphorylated
by protein kinase C activates the NADPH oxidase not only in a cell-free
system containing neutrophil membrane and cytosol, but also in a system
in which the cytosol is replaced by the recombinant proteins
p67PHOX, Rac2, and phosphorylated p47PHOX, suggesting
that neutrophil plasma membrane plus those three cytosolic proteins are
both necessary and sufficient for oxidase activation. In both the
cytosol-containing and recombinant cell-free systems, however,
activation by SDS yielded greater rates of O
2 production than
activation by protein kinase C-phosphorylated p47PHOX,
indicating that a system that employs protein kinase C-phosphorylated p47PHOX as the sole activating agent, although more
physiological than the SDS-activated system, is nevertheless incomplete.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 at the expense of
NADPH (1). The oxidase comprises multiple protein components present in
both the cytosol and the plasma membrane. The enzyme is dormant in
resting cells but becomes activated when the cells are exposed to
appropriate stimuli. Upon activation, a cytosolic complex consisting of
the oxidase components p47PHOX, p67PHOX, and
p40PHOX associates with the membrane-bound cytochrome
b558 to assemble the active oxidase (2-7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside, NADPH, ATP,
guanosine 5-O-(3-thiotriphosphate)
(GTP
S),1 guanosine
5'-O-(2-thiodiphosphate) (GDP
S), glutathione agarose and cytochrome
c from Sigma; rat brain protein kinase C, calyculin A, and
GF-109203X from Calbiochem; and the Bradford protein assay reagent
from Bio-Rad.
70 °C until use.
70 °C.
T plasmids containing cDNA inserts encoding the downstream proteins as described by Park et al. (3). The fusion
proteins were purified by affinity chromatography on
glutathione-agarose beads. Initially the culture was grown overnight at
37 °C in 100 ml of "Terrific Broth" containing 0.1% ampicillin,
then diluted into 1 liter of fresh Terrific Broth/ampicillin. The
diluted cultures were grown for an additional hour at 37 °C (for
GST-p47PHOX expression) or an additional 2.5 h at 37 °C
(for GST-Rac2 expression). Isopropyl-
-D-thiogalactopyranoside (0.1 mM)
was then added, and the cultures were grown with vigorous agitation for
an additional 3 h at 37 °C for GST-p47PHOX expression
or 30 °C for GST-Rac2 expression. At the conclusion of the
incubations, the cells were recovered by centrifugation at 2000 × g for 10 min at 4 °C. The GST-p47PHOX pellet was
suspended in 10 ml of ice-cold phosphate-buffered saline containing a
1 × mixture of protease inhibitors (Roche Molecular
Biochemicals), while the GST-Rac2 pellet was resuspended in a lysis
buffer (50 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and
1 mM phenylmethylsulfonyl fluoride). The cells were then
disrupted by sonication. The sonicates were clarified by centrifugation
at 14,000 × g for 15 min at 4 °C. The fusion
proteins were isolated from the supernatant by purification over
glutathione-agarose as described by Smith and Johnson (25). Before use,
excess glutathione was removed from the solution of purified
recombinant protein by dialysis against relaxation buffer. The
concentrations of all proteins (95-99% pure) were determined with a
Bio-Rad assay kit using bovine serum albumin as a standard.
S or GDP
S, 1 mM ATP, 250 nM calyculin A, and 105 pmol of GST-Rac2, 75 pmol of p67PHOX and 70 pmol of GST p47PHOX, phosphorylated or
unphosphosphorylated, in a final volume of 200 µl. GST-Rac2 was
reconstituted with 130 µM GTP
S by preincubation of 105 pmol of the protein with 2.6 mM EDTA for 10 min at room temperature followed by the addition of 4 mM
MgCl2 (26). Reactions were started by adding the detection
mixture (0.1 mM cytochrome c and 0.16 mM NADPH, final concentrations). Reduction was followed in
a Uvikon 941 dual beam recording spectrophotometer (Kontron Instruments, Milan, Italy), reading against a reference containing the
same components plus 150 units of superoxide dismutase. Unless otherwise indicated, the rate of O
2 production is expressed as moles of O
2/moles of cytochrome
b558/minute. Where indicated, cytosol
(107 cell equivalents) was added to the system instead of
the recombinant proteins GST-p47PHOX, p67PHOX, and
GST-Rac2. Experiments were also performed in which the recombinant proteins (p67PHOX, GST-p47PHOX, and GST-Rac2) were
individually omitted to determine which of these proteins was required
for the production of O
2 by the oxidase.
S as described previously, with the exception that unphosphorylated p47PHOX was used in place of
p47PHOXP6. After the preincubation, SDS (90 µM final concentration) was added to the reaction mixture
and incubated for 1 min. In some experiments, cytosol (107
cell eq) was used in place of the recombinant proteins as described above.
E559-540 = 21.6 mM
1 cm
1 (27). In some spectra
the height of the peak was estimated by interpolation to correct for
base-line drift.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (52K):
[in a new window]
Fig. 1.
Recombinant proteins used in these
experiments. SDS-PAGE of the purified proteins was performed as
described under "Experimental Procedures," using a 10% running
gel. Coomassie Blue staining was used to show GST-p47PHOX
(lane 1), p67PHOX (lane 2), and GST-Rac2
(lane 3).
2 is produced upon the addition of SDS
(30). In order to see if the same system could be activated by a
kinase, we conducted experiments in which O
2 production was
measured in a recombinant system that contained phosphorylated
p47PHOX (i.e. p47PHOXP6) instead
of the unphosphorylated protein. The results (Fig. 2) showed that O
2 was
produced in the complete system, but that the omission of all or any
one of the three recombinant cytosolic proteins or the omission of
membrane (not shown) essentially eliminated oxidase activity. The use
in the recombinant system of Rac2 preloaded with GDP
S led to a
marked reduction in O
2 formation as compared with a system
that employed Rac2 preloaded with GTP
S, suggesting that Rac2 had to
be in its active form for the oxidase to be activated (Fig.
3). These findings indicate that all four
components (membrane, p67PHOX, p47PHOXP6,
and Rac2) were required for activation of the NADPH oxidase, indicating
that they are both necessary and sufficient for
kinase-dependent cell-free oxidase activation.
View larger version (17K):
[in a new window]
Fig. 2.
O 2 production by
GST-p47PHOXP6
activated
leukocyte NADPH oxidase as a function of time. Incubations were
carried out as described in the text, using the cytochrome c
assay. Components were omitted from the assays as indicated. The
results shown are representative of three or more separate experiments.
The means ± S.E. for the final points (22 min) are 388 ± 46 (complete),
67 ± 4.2 (no GST-p47PHOXP6),
4 ± 8 (no p67PHOX),
50 ± 4.1 (no GST-Rac2), and
12.5 ± 4.2 (no recombinant proteins) mol of O
2/mol of
cytochrome b558/min. Differences between
O
2 production in the complete assay mixture and O
2
production in each of the omission experiments were significant at the
level of p < 0.005.
View larger version (10K):
[in a new window]
Fig. 3.
Requirement for guanine nucleotides in the
activation of the NADPH oxidase by p47PHOXP6 in the
recombinant cell-free system. Experiments were conducted as
described in the text; Rac2 was loaded with GTP S or GDP
S as
indicated. The results shown represent the mean ± S.E. of four
separate experiments.
2 production by the system (31). To see if
the same situation prevailed in the recombinant system, the rate of
O
2 production using p47PHOXP6 was compared
with the rate of O
2 production using unphosphorylated p47PHOX. The results (Fig. 4)
show that O
2 production in this system required the
phosphorylation of p47PHOX. It is possible that further
phosphorylation of p47PHOX by a membrane-associated kinase
might also be necessary for the O
2-forming activity in this
system. The addition of the protein kinase inhibitor GF-109203X to the
reaction mixture, however, had no effect on O
2 production,
indicating that phosphorylation at least by GF-109203X-inhibitable
membrane-associated kinases such as activated protein kinase C was not
a factor in these experiments.
View larger version (13K):
[in a new window]
Fig. 4.
NADPH oxidase activation requires
phosphorylated p47PHOX in the recombinant cell-free
system. The cytochrome c assay was conducted as
described in the text, except that GST-p47PHOXP6 or
unphosphorylated GST-p47PHOX was added to the assay mixtures as
indicated. The protein kinase inhibitor GF-109203X (5 µM)
was also added to some of the incubations. Results are expressed as
mean ± S.E. of three or more experiments.
2 production in the
recombinant system was next examined. In these experiments activity was
monitored at varying concentrations of the three recombinant proteins
in the presence of a constant amount of membrane (Fig. 5A) and at varying
concentrations of membrane in the presence of a constant amount of
recombinant proteins (Fig. 5B). The maximal rate of
O
2 generation by the p47PHOXP6-activated
system was 4.6 ± 0.5 nmol O
2/min/107 cell eq
of membrane (mean ± S.E., n = 6), equivalent to
190 mol of O
2/mol of cytochrome
b558/min, a value achieved using 5 × 106 cell equivalents of membrane (12 pmol of cytochrome
b558), 105 pmol of GST-Rac2, 75 pmol of
p67PHOX, and 70 pmol of GST p47PHOXP6, the
rate falling sharply at concentrations on either side of the optimum.
In order to investigate if this effect also occurred in the cytosolic
cell-free system, production of O
2 using the same
concentration of Rac2 and p67PHOX at different concentrations
of p47PHOXP6 was determined. At the concentration
of p47PHOXP6 used in this study, the activity of
the recombinant and cytosolic cell-free systems were the same (Fig.
6). Curiously, the addition of more than
70 pmol of p47PHOXP6 reduced O
2 production
in the recombinant system but not in the cytosolic cell-free system.
When the oxidase is activated, p47PHOX, p67PHOX, and
Rac2 translocate to the membrane in equimolar quantities (32).
Therefore we employed approximately equimolar concentrations of the
three recombinant cytosolic components (actual stoichiometry 1.5/1/1
for Rac2/p47PHOX/p67PHOX). Altogether, these results
indicate that an excess of either membrane or cytosolic components
inhibits O
2 production in the recombinant cell-free system.
Why this same relationship doesn't prevail in the cytosol-containing
system is a mystery.
View larger version (10K):
[in a new window]
Fig. 5.
Oxidase activity as a function of the
concentrations of the recombinant proteins and membranes. The
incubations were carried out as described under "Experimental
Procedures" using the cytochrome c assay. The reaction
mixtures contained 5 × 106 cell eq of membrane
(12 ± 1.4 pmol of cytochrome b558), plus
increasing concentrations of GST-p47PHOXP6,
p67PHOX, and GST-Rac2 (A) or 70 pmol of
GST-p47PHOXP6, 105 pmol of GST-Rac2, and 75 pmol of
p67PHOX, plus membrane at the concentration shown
(B). The values shown represent the initial rates of
O 2 production (means ± S.E.) for three or more separate
experiments.
View larger version (17K):
[in a new window]
Fig. 6.
Oxidase activity as a function of
GST-p47PHOXP6 concentration in the recombinant and
cytosolic cell-free systems. The incubations were carried out as
described under "Experimental Procedures," assaying O 2
production by cytochrome c reduction. The recombinant
reaction mixtures contained 75 pmol of p67PHOX, 105 pmol of
GST-Rac2, and GST-p47PHOXP6 at the concentrations
shown. Results are expressed as mean ± S.E. of three separate
experiments.
2 in the recombinant
and cytosolic cell-free systems (Fig.
7A). In contrast when SDS was
used as the stimulus, the rate of O
2 production in the
recombinant cell-free system was approximately 50% of the rate seen
with cytosol (Fig. 7B), and both rates were considerably greater than the rates obtained in the
p47PHOXP6-activated systems. These results strongly
suggest that while p47PHOXP6 is sufficient to
activate the oxidase at a low level, something from the cytosol that is
missing in the recombinant cell-free system is required for maximal
activation of the oxidase.
View larger version (11K):
[in a new window]
Fig. 7.
Activation of the NADPH oxidase in the
recombinant and cytosolic cell-free system by
GST-p47PHOXP6 (A)
and SDS (B). The incubations
were carried out as described under "Experimental Procedures" using
the cytochrome c assay. Results are expressed as mean ± S.E. of three or more experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S and GDP
S confirm that the activation of the
enzyme by protein kinase C also requires the activation of Rac2, as
shown previously by others (34, 35).
2 production in the
amphiphile-dependent cell-free system (21, 33), further supports the physiological role played by protein kinase
C-dependent activation of the oxidase.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grants AI-24227, AI-28479, and RR-00833.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Postdoctoral fellow of the Fundação de Amparo à Pesquisa do Estado de São Paulo (Brazil).
¶ Postdoctoral fellow of the Arthritis Foundation.
2 Protein kinase C-phosphorylated P47PHOX is designated p47PHOXP6, because it contains 6 mol of phosphate/mol of p47PHOX.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
GTPS, guanosine
5-O-(3-thiotriphosphate);
PIPES, 1,4-piperazinediethanesulfonic acid;
GST, glutathione
S-transferase;
GDP
S, guanosine 5'-O-(2-thiodiphosphate);
PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|