Vol. 169, No. 1, 1990
May 31, 1990
A PROTEIN
BIOCHEMICAL
The Department
Tel-Aviv
March
RESEARCH COMMUNICATIONS
Pages 198-202
OF NEUTROPHIL
GRANULES INTERFERES
WITH
OF NADPH OXIDASE IN A CELL-FREE SYSTEM
Irit
Received
AND BIOPHYSICAL
26,
Aviram
and Anat
ACTIVATION
Faber
of Biochemistry,
Faculty
of
University,
Tel-Aviv
69978,
Life
Sciences,
Israel
1990
A soluble
extract
of neutrophil
granules
interfered
with activation
of the NADPH oxidase
in a cell-free
system . The extract
had no effect
on superoxide
production
by preactivated
enzyme. The inhibitory
activity
was retained
during
dialysis
and was lost upon exposure
to proteinase
K
indicating
that
the
active
substance
was a protein.
The inhibitor
exhibited
a high stability
at elevated
temperatures
. Chromatography
of
granules
extract
on ion exchangers
implied
that
the inhibitor
was a
positively
charged
protein
eluting
from S Sepharose
cation
exchanger
al990
Academic Press,
Inc.
above 0.4M concentration
of NaCl.
Superoxide
kill
ingested
the superoxide
to infections
rare clinical
production
by stimulated
neutrophils
enables
the
cell
to
microorganisms
(1,Z).
The physiological
significance
of
generating
NADPH oxidase
is evidenced
by susceptibility
of patients
of chronic
granulomatous
disease
(CGD) . This
syndrome
is characterized
by a defective
oxygen
radical
production
by patients
phagocytes
(3,4).
Since
uncontrolled
release
of superoxide
ions
may damage body
tissues,
a careful
regulation
of the activity
of NADPH oxidase
is reguired.
Latency
of the enzyme in resting
cells
as well
as pathways
for
deactivation
of active
NADPH oxidase
in prestimulated
PMN (5,6)
represent different
approaches
for protection
of
body tissues
against
the
effects
of permanently
active
NADPH oxidase.
NADPH oxidase
can be activated
also in a cell-free
system consisting of
neutrophil
or macrophage
plasma membranes,
cytosolic
components
and an unsaturated
fatty
acid (e.g.arachidonate)
or SDS (7,s ). In early
reports
on cell-free
activation
of NADPH oxidase
in PMN , homogenates
or
unpurified
cellular
membranes
were employed
(7).
Later,
when only the
light
membrane fractions
obtained
by differential
( 9
) or by density
gradient
centrifugation
(10,ll
) were used for
oxidase
activation
,
specific
activities
improved
. This observation
suggested
to us that
in
the granules
fraction
present
in unpurified
homogenates
or membranes
,
an inhibitory
substance
might
have been present.
In the present
report
evidence
for the existence
of such an inhibitor
of NADPH oxidase
activation
is presented.
0006-29lX'!JO $1.50
Copyright
All rights
0 I990 by Academic
Press,
of reproduction
in any form
Inc.
reserved.
198
Vol.
169, No,, 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIALS AND METHODS
Fractionation
& neutrouhils:
Human neutrophils
were isolated
from fresh
buffy
coats
by the
standard
procedure
of dextgan
sedimentation
and
Ficoll
density
gradient
centrifugation.
Cells
(10 /ml)
in 1OmM potassium
buffered
saline
(PBS)
containing
1mM EGTA , 7 mM
phosphate
- (pH 7.0)
(PMSF) and 15 pg/ml
leupeptin
were
disrupted
by sonication
as described
(11).
Sonicates
were centr'ifuged
for 10 minutes
at BOOg and the pellets
were discarded.
Granules
were
sedimented
10 min at 10,OOOg and the
supernatants
were recentrifuged
for
45 min at 204,000
g to give
soluble
cytosol
and low density
were resuspended
in PBS / 0.34
M sucrose
membranes
. The membranes
(sucrose-PBS).
NADPH oxidase
activation
and assav:
Arachidonate-dependent
activation
was performed
in two succezve
steps as described
(11).
Membranes
and
cytosol
( 1-2~10~ and 2-4~10~
cell
equivalents
respectively)
in 0.1 ml
sucrose-PBS
containing
5 mM magnesium acetate
and arachidonate
(240-300
PM) were incubated
6 minutes
at 30°C.
After
dilution
with
0.7 ml
sucrose-PBS
/ 0.2 mM NADPH / SO pg cytochrome
c
reduction
rates
of
cytochrome
c were measured
at 550 nm before
and aft&
the addition
of 30
pg of superoxide
dismutase
(SOD).
In most experiments
oxidase
was activated
in a single
step assay
using
SDS (50 PM) . The reaction
buffer
(0.8 ml) consisted
of lo mM pH
6.7 Hepes-buffered
saline
(HBS)/
10 pM flavin
adenine
dinucleotide
/ a0
PM cytochrome
c/ 1 mM EGTA . When column fractions
eluted
at high NaCl
concentration
were added to the activation
mixture,
controls
of NADPH
oxidase
activity
at identical
salt concentration
were run in parallel.
Extraction
& aranules:
Granules
pellet
was resuspended
in 20 mM TrisHCl pH7.5 buffer/EGTA/leupeptin/PMSF
at a density
corresponding
to 2X10a
cell
equivalents/ml
and sonicated
(3x20
set).
The sonicate
was
centrifuged
15 minutes
at 27,000g
and the supernatant
representing
the
soluble
granules
extract
containing
0.9-1.3
mg/ml protein
was used as
the source of the inhibitory
material.
m
exchanae
chromatoaranhv
of the inhibitor
: Granules
extract
(1-3ml)
was applied
to a 0.8x1.5
cm DE-52
cellulose
(Whatman)
column
equilibrated
with 20 mM Tris-HCl
pH 7.5. The flow-through
containing
all
the inhibitory
activity
was loaded
on a o.axl.ocm
S Sepharose
(Sigma )
column
equilibrated
with
20 mM Hepes pH 7.0 buffer.
The column
was
washed with
the equilibration
buffer
containing
0.15 M Nacl and eluted
with a linear
0.15-0.8M
NaCl gradient
(20 ml total
volume).
Fractions
of
1.0 ml were
collected.
Protein
concentration:
was evaluated
by the method
of Lowry
( 12 ) .
Columns fractions
were monitored
at 28Onm .
RESULTS AND DISCUSSION
Inhibition
of NADPH oxidase
activation
& aranules
extract.
Arachidocell-free
activation
of
the
NADPH
oxidase
was
carried
out
nate-promoted
in two steps to permit
separation
of the activation
and activity
phases
increasing
amounts
of
the
granules
extract
were
present
(11 ). When
inhibition
of
during
the first
(activation)
phase , a dose dependent
Introduction
of the inhibitory
enzymic
activity
was observed
(Fig.1).
dose after
completion
of the activation
(6 minutes)
followed
by an
additional
6 minutes
of incubation
had no effect
on superoxide
production,
indicating
that
the inhibitory
substance
interfered
with
activation
step only and did not act as a superoxide
scavenger.
Since
otherwise
the experimental
conditions
at which the components
of oxidase
this
finding
implied
also
were exposed to the inhibitor
were identical,
199
Vol.
169, No. 1, 1990
BIOCHEMICAL
5.0
AND BIOPHYSICAL
10.0
15.0
granule
20.0
extract
25.0
RESEARCH COMMUNICATIONS
30.0
(pg)
Fig-l.
!Phe effect
of soluble
granules
extract
on the activity
of NADPH
oxrdase activated
by arachidonate
in a cell-free
system.(o)-the
extract
was present
during the initial
step (6 minutes)
of arachidonate-mediated
was added
after
the
initial
6 minutes
activation
: (0) -extract
and arachidonate
and incubated
for
preincubation
of membranes , cytosol
an additional
6 minutes before
final
dilution.
that the latter
did not act as a non specific
protease
. It follows
that
the inhibitory
substance
seems distinct
from
the
NADPH oxidaseinactivating
proteinase
of azurophilic
granules
mentioned
by
Borregaard
(13)*
Granules
extract
could
be
Pronerties
of the
inhibitory
substance.
concentrated
by ultrafiltration
(Diaflo
ultrafilter,
Amicon,
equipped
with yMl0 membrane ) and when chromatographed
on a Sephadex G-25 column,
the active
substance
eluted
in the void volume (results
not shown):
both
findings
indicated
that the inhibitor
was a macromolecule.
At pH 7.5 the inhibitor
present
in the extract
of granules
was not
retained
by a DE-52 cellulose
anion
exchange
column
. Flow-through
of
0.4
.
0.3
I
4
0.2
z
4-
0.1
0.0
0
5
10
15
FRACTION
20
25
Nr.
Fig.2.
Chromatography
of the NADPIi oxidase inhibitory
substance
on a S
Sepharose cation
exchange column. Soluble extract
of neutrophil
granules
(6.0 ml of 0.8 mg protein/ml)
was
loaded on a DE-52 cellulose
column ;
flow-through
containing
all the inhibitory
activity
( 0.5mg/ml protein)
was transferred
to
an S Sepharose column and eluted
by salt gradient.
0.1 ml of each fraction
was added to SDS-supported
NADPH oxidase
activation
assay.
200
Vol.
the
169, No,, 1, 1990
DE-52
Column
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
containing
all
activity
detected
in the soluble
extract
, was loaded
on a Sepharose
S cation
exchanger
. The inhibitor
was adsorbed
by the resin
and could be eluted
as a broad peak between
0.4-0.7
M NaCl concentration
(Fig.2
).
The inhibitory
substance
(in the crude
extract
and in partially
purified
fractions
from S Sepharose
column)
exhibited
a high stability
to heat:
its activity
was not reduced
by IO minutes
incubation
at 90°C
This property
was taken
advantage
of in experiments
devised
(Table
I).
to test
whether
it was susceptible
to cleavage
by proteinase
K, namely,
whether
it was a protein
. The granules
extract
was incubated
I5 minutes
with proteinase
K (Merck).
Before the resulting
extract
could be assayed
for its
effect
on oxidase
activation
it was necessary
to inactivate
proteinase
K, since
the latter
abolished
activation
of the oxidase
This was accomplished
by exposure
of the mixture
of granule
(Table I).
extract
and proteinase
K to 90° , a treatment
which
inactivates
proteinase
K (Table
I).
The extract
treated
with proteinase
K lost
its
inhibitory
activity,
indicating
that the active
substance
was a protein,
cleaved
by the protease.
In conclusion
t we described
a novel
activity
of neutrophil
granules
which
interferes
with
activation
of the NADPH oxidase
in a
cell-free
system.
The inhibitory
substance
is a positively
charged
highly
thermostable
protein
.
It is conceivable
that
the inhibitory
protein
might
have
been defined
previously
but its regulatory
function
with respect
to activation
of the NADPH oxidase
remained
unrecognized
The present
study gives
no indication
regarding
the mechanism
action
of the inhibitor
nor its
exact
location
within
the different
subpopulations
of granules
(I3,14)
. It is noteworthy
that
Clark
al.,
(15)
and Borregaard
(13)
claimed
that
most
of activatable,
membrane-bound
NADPH oxidase
of resting
, disrupted
neutrophils
Table
Effects
of
None
DE-52 flow-through
(17.5 fig)
heateda DE-52 flow-through
proteinase
X-treated
r heateda
proteinase
Kc
heated proteinase
Ka
:
O~(nmol/min*mg
DE-52 flow-through
inhibitor
et
is
to
protein)
450
155
160
400
30
400
'70 jbg protein
in 0.1 ml 20 mM Tris-HCl
pIi 7.5 was incubated
temperature
with
40 pg proteinase
K and heated for 10 minutes
25~1 of this mixture
was added to the
oxidase assay.
%Opg protein.
2Ul
.
of
I
heat and proteinase
X on the capacity
of the
interfere
with activation
of NADPIi oxidase
Additions
,
at room
at 90 OC.
Vol.
169, No. 1, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
located
in specific
granules
. This would
suggest
that
the inhibitor
described
and partially
charaterized
in this
study is confined
to other
compartments
of the cell e.g.
azurophilic
granules.
The existence
of a negative
regulatory
control
of activation
of the
NADPH oxidase
might have been anticipated.
Most constituents
of granules
exhibit
however
antimicrobial
activity
(14)
: in this
respect
, a
possible
antiinflammatory
activity
of a protein
activation
of the superoxide
generating
pathway
studies
will
be necessary
to define
the nature
of
mode of its activity
within
the cell.
Acknowledqment:
Binational
Science
This
research
Foundation.
was
supported
in
that
interferes
is unusual.
the inhibitor
part
by
with
Further
and the
U.S.-Israel
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202