Vol.
167,
March
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
16, 1990
790-797
PROPERTIES OF THE NADPH DEHYDROGENASE COMPONENT OF THE OXIDASE COMPLEX FROM RABBIT PERITONEAL NEUTROPHILS: RECONSTITUTION OF AN OXIDASE ACTIVITY WITH THE DEHYDROGENASE COMPONENT AND A MEMBRANE EXTRACT Francois
LAPORTE,
Jacques
DOUSSIERE and Pierre
V. VIGNAIS
LBIO/Laboratoire Fondamentale, Centre
de Biochimie, Departement de Recherche d'btudes Nucleaires, 85X, 38041 Grenoble France
Received
1990
January
26,
cedex,
Summary: A flavin-linked NADPH cytochrome c oxido-reductase of molecular mass 77-kDa was extracted from membranes of rabbit peritoneal neutrophils and purified in the presence of Triton X-100. The redox properties of this enzyme were examined. By some criteria including its high sensitivity to mersalyl, and its relatively high specificity for NADPH compared to NADH, the rabbit neutrophil NADPH cytochrome c reductase resembled NADPH-cytochrome P-450 reductase. Limited proteolysis generated water soluble fragments, with molecular masses of 67-kDa and 57-kDa, which were still endowed with a substantial reductase activity. When added to a lysate of neutrophil membranes in octylglucoside, in the presence of an oxidase activation medium consis2t+ing of rabbit neutrophil cytosol, GTP-y-S, arachidonic acid and Mg , the purified reductase enhanced the production of 027, suggesting that it forms part of the 02; generating oxidase. 01990 Academic mess, Inc. At least
two redox
flavodehydrogenase cytochrome in
the
the from
thought
membrane
respiratory
component,
(8). from
This
(for (6),
and responsible has been
bovine
(71,
neutrophils
showed However,
this
In early
studies,
no attempt
oxidase
activity
with
purified
ability
some
the
molecule
located for
The NADPH dehydrogenase
l-5).
itself.
the
complex
mass of 67-65-kDa,
molecule with
termed
b,
oxidase
neutrophils
review
NADPH to 02.
to be compatible
a NADPH-dependent
cytochrome
to make up the
a molecular
human neutrophils
electrons
namely
of activated
burst with
neutrophils low
are
b558,
plasma
components,
and a low potential
oxidase acting
purified
and pig to transfer
activity as the
was too oxidase
was made to reconstitute reductase.
In this
paper
the we
Abbreviations: PBS: phosphate buffer saline consisting of 2.7 mu KCl, 136.7 mM NaCl, 1.5 mM KH2P04 and 8.1 mM Na2HP04, pH 7.4 ; PMA: Phorbol myristate acetate ; DFP: diisopropyl fluorophosphate ; SOD: superoxide dismutase ; EMP: medium consisting of 20% ethyleneglycol (w/v), 0.1 M mannitol, and 10 mM Na phosphate pH 7.4 ; EMPT: EMP supplemented with 0.2% Triton X-100 ; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis. 0006-291X/90
Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
790
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
describe
some of the
properties
of a NADPH cytochrome
purified
from
neutrophil
membranes,
that
the
strongly
MATERIALS
rabbit
addition
of this
enhances
the
AND
rate
reductase
of production
c reductase
and we present
to lyzed
neutrophil
COMMUNICATIONS
evidence membranes
of 02:.
METHODS
The following chemicals and enzymes were obtained from the companies indicated. Sigma: ferricytochrome c (horse heart, type dismutase, oyster glycogen type II, Na deoxycholate, VI) I superoxide Triton X-100, diisopropyl fluorophosphate ; Boehringer: NADPH ; sucrose ; Whatman: DE52 cellulose ; Pharmacia: 2'-5'-ADP Merck: Sepharose 4B. A purified preparation of bovine adrenal NADPH cytochrome P-450 reductase was a kind gift from Dr. G. Defaye. Peritoneal neutrophils were elicited in rabbits with oyster glycogen. Three hundred ml of sterile 0.9% NaCl solution containing 0.1% oyster glycogen were injected into the peritoneal cavity of rabbits. Neutrophils were collected after 12-14 h. After hypoosmotic lysis of contaminant erythrocytes, the neutrophils were washed with PBS and suspended at a final concentration of 5~10~ cells x ml-' of PBS. The final cell preparation contained more than 95% neutrophils. NADPH cytochrome c reductase was assayed at 25'C in PBS supplemented with 0.1% Triton X-100, 100 v cytochrome c and 200 PM NADPH, final volume 1 ml. The absorption coefficient of reduced cytochrome c at 550 nm was 21.1 m~-l.cm-l. 02: was assayed by the SOD inhibitable reduction of cytochrome c. FAD and FMN were determined by the method of Faeder and Siegel (9). The NADPH cytochrome c reductase was purified from rabbit neutrophil membranes by a three-step procedure. Rabbit neutrophils supplemented with 10 mu glucose were activated with 0.5 pg PMA x mg protein-l. After a 5 min incubation at 37'C, the neutrophils were pelleted by centrifugation at 300g for 5 min and resuspended in cold 0.25 b4 sucrose, 50 mM Mops pH 7.4 and 1 mM DFP at the final concentration of 10 mg protein x ml-'. ~11 subsequent operations were carried out at 2-4-C. The cells were disrupted by sonic irradiation for 2x20 set, using a Branson sonifier at 30-40 W Unbroken cells, cell debris and lysosomal granules were output. discarded by sedimentation after centrifugation at 2500g for 10 min. The membrane pellet recovered after centrifugation of the supernatant at 140000g for 1 h was resuspended in the EMP medium supplemented with 0.4% Na deoxycholate and 0.2% Triton X-100, and gently stirred for 15 min. The lysate was centrifuged at 140000g for 1 h. The supernatant contained more than 60 percent of the NADPH cytochrome c reductase activity assayed in the membrane fraction prior to extraction with detergent. The extract was chromatographed on a DE52 cellulose column equilibrated with the EMP medium washing the column supplemented with 0.2% Triton X-100 (EMPT). After with 2 Vol of EMPT, a continuous gradient of 0-0.25~ sodium sulfate Two peaks of reductase activity referred to as in EMPT was applied. DE I
167,
March
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
16, 1990
790-797
PROPERTIES OF THE NADPH DEHYDROGENASE COMPONENT OF THE OXIDASE COMPLEX FROM RABBIT PERITONEAL NEUTROPHILS: RECONSTITUTION OF AN OXIDASE ACTIVITY WITH THE DEHYDROGENASE COMPONENT AND A MEMBRANE EXTRACT Francois
LAPORTE,
Jacques
DOUSSIERE and Pierre
V. VIGNAIS
LBIO/Laboratoire Fondamentale, Centre
de Biochimie, Departement de Recherche d'btudes Nucleaires, 85X, 38041 Grenoble France
Received
1990
January
26,
cedex,
Summary: A flavin-linked NADPH cytochrome c oxido-reductase of molecular mass 77-kDa was extracted from membranes of rabbit peritoneal neutrophils and purified in the presence of Triton X-100. The redox properties of this enzyme were examined. By some criteria including its high sensitivity to mersalyl, and its relatively high specificity for NADPH compared to NADH, the rabbit neutrophil NADPH cytochrome c reductase resembled NADPH-cytochrome P-450 reductase. Limited proteolysis generated water soluble fragments, with molecular masses of 67-kDa and 57-kDa, which were still endowed with a substantial reductase activity. When added to a lysate of neutrophil membranes in octylglucoside, in the presence of an oxidase activation medium consis2t+ing of rabbit neutrophil cytosol, GTP-y-S, arachidonic acid and Mg , the purified reductase enhanced the production of 027, suggesting that it forms part of the 02; generating oxidase. 01990 Academic mess, Inc. At least
two redox
flavodehydrogenase cytochrome in
the
the from
thought
membrane
respiratory
component,
(8). from
This
(for (6),
and responsible has been
bovine
(71,
neutrophils
showed However,
this
In early
studies,
no attempt
oxidase
activity
with
purified
ability
some
the
molecule
located for
The NADPH dehydrogenase
l-5).
itself.
the
complex
mass of 67-65-kDa,
molecule with
termed
b,
oxidase
neutrophils
review
NADPH to 02.
to be compatible
a NADPH-dependent
cytochrome
to make up the
a molecular
human neutrophils
electrons
namely
of activated
burst with
neutrophils low
are
b558,
plasma
components,
and a low potential
oxidase acting
purified
and pig to transfer
activity as the
was too oxidase
was made to reconstitute reductase.
In this
paper
the we
Abbreviations: PBS: phosphate buffer saline consisting of 2.7 mu KCl, 136.7 mM NaCl, 1.5 mM KH2P04 and 8.1 mM Na2HP04, pH 7.4 ; PMA: Phorbol myristate acetate ; DFP: diisopropyl fluorophosphate ; SOD: superoxide dismutase ; EMP: medium consisting of 20% ethyleneglycol (w/v), 0.1 M mannitol, and 10 mM Na phosphate pH 7.4 ; EMPT: EMP supplemented with 0.2% Triton X-100 ; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis. 0006-291X/90
Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
790
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
describe
some of the
properties
of a NADPH cytochrome
purified
from
neutrophil
membranes,
that
the
strongly
MATERIALS
rabbit
addition
of this
enhances
the
AND
rate
reductase
of production
c reductase
and we present
to lyzed
neutrophil
COMMUNICATIONS
evidence membranes
of 02:.
METHODS
The following chemicals and enzymes were obtained from the companies indicated. Sigma: ferricytochrome c (horse heart, type dismutase, oyster glycogen type II, Na deoxycholate, VI) I superoxide Triton X-100, diisopropyl fluorophosphate ; Boehringer: NADPH ; sucrose ; Whatman: DE52 cellulose ; Pharmacia: 2'-5'-ADP Merck: Sepharose 4B. A purified preparation of bovine adrenal NADPH cytochrome P-450 reductase was a kind gift from Dr. G. Defaye. Peritoneal neutrophils were elicited in rabbits with oyster glycogen. Three hundred ml of sterile 0.9% NaCl solution containing 0.1% oyster glycogen were injected into the peritoneal cavity of rabbits. Neutrophils were collected after 12-14 h. After hypoosmotic lysis of contaminant erythrocytes, the neutrophils were washed with PBS and suspended at a final concentration of 5~10~ cells x ml-' of PBS. The final cell preparation contained more than 95% neutrophils. NADPH cytochrome c reductase was assayed at 25'C in PBS supplemented with 0.1% Triton X-100, 100 v cytochrome c and 200 PM NADPH, final volume 1 ml. The absorption coefficient of reduced cytochrome c at 550 nm was 21.1 m~-l.cm-l. 02: was assayed by the SOD inhibitable reduction of cytochrome c. FAD and FMN were determined by the method of Faeder and Siegel (9). The NADPH cytochrome c reductase was purified from rabbit neutrophil membranes by a three-step procedure. Rabbit neutrophils supplemented with 10 mu glucose were activated with 0.5 pg PMA x mg protein-l. After a 5 min incubation at 37'C, the neutrophils were pelleted by centrifugation at 300g for 5 min and resuspended in cold 0.25 b4 sucrose, 50 mM Mops pH 7.4 and 1 mM DFP at the final concentration of 10 mg protein x ml-'. ~11 subsequent operations were carried out at 2-4-C. The cells were disrupted by sonic irradiation for 2x20 set, using a Branson sonifier at 30-40 W Unbroken cells, cell debris and lysosomal granules were output. discarded by sedimentation after centrifugation at 2500g for 10 min. The membrane pellet recovered after centrifugation of the supernatant at 140000g for 1 h was resuspended in the EMP medium supplemented with 0.4% Na deoxycholate and 0.2% Triton X-100, and gently stirred for 15 min. The lysate was centrifuged at 140000g for 1 h. The supernatant contained more than 60 percent of the NADPH cytochrome c reductase activity assayed in the membrane fraction prior to extraction with detergent. The extract was chromatographed on a DE52 cellulose column equilibrated with the EMP medium washing the column supplemented with 0.2% Triton X-100 (EMPT). After with 2 Vol of EMPT, a continuous gradient of 0-0.25~ sodium sulfate Two peaks of reductase activity referred to as in EMPT was applied. DE I
