Journal of Leukocyte Biology 36:751-759 (1984)

Activation of a NADPH Oxidase From Horse

Polymorphonuclear Leukocytes in a Cell-
Free System

R.A. Heyneman and R.E. Vercauteren

Laboratory of Physiological Chemistry, Faculty of Veterinary Medicine, University of
Ghent, Ghent, Belgium

A postnuclear cell fraction from resting horse polymorphonuclear (PMN) leu-

kocytes incubated with fatty acid-salt ions such as oleate or linoleate generated
a NADPH-dependent oxygen consumption and superoxide production. Oxida-
tive activity was negligible or absent in the postnuclear fraction from mononu-
clear leukocytes, p-chloromercuribenzene sulfonic acid-treated granulocytes,
and granulocytes from a patient with chronic granulomatous disease. Although
consistently associated with the membrane fraction from resting PMN leuko-
cytes, the superoxide-generating activity was shown to be dependent on a thus
far unknown cytosolic constituent. The apparent Km’5 for NADPH and NADH
(66 and 1,600 M, respectively), the pH optimum for the reaction (7.0), the
cyanide insensitivity, and transient nature of the reaction together with the
stoichiometric relationship between oxygen uptake and NADPH oxidation led
to the conclusion that in the presence of cytosol a cell-free latent respiratory
burst oxidase can be converted into an active enzyme by interaction with
oleate micelles.

Key words: PMN leukocytes, NADPH oxidase, activation, respiratory burst

INTRODUCTION

During phagocytosis or upon perturbation of the plasma membrane with a

suitable agent, polymorphonuclear (PMN) leukocytes demonstrate a considerable
cyanide insensitive increase in their oxygen consumption accompanied by a produc-
tion of superoxide anions and hydrogen peroxide. The initiation of this respiratory
phenomenon is generally attributed to activation of a latent NAD(P)H oxidase local-
ized on the plasmalemma of the cell [8,12,16]. Most investigators have shown, either
biochemically [1] or cytochemically [7], that the NAD(P)H oxidase is expressed only

Received May 9, 1983; accepted April 20. 1984.

Reprint requests: R. Heyneman, Laboratory of Physiological Chemistry, Casinoplein 24, B-9000 Ghent,
Belgium.

© 1984 Alan R. Liss, Inc.

752 Heyneman and Vercauteren

after appropriate activation of intact cells. Recently, however, Dechatelet et al [10]

reported that a particular fraction derived from normal unstimulated cells may be
activated by dialysis to mimic the respiratory activity of a similar cell fraction derived
from phagocytosing cells.
In this paper, we report the activation in a cell-free system of a granulocyte
NADPH oxidase by a micellar solution of sodium oleate. Oeneration of oxidative
activity by the membrane fraction of the cell is shown to depend on the presence of
an unknown cytosolic constituent. The characteristics of the reaction provide good
evidence for the identity of the investigated oxidase with the respiratory burst enzyme.

MATERIALS AND METHODS

Preparation and Fractionation of Leukocytes

Venous blood was obtained from healthy horses, and leukocytes were isolated
essentially as described by the Boyum technique 15]. All steps were performed at 0-
4#{176}C.The separated mononuclear and PMN leukocytes were suspended in 0.34 M
sucrose buffered at pH 7.4 with 3 mM Tris-HCI (homogenization medium) at a
concentration of 5 x 108 cells/ml. The PMN leukocyte preparations routinely yielded
85-91 % neutrophils, 2-8% eosinophils, and 2-10% mononuclears.
The cells were homogenized in a minimal volume of homogenization medium
with the aid of a chilled Potter-Elvehjem homogenizer with a motor-driven Teflon
pestle until more than 90% cell breakage was reached. Nuclei and unbroken cells
were pelleted by centrifugation at 400g for 10 mm. The postnuclear supernatant was
removed and immediately assayed for NADPH oxidase activity. To prepare a partic-
ulate fraction freed of the cytosolic constituents, the postnuclear supernatant was
centrifuged at lOO,000g for 60 mm in a Spinco ultracentrifuge at 4#{176}C.
The supernatant
(cytosol fraction) was removed and the pellet (particulate fraction) washed once in
homogenization medium. The resuspended particulate fraction was layered over a
discontinuous sucrose gradient containing equal volumes of a 25%, 45%, and 60%
sucrose solution. Centrifugation was carried out for 120 mm in a Beckman SW 25.1
rotor at 60,000g. The gradients were fractionated and the sucrose interface bands (I,
II, and III) immediately stored in liquid nitrogen until analyzed.

Biochemical Assays

Oxygen consumption was measured amperometrically in a microvessel accord-

ing to the model designed by Rasmussen [22]. The originally described paraffin wax
seals were replaced by small rubber 0-rings. Using a Yellow Springs Instrument
Clark electrode as a vessel wall the total volume of the chamber was less than 0.2 ml.
Reagents were added during incubation with the aid of tygon tubing fixed on a
microsyringe through a capillary bore in the upper perspex plate. The reaction vessel
was immersed in a constant temperature water bath at 25#{176}C.The reaction mixture
contained 10 mM phosphate buffer pH 7.2, 100 mM NaCI, 1 mM MgCl2, 1 mM
KCN, 2 mM sodium oleate, and approximately 0.20
mg of postnuclear supernatant
protein. A stock solution of 100 mM sodium oleate was previously made up in warm
0.9% NaCI and diluted before use. Reaction was initiated by the addition of 0.5 mM
NADPH. Initial rates of oxygen consumption were used for calculation.
Superoxide production was quantitated by following spectrophotometrically the
reduction of cytochrome c at 25#{176}C.To minimize turbidity by oleate, small amounts
of enzyme protein were incubated in a cuvette with 2 mM sodium oleate in 0.2 ml 10
mM phosphate buffer (pH 7.2), 100 mM NaCl and 1 mM MgCl2. After 1 mm, 25
 Activation of a Granulocyte NADPH Oxidase 753

jM cytochrome c, 0.5 mM KCN, and buffer were added, up to a final volume of 2

ml. The assays were started by the addition of 0.5 mM NADPH, and the absorbance
change at 550 nm was followed. After 3 mm, 20 g of superoxide dismutase (SOD)
was added to the assay and the absorbance change versus time was further recorded.
Of-dependent cytochrome c reduction was calculated by subtracting the initial reac-
tion rate after SOD addition from the former value.
Other biochemical assays were identical with those described previously [15].
Alkaline phosphatase was assayed with p-nitrophenylphosphate as a substrate. 13-
Olucuronidase was estimated fluorometrically with 4-methylumbelliferyl-/3-D-glucu-
ronide as a substrate. Protein, lysozyme, lactate dehydrogenase, and 5’-nucleotidase
were assayed according to published methods that were found to give good results
with leukocyte preparations [6, 1 1] . The formation of lipid peroxides were estimated
by measuring the production of malondialdehyde with the thiobarbituric acid as-
say [17].

Materials

NADH and NADPH were the highest grade available and were obtained from
Boehringer-Mannheim Corp. (Munich, Oermany); superoxide dismutase and sodium
oleate and linoleate were purchased from Sigma (USA); cytochrome c and p-chloro-
mercuribenzene sulfonic acid (PCMBSA) were from Serva (Heidelberg, Oermany),
and tetranitromethane was from Aldrich Europe.

RESULTS

In the presence of a micellar solution of sodium oleate, the postnuclear super-

natant fraction from resting horse PMN leukocytes showed a NADPH-dependent and
cyanide-insensitive oxygen consumption. The dependence of this activity on oleate
concentration and on pH of the medium is depicted in Figure 1 . A postnuclear cell
fraction prepared from PMN leukocytes preincubated with 1 mM PCMBSA, a
nonpenetrating sulthydryl reagent 1261 and inhibitor of the leukocyte oxidative activity
[27], lacked this O2-consuming activity. A comparable cell fraction from mononuclear
leukocytes showed a slight O2-uptake, which can most probably be ascribed to
contaminating neutrophils and to monocytes in which a respiratory burst has been
described 119].
We have previously shown that a micellar solution of sodium oleate activates
the cyanide-resistant and NADPH-dependent oxidase situated at the plasma membrane
of horse PMN leukocytes [14,161. Consistent with reports on human [1,12] and
guinea pig [8] PMN leukocytes, the membrane-bound oxidase catalyzes the reduction
of oxygen to superoxide anions. The results shown in Table I demonstrate the ability
of oleate to initiate a NADPH-dependent superoxide production in a cell-free prepa-
ration of resting granulocytes. The generation of O is proved either directly by the
SOD-inhibitable reduction of cytochrome c or indirectly by the effect of tetranitro-
methane, a scavenger of O. Addition of tetranitromethane resulted in a considerable
loss of cytochrome c reduction and apparent oxygen consumption. In the presence of
superoxide dismutase, which recycles half of the O back to atmospheric oxygen, a
decrease in oxygen uptake of approximately 40% is observed. Omission of either
oleate or reduced NADP or boiling the sample resulted in a complete loss of activity.
Both oxygen consumption and superoxide anion production by the postnuclear
cell fraction from unstimulated PMN leukocytes showed a linear relationship to
protein concentration in the presence of 2 mM oleate. Lineweaver-Burk plots of
754 Heyneman and Vercauteren

20

A B
. .

2
0.
00 00
E
.E 10
‘ .

ri ‘

.2
0
E

1 2 3 4 5 6 7 8
oleate (mM) pH

Fig. I. Rates of oxygen consumption by the postnuclear supernatant fraction from resting PMN (0),
PCMBSA-treated PMN (#{149}).and mononuclear leukocytes () incubated at 25#{176}Cin a medium contain-
ing 10 mM phosphate buffer, 100 mM NaCI, 1 mM MgCl2. 1 mM KCN. and 0.5 mM NADPH.
Mononuclear leukocyte preparations contained on average 77% lymphocytes. 19% monocytes. and 4%
neutrophils. Cells treated with PCMBSA were preincubated with 1 mM inhibitor at 25#{176}Cfor 5 mm and
thoroughly washed four times with the homogenization medium prior to cell breakage. A. Effect of
oleate concentration on oxygen uptake assayed at pH 7.2. B. pH optimum of oxygen consumption in the
presence of 2 mM oleate. Points represent the mean ± SD of three experiments.

TABLE 1. Oleate-Stimulated and NADPH-Dependent Oxygen Consumpti on and 02 -Production

by the Postnuclear Fraction From Resting PMN Leukocytesa

Conditions 02 consumption O production

Complete system 17.1 ± 2.3 13.3 ± 3.6

Plus SOD (30 zg/ml) 10.6 ± 2.6 -

Plus boiled SOD 15.2 ± 2.1 -

Plus tetranitromethane (0. 1 mM) 3. 1 ± 1. 1 1.7 ± 0.5

KCN omitted 12.2 ± 3.3 12.4 ± 2.8
Oleate omitted 0.6 ± 0.3 0.3 ± 0.2
NADPH omitted 0.3 ± 0.3 0
Boiled sample” 0 0

“The assays were conducted as described in the Methods section, with modifications as indicated.
Activities are expressed as nmol’ mm mg protein and values are mean ± standard deviation for
four experiments. Experiments with tetranitromethane. dissolved in ethanol (final concentration 0.5%).
were run with appropriate blanks.
“Heated for I mm in boiling water.

pyridine nucleotide concentration versus 02 consumption were linear with NADH

and NADPH. The apparent Km values were 61 ± 11 zM (mean ± SE, n = 4) for
NADPH and 1,600 ± 350 ptM (mean ± SE, n = 3) for NADH. Following the
production of O in relation to coenzyme concentration gives very similar results.
The effect of various cations is shown by the results in Table 2. The oxidative activity
is considerably elevated at higher ionic strengths (0.2). Addition of 1 mM Mg +

stimulates the oxygen consumption either at low or at high ionic strength. Equal
concentrations of Ca2 ± have no effect. Substituting oleate by 2 mM linoleate in the
assay gives a lowering of 15-20% of the observed activity. Crude phospholipid from
 Activation of a Granulocyte NADPH Oxidase 755

TABLE 2. Effect of Cations on the Oleate-Stimulated and NADPH-Dependent

Oxygen Consumption by the Postnuclear Fraction of Resting PMN
Leukocyt&

Cations Concentration Oxygen consumption

(as chloride) (mM) (nmol/min/mg protein)

- - 5.8 ± 1.3
Na 100 10.2 ± 0.9
100 9.6 ± 3.1
Ca2 I 4.0 ± 2.0
Mg2 I 13.5 ± 1.5
Na + Mg2 100 + 1 18.2 ± 2.1

Assays were conducted as described under Materials and Methods, except that the
assay mixtures were supplemented with salts at the concentrations indicated. The
data presented are the mean ± SD of three experiments.

TABLE 3. Stoichiometry of the NADPH-Dependent Oxygen Consumption by the Oleate-Activated

Postnuclear Fraction Oxidasea

02 consumption NADPH oxidized

Additions (nmol/3 mm) (nmol/3 mm) Ratio 02/NADPH

None 22.6 13.8 1.64 ± 0.26 (4)

SOD (30g/ml) 13.9 11.9 1.17 ± 0.22 (4)

“Oxygen measurements were performed in the presence of2 mM sodium oleate as described in Methods.
The amount of protein introduced into the microvessel was adjusted to 0.40 mg. After 0 and 3 mm of
registration, 0. 1 ml of the assay mixture was rapidly picked up from the electrode vessel and injected
into a cuvette containing I .4 ml of I mM NaOH. The absorbance was read at 340 nm fr NADPH
estimation. Superoxide dismutase was added prior to NADPH. Ratios are expressed as mean ± SD.
with the number of experiments in parentheses.

brain [13] causes little or no oxidative activity. The ability of the postnuclear fraction
to be stimulated by oleate decreases rapidly even at 0#{176}C
(t112 = 20 h), but can be
kept for several months at - 195#{176}C.
To evaluate a possible participation of a Of-driven nonenzymatic free radical
chain reaction involving the oxidation of NADPH to NADP, the influence of super-
oxide dismutase on the stoichiometry between 02 and NADPH was quantified. The
observed decrease of the ratio 02/NADPH from 1 .64 to 1 . 17 (Table 3) was substan-
tially due to a decline of the apparent oxygen consumption and not to a lowering of
NADPH oxidation. Such a decrease of NADPH oxidation would be expected if a
nonenzymatic chain reaction is operative [9] . However, the conditions at which the
experiments were conducted (pH 7.2 with 0.5 mM NADPH) are unfavorable for
generation of a chain reaction [4]. Further evidence arguing against a O -propagated
chain reaction such as a NADPH-dependent lipid peroxidation [211 is provided by the
fact that the formation of malondialdehyde, a general matabolite of lipid peroxidation,
is only demonstrated after incubation with Fe3-ADP, a known initiator of lipid
peroxidation [25]. The absorbances ± SD (n = 3) obtained with the thiobarbituric
acid assay after 10 mm at 37#{176}Cwere respectively 0.027 ± 0.005 without oleate,
0.030 ± 0.008 in the presence of 2 mM oleate, and 0.061 ± 0.010 in the presence
of 50 1tM Fe3 + ADP
756 Heyneman and Vercauteren

TABLE 4. Distribution of Enzymes in Subcellular Fr actions From R esting Horse Granulocytesa

Band I Band II Band III

Cytosol (11-25%) (25-45%) (45-60%)

NADPH-dependent 02 production
- cytosol 0 0 0 0
+ cytosol - S ± 2 52 ± 12 13 ± 3
Lactate dehydrogenase 98 ± 6 3 ± 2 1 ± I 3 ± I
5’-Nucleotidase 12 ± 2 18 ± 4 56 ± 10 Il ± 4
Alkaline phosphatase 21 ± 5 24 ± 5 46 ± 7 14 ± 6
Lysozyme 6 ± 3 2 ± 0.5 40 ± 6 58 ± 12
Peroxidase 5 ± 4 13 ± 6 6 ± 2 80 ± 21
Protein 48±6 5±2 14± 3 29± 4

“Results are given in percentages of values fbund in the postnuclear supernatant (mean ± SD of three
independent experiments). Bands refer to the interface fractions collected from a discontinuous sucrose
density gradient as described under Methods with the sucrose boundary concentrations between brackets.
Oleate-activated superoxide-forming activity was quantified in the absence and presence of the cytolic
fraction (0.5 mg of cytosolic protein).

On a single occasion, we had the opportunity to determine the oxidative activity

of PMN leukocytes from a 17-year-old boy with chronic granulomatous disease
(COD). In contrast to the postnuclear fraction from normal human granulocytes,
which consume on average 24 nmol 02/min/mg protein, a similar cell fraction from
resting CGD leukocytes was shown to be totally incapable of consuming oxygen in
the presence of cyanide, NADPH, and oleate (lower limit of detection: 0.2 nmol
0,/mm).
To further localize the oxidative activity, the postnuclear supernatant from
resting horse granulocytes was fractionated into a cytosol fraction and a particulate
fraction that was centrifuged on a discontinuous sucrose gradient. After this, oxidative
activity was no longer perceptible in any of the isolated fractions (Table 4). However,
recombination of the gradient fractions with cytosol revealed that a SOD-inhibitable
and NADPH-dependent 02 production could be recovered in the 25-45 % sucrose
interface fraction (band II). This activity coincides with the major peaks of 5’-
nucleotidase and alkaline phosphatase activity, two enzymes previously shown to be
localized in the plasma membrane fraction of horse neutrophils [15]. Most of the
myeloperoxidase and lysozyme activity, both granular marker enzymes, equilibrate at
a higher sucrose density.

DISCUSSION

Among a number of substances, saturated [20] and polyunsaturated [2] fatty

acids have been shown to activate the NADPH oxidase from intact PMN leukocytes.
Recently we have demonstrated that the dormant NADPH oxidase from resting horse
granulocytes is stimulated by sodium oleate [141. The resulting activity is mainly
recovered in the plasma membrane fraction of these cells [16]. The present commu-
nication reports the activation of a NADPH dependent oxygen consumption in the
postnuclear cell fraction of resting horse granulocytes by a micellar solution of sodium
oleate. Evidence for the identity of the latter oxidative activity with that of the
NADPH oxidase derived from activated cells is based on the following considera-
tions: 1) In either system, the oxidative activity is insensitive to azide or cyanide. 2)
 Activation of a Granulocyte NADPH Oxidase 757

As in whole cells [18] , both the capability to activate the oxidative process(es) in a
cell-free system and the resulting activity are transient. Consequently, freshly pre-
pared cell fractions from viable cells must be used. 3) Negligible oxygen consumption
is monitored in the postnuclear fraction from lymphocytes that lack the respiratory
burst enzyme. Oxidative activity was totally absent in the postnuclear fraction of
granulocytes from a patient with chronic granulomatous disease, an inherited condi-
tion in which granulocytes are unable to express a respiratory burst [3] . 4) The
observed apparent Kni values for NADH and NADPH and the pH optimum of the
reaction are very similar to those previously reported for the oxidase from stimulated
horse [16], human [1], and guinea pig [8] PMN leukocytes. 5) At least part of the
oxygen consumed is reduced univalently to O . Oeneration of this oxygen radical is
one of the features of the respiratory burst enzyme in stimulated granulocytes or in
derived cell fractions during oxidation of NADPH.
There is good evidence that phospholipid fatty acid side chains might be
involved in the activation process of the latent NADPH oxidase and in the subsequent
production of superoxide anions. Treatment of granulocytes with phospholipase C
largely prevented stimulation with phorbol myristate acetate [24], whereas addition
of phosphatidylethanolamine significantly increased 0 production of the activated
and “solubilized” oxidase [13]. So it is tempting to speculate that a micellar solution
of fatty acid such as oleate might contribute to the process in a twofold manner. First,
by a perturbation of the membrane vesicles resulting in an activation of the oxidative
system through a still undefined mechanism. Second, by facilitating the binding of a
cytoplasmic component required for O production or at least by creating a lipid
environment that intensifies the rate of O production.
The dependence of the oxidative activity on the association of a particulate and
a cytosolic constituent is clearly shown in Table 4. In the presence of a minimally
diluted cytosol fraction, formation of O is greatly recovered in a particulate gradient
fraction characterized by the presence of plasma membrane enzymes (5 ‘-nucleotidase,
alkaline phosphatase) [15]. Since it is largely accepted that the superoxide-producing
enzyme from stimulated neutrophils is situated at the plasmalemma of the cell, it
seems reasonable to assume that a dormant oxidase on the plasma membrane is
converted into an active enzyme by the lipid activator in the presence of a cytosolic
constituent. The nature of the cytoplasmic factor and the mechanism by which it
interferes in the triggering process or in the enzymatic catalysis are unknown. Its
necessity in relatively concentrated form may be a reason why attempts to activate a
repiratory burst enzyme in isolated cell fractions have failed thus far. This finding
probably also explains why plasma membrane vesicles prepared from resting PMN
and containing cytosol (cytoplasts) [23] could be stimulated to generate superoxide
radicals, whereas other cell-derived vesicles (podosomes) [8], which have lost a great
deal of their cytoplasmic content during the ultrasonic preparation procedure, failed
to be turned on.
A significant problem that confronts all investigations on plasma membrane
vesicles is membrane sidedness. In intact granulocytes the physiological substrate,
solubilized in the cytosol, and the activator face opposite sites of the plasma mem-
brane. Membrane vesicles prepared by disruption of cells may be in a right-side or
inside-out form, and therefore inaccessible for either the activator or the substrate and
cytosolic factor. However, fatty acid-salt ions such as oleate are well-known deter-
gents, which may considerably alter the permeability of cellular membranes. We
758 Heyneman and Vercauteren

believe that through oleate treatment both sides of the membrane vesicles become
accessible to the constituents of the assay mixture, leading to the formation of an
active NADPH oxidase.

ACKNOWLEDGMENTS

We would like to thank Professor Dr. R. Van Furth for supplying blood samples
from a patient with COD. We are indebted to Mrs. D. Bauwens and Mrs. M. Van De
Oehuchte for their valuable technical and secretarial assistance.

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