ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 280, No. 1, July, pp. 74-79, 1990

Involvement of Membrane Charges in Constituting Active Form of NADPH Oxidase in Guinea Pig Polymorphonuclear Leukocytes’ Toshiaki Sadahiko Department

Ohtsuka,’ Ishibashi

Masanori

of Physiological

Hiura,

Chemistry,

Masaki

Hiroshima

Received October 20,1989, and in revised form February

Ozawa, Naoki

University

Okamura,

the

Mayumi

Nakamura,

School of Medicine, Kasumi, Minami-ku,

and

Hiroshima

734, Japan

23,199O

NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)stimulated guinea pig polymorphonuclear leukocytes (PMNL) was inhibited by positively charged myristylamine. The inhibitory effect of myristylamine was significantly suppressed by simultaneous addition of a negatively charged fatty acid, such as myristic acid. However, the suppression by myristylamine was not sufficiently restored when myristic acid was added later. On the other hand, pretreatment of PMA-stimulated PMNL with glutaraldehyde, a protein crosslinking reagent, stabilized NADPH oxidase activity against inhibition by myristylamine, but not against that by pchloromercuribenzenesulfonic acid. In a cell-free system of reconstituted plasma membrane and cytosolic fractions prepared from unstimulated PMNL, arachidonic acid-stimulated NADPH oxidase activity was also inhibited by myristylamine. During the activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system, cytosolic activation factor(s) translocated to plasma membranes. The bound cytosolic activation factor(s) was released from the membranes by myristylamine, accompanied by a loss of NADPH oxidase activity. It is plausible from these results that the inhibitory effect of alkylamine on NADPH oxidase is due to induction of the decoupling and/or dissociation of the cytosolic activation component(s) from the activated NADPH oxidase complex by increments of positive charges in the membranes, and that the glutaraldehyde treatment prevents the dissocic 1990 Academic PMS, h. ation of component(s).

’ This work was supported by Grants-in-Aid for Cooperative search and Scientific Research from the Ministry of Education, ence and Culture of Japan. ’ To whom correspondence should be addressed.

ReSci-

A cyanide-insensitive respiratory burst is observed in polymorphonuclear leukocytes (PMNL)3 which produces superoxide anion (0,) and then other active oxygen metabolites (l-3), when PMNL are stimulated by phagocytic stimulation or by exposure to soluble membrane stimulants such as phorbol 12-myristate 13-acetate (PMA) and formyl-methionyl-leucyl-phenylalanine. Plasma membrane-bound NADPH oxidase is responsible for the production of 0; (4), but the enzyme is dormant unless the PMNL are stimulated. Though precise regulatory mechanisms for the activation of this enzyme activation have not been clarified, it has been reported that a guanine nucleotide-binding protein (5, 6), phosphatidylinositol turnover (6-8), intracellular calcium ion concentration (9), and protein kinase C activity (10-12) are involved, more or less, in the activation process. NADPH oxidase is supposed to be a multicomponent electron transport system consisting of a flavoprotein (13,14) and cytochrome bss8(15,16). In addition, involvement of undefined cytosolic component(s) was recently reported in a cell-free O,-producing system (17-24). Intracellular localization and translocation of these components are drawing great attention as a molecular mechanism to elucidate the process for the activation of NADPH oxidase (25-27). Recently, it was reported that 0, production in intact PMNL and in the cell-free system was controlled by the change in the membrane charge by a balance between lipophilic ions, such as positively charged alkylamines and negatively charged SDS and fatty acids (28, 29). In the present study, we have utilized alkylamine and fatty acids to elucidate the mechanism for the activation of 3 Abbreviations used: PMNL, polymorphonuclear leukocytes; 02, superoxide anion; PMA, phorbol12-myristate 13.acetate; PCMBS, pchloromercuribenzenesulfonic acid; GTP[S], guanosine 5’.(3.O-thio)triphosphate; SDS, sodium dodecyl sulfate; Pipes; 1,4-piperazinediethanesulfonic acid.

74 All

0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.

ACTIVE

FORM

OF GUINEA

oxidase. We found that NADPH oxidase activity in the membrane fraction was inhibited by alkylamine, and that the inhibition was efficiently suppressed by the pretreatment of PMNL with glutaraldehyde, a protein crosslinking reagent. We also found that the translocated cytosolic activation factor(s) was released from the membranes by alkylamine, accompanied by a loss of NADPH oxidase activity. The effects of alkylamine were modified by fatty acid. From these results, we discuss the significance of the membrane charge in the association of component proteins of NADPH oxidase. NADPH

MATERIALS

AND

METHODS

Materials. Ferricytochrome c (horse heart type III), superoxide dismutase, catalase, and GTP[S] were purchased from Sigma Chemical Co. (St. Louis, MO); NADPH from Kohjin Co., Ltd. (Tokyo, Japan); PMA and arachidonic acid from Funakoshi Pharmaceutical Co. (Tokyo, Japan); palmitylamine, myristylamine, glutaraldehyde, and p-chloromercuribenzenesulfonic acid (PCMBS) from Nacalai Tesque (Kyoto, Japan); laurylamine and myristic acid from Wako Pure Chemical Ind. (Osaka, Japan). All other chemicals were of reagent grade from standard commercial sources. Preparation of polymorphonuclear leukocytes. PMNL were obtained from the peritoneal cavities of female guinea pigs of the Hartley strain as reported previously (30). Rat peritoneal PMNL were prepared according to the same procedure. The content of PMNL in the preparation was more than 90%. Activation of PMNL by PMA and treatment of PMNL with glutaraldehyde. PMNL (2 X lo7 cells/ml) were suspended in Hanks’Pipes buffer (pH 7.3) containing 8 mM Pipes, 137 mM NaCl, 5.4 mM KCl, 0.81 mM MgS04, 1.28 mM CaCl,, 0.43 mM Na,HPO,, 0.44 mM KH,PO,, and 5.5 mM glucose, and preincubated at 37°C for 5 min. Then, 320 nM PMA was added and the mixture was incubated for 3 min. After the PMA stimulation, PMNL were treated, if indicated, with 0.05% (W/V) glutaraldehyde at 37°C for 10 s. The glutaraldehyde reaction was terminated by addition of 50 mM ammonium acetate. Then, PMNL were placed on an ice-water bath, washed, collected by centrifugation at 12Og and 4°C for 5 min, and resuspended to 4 X lo7 cells/ml in either 0.34 M sucrose-10 mM Tris-HCl buffer (pH 7.4) or a relaxation buffer containing 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl,, 1.25 mM EGTA, and 10 mM Pipes (pH 7.3). PMNL stimulated in the same manner but without addition of glutaraldehyde were used as the control. Subcellular fractionations of PMNL. The PMA-activated membrane fraction was prepared according to the reported method (31) with slight modifications. PMNL were disrupted by sonication (3 A, 10 s) twice at 0°C using an Ohtake sonicator type 5202 equipped with a microtip. The disruption was confirmed with microscopic observation. The homogenate was centrifuged at 480g and 4°C for 10 min to remove cell debris and intact cells. The postnuclear supernatant obtained was further centrifuged at 100,OOOgand 4°C for 30 min. The pellet showing NADPH oxidase activity was resuspended in the same buffer and used as the activated membrane fraction. For cell-free activation experiments, resting PMNL were suspended in the relaxation buffer (pH 7.3), and disrupted by sonication (2 A, 15 s) twice at 0°C. The homogenate was subjected to fractionation on a discontinuous Percoll gradient according to a reported procedure (32) with some modifications, and plasma membranes were obtained by centrifugation of the pooled y-fraction (32) at 100,OOOgand 4°C for 1 h. Aliquots of prepared plasma membrane suspension in the same buffer (lOa cell es/ml) and cytosolic fraction (5 X IO7 cell es/ml) were stored at ~80°C until use.

PIG NADPH

OXIDASE

75

Determination of the NADPH oxidase activity and protein. NADPH oxidase activity was measured as superoxide dismutase-sensitive reduction of cytochrome c (33) at 25°C. To measure the activity in the activated membrane fraction, the sample preparation was added to the reaction mixture consisting of 500 PM NADPH, 80 PM cytochrome c, and 10 unit/ml catalase in Hanks’-Pipes buffer. One hundred units per milliliter superoxide dismutase was added to a reference cuvette. The reaction was started by the addition of the sample, and the change in an absorption at 550 nm corresponding to reduction of ferricytochrome c was followed. To see the effect of alkylamine, it was dissolved in ethanol and added directly to the reaction mixture. The final concentration of ethanol in the assay system was 0.5% throughout. If indicated, myristylamine was added to the postnuclear supernatant fraction (2 X 107 cell es/ml) of control and PMA-stimulated PMNL, and the mixture was incubated for 3 min at 25°C in the relaxation buffer. Following separation of the mixture into soluble and membrane fractions by centrifugation at 100,OOOg and 4°C for 1 h, NADPH oxidase activity in each fractions was measured in the relaxation buffer as described above. In the cell-free activation system, both cytosol and plasma membranes equivalent to 2.5 X 10’ PMNL/ml were added to an assay mixture consisting of 100 KM cytochrome c and 20 1M GTP[S] in the relaxation buffer (pH 7.3). Fifty micromolar arachidonic acid, which maximally stimulated NADPH oxidase activity, was used as a stimulant. The reference cuvette contained 100 unit/ml of superoxide dismutase. The mixture was preincubated for 3 min at 25”C, and the reaction was initiated by the addition of 200 PM NADPH to measure NADPH oxidase activity as above. If indicated, the same amounts of the cytosol and plasma membranes were incubated first with or without 50 PM arachidonic acid for 5 min at 25”C, and then with or without 50 PM myristylamine for 3 min at 25°C in the relaxation buffer (pH 7.3). After centrifugation of the mixture at 100,OOOgand 4°C for 1 h, each of the soluble and membrane fractions was assayed for NADPH oxidase activity in the relaxation buffer (pH 7.3) as described above. For measurement of the activity of cytosolic activation factor(s) in the soluble fraction obtained above for the activation of NADPH oxidase activity, the fraction (1.25-2.5 X lo6 cell es/ml) was combined with the fresh plasma membranes (2.5 X lo6 cell es/ml) from untreated resting PMNL, and the mixture was preincubated for 3 min at 25°C in the presence of 100 PM ferricytochrome c, 20 FM GTP[S], and 50 PM arachidonic acid. Then, the reaction was initiated by the addition of 200 FM NADPH, and the superoxide dismutase-inhibitable reduction of ferricytochrome c was followed. The activity of cytosolic activation factor(s) was expressed as nmol O;/min/mg protein or nmol O,/min/lO’cell eq of the soluble fraction. Protein contents were determined using bovine serum albumin as a standard (34).

RESULTS

Effect of Alkylamine and Fatty Acid on PMA-Stimulated NADPH Oxidase in PMNL In an attempt to elucidate the possible involvement of the membrane charge in NADPH oxidase activity, a plasma membrane fraction with NADPH oxidase activity was prepared from PMA-stimulated PMNL. 0, production by the membrane fraction was inhibited by the addition of myristylamine in a concentration-dependent manner (Fig. 1). It is unlikely that myristylamine scavenges 02, since reduction of cytochrome c with a xanthine oxidase system examined by a standard procedure (35) was little affected by myristylamine up to 100 PM (data not shown). The result indicates that NADPH oxidase activity in the membrane fraction is sensitive to al-

*

76

%

OHTSUKA

s a0

-e

0 Myristylamine

(pb.4)

FIG. 1. Effects of alkylamine and fatty acid on NADPH oxidase activity in the membrane fraction from PMA-stimulated PMNL. The activity was measured in the presence of indicated concentrations of myristylamine as described under Materials and Methods. To the fractions, 0 KM (O), 50 pM (A) or 100 fiM (0) of myristic acid was added either simultaneously (solid lines) or 2 min after the addition of myristylamine (broken lines). The activity is expressed as a percentage of control values, which are 66.7,70.7, and 76.9 nmol O;/min/mgprotein for 0 FM, 50 FM, and 100 KM myristic acid in solid lines, respectively, and 80.3,87.6, and 95.1 nmol O;/min/mg protein for 0 PM, 50 pM, and 100 FM myristic acid in broken lines, respectively. Results are expressed as means ? SE (n = 3). No bar means that the SE is within the range of the symbol. The t test was used for statistical analysis. *Effect of simulta neous addition of myristic acid is significant; P < 0.01.

kylamine as reported previously for the activity in intact PMNL (28). Though a negatively charged lipophilic agent, myristic acid, had little effect by itself on NADPH oxidase activity, the inhibitory effect of myristylamine on the activity was significantly suppressed by myristic acid, when myristic acid and myristylamine were added simultaneously to the assay system for 0; production (Fig. 1, solid lines). The results suggest that the inhibitory effect of alkylamine is related to its positive charge. However, the inhibition was not sufficiently restored by myristic acid when myristic acid was added after the myristylamine treatment (Fig. 1, broken lines), indicating that NADPH oxidase activity in the membrane fraction did not depend simply on the membrane charge. These results were reproduced in a separate experiment with rat peritoneal PMNL, though NADPH oxidase activity in rat PMNL was about l/5 that of guinea pig PMNL (data not shown). Decrease in Sensitivity of NADPH Oxidase to Alkylamine by Pretreatment of PMNL with Glutaraldehyde It has been reported that the NADPH oxidase in the membranes isolated from PMA-stimulated PMNL is stabilized by treatment of the membranes with glutaraldehyde (31). Since NADPH oxidase has been assumed

ET AL.

to be a multicomponent electron transport system (4, 36), this stabilizing effect of glutaraldehyde may be due to the formation of a more stable, undissociable NADPH oxidase complex including cytosolic activation factors. Recently, we reported that NADPH oxidase activity of PMA-stimulated PMNL was well retained throughout the disruption of PMNL and preparation of the membrane fraction when the PMNL was treated with glutaraldehyde immediately after the PMA treatment (37). So, we have examined if the above-mentioned effect of alkylamine on NADPH oxidase activity in membrane fractions from PMA-stimulated PMNL would be modified by the glutaraldehyde treatment. Figure 2 shows the changes by the glutaraldehyde treatment in the inhibitory effect of various alkylamines with different chain length on NADPH oxidase activity. By the treatment of the PMA-stimulated PMNL with glutaraldehyde, the inhibitory effects of alkylamines were fairly decreased. In marked contrast, the inhibitory effect of PCMBS on NADPH oxidase activity was not affected by the treatment of the PMNL with glutaraldehyde (Fig. 2). We also compared the protein contents in the membrane fractions between the glutaraldehyde-treated and untreated PMNL and found no significant difference. Effect of Alkylamine on the Intracellular Distribution of Cytosolic Activation Factor(s) in PMA-Stimulated PMNL To examine for possible mechanisms of the inhibitory effect of alkylamine on NADPH oxidase, myristylamine 1

I

1 rM’

FIG. 2. The effect of treatment of PMNL with glutaraldehyde on NADPH oxidase activity in the membrane fraction from PMA-stimulated PMNL in relation to the inhibitory effects of alkylamines and PCMBS on the oxidase activity. After the stimulation of PMNL by PMA, the PMNL were treated with (0) or without (0) 0.05% glutaraldehyde, and then the membrane fraction was obtained as described under Materials and Methods. OF-producing activity was measured in the presence of the indicated concentrations of alkylamines or PCMBS, as described under Materials and Methods. The activity is expressed as a percentage of the respective control value. Control values, i.e., those without the inhibitor, in glutaraldehyde-untreated and -treated membranes in the alkylamine experiments are 83.8 -t 0.6 and 113.9 f 1.6 nmol OB/min/mg protein (n = 3), respectively, and those of in the PCMBS experiments are 72.9 & 1.4 and 98.7 + 1.4 nmol O,/ min/mg protein (n = 3), respectively. No bar means that SE is within the range of the symbol.

ACTIVE TABLE

FORM

OF GUINEA

I

Effects of Myristylamine on NADPH Oxidase Activity in and on Release of Cytosolic Activation Factor(s) from Membranes Prepared from PMA-Stimulated PMNL 0; production (nmol/min/mg protein of membrane or soluble fraction) Resting Experiment

Control

A, NADPH oxidase activity Membrane 1.6 k 0.1 Soluble 1.6 + 0.2 B, activity

Myristylamine

PMA-stimulated Control

Myristylamine

89.3 f 2.8 1.7 + 0.4

5.4 + 2.4 1.8 f 0.4

of cytosolic activation factor(s) 17.8 f 0.3 13.3 + 0.2* 18.5 + 0.3

18.2 k 0.8

0.8 + 0.6 0.5 f 0.3

Note. In Experiment A, postnuclear supernatant equivalent to 2 X lo7 PMNL/ml of resting and PMA-stimulated PMNL were treated with or without 50 pM myristylamine for 3 min at 25°C. After centrifugation of the mixture, the membrane and soluble fractions obtained were assayed for NADPH oxidase activity. In Experiment B, activity of cytosolic activation factor(s) in the soluble fractions (2.5 X lo6 cell eq/ml) obtained in Experiment A was determined in the presence of fresh untreated plasma membranes (2.5 X lo6 cell eq/ml) and 50 pM arachidonic acid. Results are expressed as means f SE (n = 3). The t test was used for statistical analysis: *P < 0.01 relative to resting control.

PIG NADPH

tar(s) in the soluble fraction was fairly decreased by the glutaraldehyde treatment (37). Effect of Alkylamine on Arachidonic Acid-Stimulated NADPH Oxidase in the Cell-Free Activation System We have also examined the effect of myristylamine on arachidonic acid-induced NADPH oxidase activation in a cell-free system consisting of plasma membrane and cytosolic fractions prepared from resting PMNL (Fig. 3). The cell-free activation of NADPH oxidase by arachidonic acid was also suppressed by myristylamine in a concentration-dependent manner (Fig. 3a). Pretreatment of the plasma membrane and cytosolic fractions with myristylamine effectively inhibited the activation (Fig. 3b). These results indicate that alkylamine inhibits not only the activated NADPH oxidase itself but also cell-free activation of the enzyme by arachidonic acid. Next, the mixture of the plasma membrane and cytosol fraction was first activated by arachidonic acid in this system, and then treated with myristylamine. Following the separation of the mixture into membrane and soluble fractions by centrifugation, NADPH oxidase activity in both fractions and the cytosolic activation factor(s) activity in the soluble fraction were measured (Table II). The results were very similar to those from the PMAstimulated PMNL, as shown in Table I. Thus, these results indicate that alkylamine treatment induces disso-

was added to the postnuclear supernatant of control and PMA-stimulated PMNL. Then the mixture was separated into membrane and soluble fractions by centrifugation, and NADPH oxidase activity in each of the frac-

tions was measured (Table I, Experiment A). NADPH oxidase resided exclusively in the membrane fraction, and the activity was almost suppressed by the treatment with myristylamine (Table I, Experiment A). On the other hand, results of Experiment B show the effectiveness of the above-mentioned soluble fraction for the activation of NADPH oxidase. NADPH oxidase activity was measured by incubation of the soluble fraction with a mixture of a fresh plasma membrane and arachidonic acid to assess the activity of the cytosolic activation factor(s) existing in the soluble fraction. In accordance with an increase in NADPH oxidase activity in the membrane fraction, the activity of cytosolic activation factor(s) in the soluble fraction disappeared partly, suggesting the translocation of the effective component(s) from the fraction to the membranes. Furthermore, when the postnuclear supernatant of PMA-stimulated PMNL was treated with myristylamine, the NADPH oxidase-stimulating activity in the soluble fraction increased in accordance with a decrease in NADPH oxidase activity in the membrane fraction (Table I, Experiments A and B). The same type of experiment was not applicable to the glutaraldehyde-treated PMNL, since the activity of cytosolic activation fac-

77

OXIDASE

Myristylomine(pM)

!

(a)

03-

L, ' 0

,o

Myristylamine

(b)

NADPH I 5

I 10

(PM)

I ,, 15 TIME (mln)

NAbPH I 0

I 5

I 10

I 15

FIG. 3. The effect of myristylamine on NADPH oxidase activity in the cell-free activation system. 0; production was measured as described under Materials and Methods, using cytosolic and plasma membrane fractions equivalent to 2.5 X lo6 cells. Fifty micromolar arachidonic acid (ARA), 200 pM NADPH, and various concentrations of myristylamine (MAM) were added to the reaction mixtures as indicated by the arrows.

78

OHTSUKA TABLE

II

Effects of Myristylamine on NADPH Oxidase Activity in and on Release of Cytosolic Activation Factor(s) from Membranes Activated by Arachidonic Acid in a Cell-Free System 0, production (nmol/min/107 cell eq of membrane or soluble fraction) Unactivated Experiment

Control

A, NADPH oxidase activity Membrane 0.2 & 0.1 Soluble 1.2 * 0.1 B, activity

Myristylamine

0.1 * 0.1 1.3 * 0.2

of cytosolic activation factor(s) 15.7 iz 0.6 14.4 k 1.6

Arachidonic

acid-activated

Control

Myristylamine

20.1 ix 0.4 1.3 k 0.1 8.6 + 1.6*

0.2 * 0.4 1.8 f 0.1 13.7 + 1.8

Note. In Experiment A, a mixture of the cytosol and plasma membranes equivalent to 2.5 X lOa PMNL/ml was treated first with or without 50 pM arachidonic acid for 5 min at 25”C, and then with or for 3 min at 25°C. After centrifugation without 50 pM myristylamine of the mixture, each of the membrane and soluble fractions separated again was assayed for NADPH oxidase activity. In Experiment B, activity of cytosolic activation factor(s) in the soluble fractions (1.25 X lo6 cell eq/ml) obtained in Experiment A was determined in the presence of fresh untreated plasma membranes (2.5 X 10s cell eq/ml), 20 j&M GTP[S] and 50 pM arachidonic acid. Results were expressed as means + SE (n = 4). The t test was used for statistical analysis: *P < 0.01 relative to unactivated control.

ciation of the cytosolic activation factor(s) from the active form of NADPH oxidase in the plasma membranes and release of component(s) to the medium, resulting in the loss of the NADPH oxidase activity. DISCUSSION

NADPH oxidase activity in the membrane fraction prepared from PMA-stimulated PMNL was inhibited by positively charged alkylamines in a concentration-dependent manner. Though negatively charged myristic acid had little effect by itself on NADPH oxidase activity, it significantly suppressed the inhibitory effect of myristylamine, when both were added at the same time to the assay system. The results suggest that the inhibitory effect of alkylamines is related to positive charges of alkylamines. However, the inhibition by myristylamine was not sufficiently restored by the addition of myristic acid, when the latter was added later. On the other hand, the treatment of PMA-stimulated PMNL with low concentrations of glutaraldehyde, a protein crosslinking reagent, protected the NADPH oxidase activity from the inhibition by alkylamines but not from that by PCMBS. Since NADPH oxidase is assumed to be composed of a flavoprotein (4), cytochrome bs5s (36), and undefined cytosolic activation factor(s), glutaraldehyde may stabilize the oxidase through the

ET AL.

formation of a more stable and undissociable complex as has been suggested previously (31, 37). These results suggest that the inhibitory effect of alkylamine on NADPH oxidase is due to decoupling and/or dissociation of the component(s) from the enzyme by the increase in the positive charge in the membranes. We also found that the cytosolic activation factor(s) in the cytosol translocated to the membranes during activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system. On the other hand, the membrane-bound factor(s) was released by the treatment with alkylamine, accompanied by a loss of NADPH oxidase activity in the membranes. In other words, the inhibitory effect of alkylamine on NADPH oxidase may be due to the suppression of an interaction between the enzyme components in the plasma membranes and the cytosolic activation factor(s). The finding also suggests that changes in the electrostatic charges of the cell membranes may cause the redistribution of the cytosolic activation factor(s) between cytosol and plasma membranes and regulate the NADPH oxidase activity. At present, it is unclear how such a charge-dependent interaction between the cytosolic factor(s) and the membrane component(s) controls 0; production in intact PMNL. Recent reports showed that (one of) the cytosolic activation factor(s) was a 47-kDa protein (38, 39), which was identified as a 46- to 48-kDa protein that underwent stimulus-dependent phosphorylation by protein kinase C in normal but not chronic granulomatous disease neutrophils (40, 41). Furthermore, we (42) and others (43) have found that this protein translocates from the cytosol to the cell membranes after phosphorylation in parallel with the stimulation of 0, production. Though the role of the protein kinase C-dependent phosphorylation is unknown, it may be possible that phosphorylation of cytosolic activation factor(s) causes charge-dependent association with the membrane. REFERENCES 1. Repine, J. E., White, J. G., Clawson, C. C., and Holmes, B. M. (1974) J. Lab. Clin. Med. 83,911-920. 2. Kakinuma, K. (1978) Biochem. Biophys. Acta 538,50-59. 3. Zabucchi, G., Berton, G., and Soranzo, M. R. (1981) FEBS Lett. 127,4-8. 4. Babior, B. M. (1984) Blood 64,959-966. 5. Okajima, F., and Ui, M. (1984) J. Biol. Chem. 259, 13,863-13,871. 6. Smith, C. D., Lane, B. C., Kusaka, I., Verghese, M. W., and Snyderman, R. (1985) J. Biol. Chem. 260,5875-5878. 7. Bennett, J. P., Cockcroft, S., and Gomperts, B. D. (1980) Biochim. Biophys. Acta 601,584-591. 8. Volpi, M., Yassin, R., Naccache, P. H., and Sha’afi, R. I. (1983) Biochem. Biophys. Res. Commun. 12’7,450-457. 9. Nakagawara, M., Takeshige, K., Sumimoto, H., Yoshitake, J., and Minakami, S. (1984) Biochim. Biophys. Acta 805,97-103. S. (1984) Bio10. Fujita, I., Irita, K., Takeshige, K., and Minakami, them. Biophys. Res. Commun. 120,318-324.

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OF GUINEA

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Involvement of membrane charges in constituting the active form of NADPH oxidase in guinea pig polymorphonuclear leukocytes.

NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig polymorphonuclear leukocytes (...
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