Comp. Biochem. Physiol. Vol. 9611,No. 3, pp. 461--464,1990
0305-0491/90 $3.00 + 0.00 © 1990PergamonPress pie
Printed in Great Britain
A MICROTECHNIQUE FOR NEUTROPHIL RESPIRATORY BURST OXIDASE IN A CELL-FREE SYSTEM-CHARACTERIZATION OF OXIDASE ACTIVATION SYSTEM SHIGENOBUUMEKI* and DOROTHY M. DELISLEt *Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-01, Japan; and i'Department of Basic and Clinical Research, BCR-7, Research Institute of Scripps Clinic, La Jolla, CA 92037, USA (Received 14 November 1989)
Abstract--l. A microtechnique for quantitating human neutrophil NADPH oxidase in a cell-free system is described. 2. This spectrophotometric discontinuous (fixed time) method is less material-consuming than existing methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples. 3. Measurement of NADPH oxidase using the new method can be accomplished in a final vol of 0.15 ml. 4. In the assay, neutrophil membranes solubilized with deoxycholate were incubated for 3 min with cytosolic fractions, magnesium, sodium dodecyl sulfate, and cytochrome c in the absence of NADPH to preincubate the oxidase before the addition of the reducing agent. 5, The reaction was started by adding NADPH and 2 min later terminated by adding superoxide dismutasc. 6, The apparent K~ for NADPH obtained by the new method was almost the same as that by the authorized method (39.2 + 3.1 SD vs 36.8 _ 1.6). Activation of neutrophil NADPH oxidase was characterized using the new assay method.
INTRODUCTION Of the body's host defence mechanisms, one is enzymatic conversion by phagocytic cells of oxygen to several toxic metabolites, including the superoxide anion and hydrogen peroxide (Babior, 1978). The enzyme system responsible for this respiratory burst appears to be an N A D P H oxidase (EC 1.6.99.6) that is inactive until the cells have been stimulated by a wide variety of phagocytic, inflammatory, and surface-reactive agents (Babior, 1978; McPhail and Snyderman, 1984). Although the mechanisms that regulate the activation of this enzyme are not well understood, it has recently been reported that the N A D P H oxidase activity is associated with a low-potential cytochrome b and a flavoprotein (Henderson et al., 1987), and is therefore likely to comprise an electron-transport chain composed of a hydrogen acceptor, tlavin adenine dinucleotide (FAD), followed by a purely electron acceptor, cytochrome b (Rossi and Zatti, 1964). For a better understanding of the precise mechanisms that regulate this enzyme, many kinds of cell-free systems for N A D P H oxidase activation have recently been developed (Bromberg and Pick, 1985; McPhail et al., 1985; Curnutte et al., 1987, Abbreviations used--PiPES, 1,4-piperazinediethanesulfonic
acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; FAD, flavin adenine dinucleotide; PBS, phosphate buffered saline; SOD, superoxide dismutase; DIP, diisopropyl fluorophosphate. 461
1989; Umeki, 1990). Existing standard techniques require a large volume of materials, such as solubilized membranes, cytosolic fractions, cytochrome c, superoxide dismutase, N A D P H and others. In this manuscript, a microassay that is less materialconsuming than authorized methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples is described.
MATERIALS AND
METHODS
Horse heart cytochrome c (type III), bovine erythrocyte superoxide dismutase (SOD), fl-NADPH (type I), sodium deoxycholate, diisopropyl fluorophosphate (DFP), ATP, EGTA, PIPES, glycerol, sucrose, FAD, dextran (average tool. wt 78,000) and Dulbecco's calcium- and magnesiumfree phosphate buffered saline (D-PBS) were obtained from Sigma Chemical Company (St Louis, MO). Sodium deoxycholate was recrystallized from ethanol before use. FicollPaque and Percoll were purchased from Pharmacia P-L Biochemicals (Piscataway, N J). Other chemicals were of the highest purity available from commercial sources. Human neutrophils of ~>98% purity were prepared as previously described (McPhail et al., 1985) using acid citrate dextrose as the anticoagulant, dextran to sediment erythrocytes, and Ficoll-Paque to separate mononuclear cells from neutrophils. The cells were treated with DFP by the method of Crowley et al. (1980), except that phosphate was omitted from the buffer and the DFP concentration was reduced to 2.5 raM. DFP-treated cells were washed two times with D-PBS. The resulting cells were homogenated by nitrogen cavitation and fractionated on a discontinuous Pereoll
462
SHIGENOBU UMEKI and
gradient by a modification (Umeki and Soejima, 1990) of the method of Borregaard et al. (1983). Membranes were obtained from the Percoll gradient by pooling the 7-fraction (Borregaard et al., 1983) with a high-speed (230,000g for 2 hr at 4°C) pellet from the cytosolic fractions. The pooled membranes were suspended at a concentration of 3 x 10Scell eq/ml ( 1 . 2 + 0 . 1 S D mg prot/ml, n = 3 ) in the half-strength Borregaard's relaxation buffer [50 mM KCI, 1.5 mM NaCI, 5 mM PIPES (pH 7.3), 1.75 mM MgC12, 0.5 mM ATP, 0.62 mM EGTA (Borregaard et al., 1983)] containing 0.34 M sucrose. The suspension was divided into aliquots and stored at -70°C. The supernatant from the 230,000 g spin [designated "cytosol", which contained 7.5 × 107cell eq/ml (1.4±0.1 SD nag prot/ml, n = 3)] was divided into aliquots and stored at -70°C; this material served as the source of cytosolic activation factor. Membranes were solubilized by the procedure described by Curnutte et al. (1987). The membrane suspension thawed at room temperature was mixed with an equal volume of extraction buffer [1 mM NAN3, 0.0017 mM CaCI 2, 20 mM sodium glycinate (pH 8.0), 2.33% sodium deoxycholate (w/v), and 50% glycerol (v/v)], vortexed briefly, incubated on ice for 30 min with occasional agitation, and finally centrifuged at 435,000g for 1 hr at 4~C in a Beckman TL-100 tabletop ultracentrifuge with a TL-100.2 rotor, using polycarbonate tubes. The dormant oxidase was found in the supernatant, whose protein concentration was 0.53 + 0.11 SD mg prot/ml (n = 3). Superoxide production was determined by a discontinuous assay in which SOD-inhibitable cytochrome c reduction was followed by scanning between 530 and 570 nm in a double-beam spectrophotometer (Umeki, 1990). Assay mixtures contained 0.1 mM cytochrome c, 3.6raM MgC12, 89mM KCI, 2.TmM NaCI, 0.5mM PIPES (pH 7.3), 0.9mM ATP, 1.2raM EGTA, 1.2 × 106cell eq of cytosol, 3 × l0 Scell eq of solubilized membranes, 0.04 mM sodium dodecyl sulfate and 0.16 mM NADPH, with alterations as noted in the Table l and Fig. legends, in a total vol of 0.15 ml. The reference cuvette contained 8 p I of SOD. Basically, reaction mixtures without NADPH were preincubated at 24°C for 3 min after the addition of 10#l of sodium dodecyl sulfate. Then the reactions were initiated at 24°C by adding 5 pl of NADPH and 2 min later the reaction of a sample cuvette was terminated by adding 5 pl of SOD (1 mg/ml of distilled water). After adding 600 ,ul of distilled water into the reference cuvette and 595 #1 of water to the sample cuvette, measurement of the cytochrome c reduction
6i
"E
DOROTHY
M. DELISLE
was done by scanning between 530 and 570 nm. The height of the peak of 5 5 0 n m represents the absorbance due to superoxide-dependent cytochrome c reduction. 02- production was calculated on the basis of the height of the peak at 550 nm using an extinction coefficient of 19.6 m M - ~ cm -t (Yonetani, 1965). The protein concentration was determined by the method of Smith et al. (1985), using the bicinchoninic acid protein assay reagent from Pierce Chemical Company 'Rockford,
IL),
RESULTS
AND
DISCUSSION
This new discontinuous assay is suitable for investigating t e m p e r a t u r e - d e p e n d e n t changes in N A D P H oxidase because of the use of a n i n c u b a t o r with thermometer. Figure 1 shows temperatured e p e n d e n t changes in activation of neutrophi] N A D P H oxidase evaluated using this new disc o n t i n u o u s microassay. Activation of N A D P H oxidase in a cell-free system was the highest at 24°C in the presence or absence o f 0.5 # M F A D . In addition to the presence o f an optimal t e m p e r a t u r e (24°C) in activation (reconstitution) o f the N A D P H oxidase in a cell-free system, activations of the oxidase at 3 T C , b o t h in the presence a n d absence of F A D , were very low; the same as those at 0°C. In order to further investigate reduced activation of" the oxidasc at a higher t e m p e r a t u r e (37°C), the stability o f solubilized m e m b r a n e s was evaluated. Figure 2 shows time-dependent changes in activation o f the oxidase after the p r e i n c u b a t i o n o f only solubilized m e m b r a n e s at 37°C, but when the reactions were carried o u t at 24~C in the presence (A) or absence (B) of F A D . Activation of the N A D P H oxidase at 24°C logarithmically decreased with increasing preincub a t i o n time o f the solubilized m e m b r a n e s at 37°C. In replotting the initial four or five data of each curve o n a log-log scale, linear regression analysis revealed lines with slopes , 0 . 7 6 a n d - 0 . 7 3 in the presence (A) and absence (B) o f F A D , respectively (data not
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0305-0491/90 $3.00 + 0.00 © 1990PergamonPress pie
Printed in Great Britain
A MICROTECHNIQUE FOR NEUTROPHIL RESPIRATORY BURST OXIDASE IN A CELL-FREE SYSTEM-CHARACTERIZATION OF OXIDASE ACTIVATION SYSTEM SHIGENOBUUMEKI* and DOROTHY M. DELISLEt *Division of Respiratory Diseases, Department of Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-01, Japan; and i'Department of Basic and Clinical Research, BCR-7, Research Institute of Scripps Clinic, La Jolla, CA 92037, USA (Received 14 November 1989)
Abstract--l. A microtechnique for quantitating human neutrophil NADPH oxidase in a cell-free system is described. 2. This spectrophotometric discontinuous (fixed time) method is less material-consuming than existing methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples. 3. Measurement of NADPH oxidase using the new method can be accomplished in a final vol of 0.15 ml. 4. In the assay, neutrophil membranes solubilized with deoxycholate were incubated for 3 min with cytosolic fractions, magnesium, sodium dodecyl sulfate, and cytochrome c in the absence of NADPH to preincubate the oxidase before the addition of the reducing agent. 5, The reaction was started by adding NADPH and 2 min later terminated by adding superoxide dismutasc. 6, The apparent K~ for NADPH obtained by the new method was almost the same as that by the authorized method (39.2 + 3.1 SD vs 36.8 _ 1.6). Activation of neutrophil NADPH oxidase was characterized using the new assay method.
INTRODUCTION Of the body's host defence mechanisms, one is enzymatic conversion by phagocytic cells of oxygen to several toxic metabolites, including the superoxide anion and hydrogen peroxide (Babior, 1978). The enzyme system responsible for this respiratory burst appears to be an N A D P H oxidase (EC 1.6.99.6) that is inactive until the cells have been stimulated by a wide variety of phagocytic, inflammatory, and surface-reactive agents (Babior, 1978; McPhail and Snyderman, 1984). Although the mechanisms that regulate the activation of this enzyme are not well understood, it has recently been reported that the N A D P H oxidase activity is associated with a low-potential cytochrome b and a flavoprotein (Henderson et al., 1987), and is therefore likely to comprise an electron-transport chain composed of a hydrogen acceptor, tlavin adenine dinucleotide (FAD), followed by a purely electron acceptor, cytochrome b (Rossi and Zatti, 1964). For a better understanding of the precise mechanisms that regulate this enzyme, many kinds of cell-free systems for N A D P H oxidase activation have recently been developed (Bromberg and Pick, 1985; McPhail et al., 1985; Curnutte et al., 1987, Abbreviations used--PiPES, 1,4-piperazinediethanesulfonic
acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; FAD, flavin adenine dinucleotide; PBS, phosphate buffered saline; SOD, superoxide dismutase; DIP, diisopropyl fluorophosphate. 461
1989; Umeki, 1990). Existing standard techniques require a large volume of materials, such as solubilized membranes, cytosolic fractions, cytochrome c, superoxide dismutase, N A D P H and others. In this manuscript, a microassay that is less materialconsuming than authorized methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples is described.
MATERIALS AND
METHODS
Horse heart cytochrome c (type III), bovine erythrocyte superoxide dismutase (SOD), fl-NADPH (type I), sodium deoxycholate, diisopropyl fluorophosphate (DFP), ATP, EGTA, PIPES, glycerol, sucrose, FAD, dextran (average tool. wt 78,000) and Dulbecco's calcium- and magnesiumfree phosphate buffered saline (D-PBS) were obtained from Sigma Chemical Company (St Louis, MO). Sodium deoxycholate was recrystallized from ethanol before use. FicollPaque and Percoll were purchased from Pharmacia P-L Biochemicals (Piscataway, N J). Other chemicals were of the highest purity available from commercial sources. Human neutrophils of ~>98% purity were prepared as previously described (McPhail et al., 1985) using acid citrate dextrose as the anticoagulant, dextran to sediment erythrocytes, and Ficoll-Paque to separate mononuclear cells from neutrophils. The cells were treated with DFP by the method of Crowley et al. (1980), except that phosphate was omitted from the buffer and the DFP concentration was reduced to 2.5 raM. DFP-treated cells were washed two times with D-PBS. The resulting cells were homogenated by nitrogen cavitation and fractionated on a discontinuous Pereoll
462
SHIGENOBU UMEKI and
gradient by a modification (Umeki and Soejima, 1990) of the method of Borregaard et al. (1983). Membranes were obtained from the Percoll gradient by pooling the 7-fraction (Borregaard et al., 1983) with a high-speed (230,000g for 2 hr at 4°C) pellet from the cytosolic fractions. The pooled membranes were suspended at a concentration of 3 x 10Scell eq/ml ( 1 . 2 + 0 . 1 S D mg prot/ml, n = 3 ) in the half-strength Borregaard's relaxation buffer [50 mM KCI, 1.5 mM NaCI, 5 mM PIPES (pH 7.3), 1.75 mM MgC12, 0.5 mM ATP, 0.62 mM EGTA (Borregaard et al., 1983)] containing 0.34 M sucrose. The suspension was divided into aliquots and stored at -70°C. The supernatant from the 230,000 g spin [designated "cytosol", which contained 7.5 × 107cell eq/ml (1.4±0.1 SD nag prot/ml, n = 3)] was divided into aliquots and stored at -70°C; this material served as the source of cytosolic activation factor. Membranes were solubilized by the procedure described by Curnutte et al. (1987). The membrane suspension thawed at room temperature was mixed with an equal volume of extraction buffer [1 mM NAN3, 0.0017 mM CaCI 2, 20 mM sodium glycinate (pH 8.0), 2.33% sodium deoxycholate (w/v), and 50% glycerol (v/v)], vortexed briefly, incubated on ice for 30 min with occasional agitation, and finally centrifuged at 435,000g for 1 hr at 4~C in a Beckman TL-100 tabletop ultracentrifuge with a TL-100.2 rotor, using polycarbonate tubes. The dormant oxidase was found in the supernatant, whose protein concentration was 0.53 + 0.11 SD mg prot/ml (n = 3). Superoxide production was determined by a discontinuous assay in which SOD-inhibitable cytochrome c reduction was followed by scanning between 530 and 570 nm in a double-beam spectrophotometer (Umeki, 1990). Assay mixtures contained 0.1 mM cytochrome c, 3.6raM MgC12, 89mM KCI, 2.TmM NaCI, 0.5mM PIPES (pH 7.3), 0.9mM ATP, 1.2raM EGTA, 1.2 × 106cell eq of cytosol, 3 × l0 Scell eq of solubilized membranes, 0.04 mM sodium dodecyl sulfate and 0.16 mM NADPH, with alterations as noted in the Table l and Fig. legends, in a total vol of 0.15 ml. The reference cuvette contained 8 p I of SOD. Basically, reaction mixtures without NADPH were preincubated at 24°C for 3 min after the addition of 10#l of sodium dodecyl sulfate. Then the reactions were initiated at 24°C by adding 5 pl of NADPH and 2 min later the reaction of a sample cuvette was terminated by adding 5 pl of SOD (1 mg/ml of distilled water). After adding 600 ,ul of distilled water into the reference cuvette and 595 #1 of water to the sample cuvette, measurement of the cytochrome c reduction
6i
"E
DOROTHY
M. DELISLE
was done by scanning between 530 and 570 nm. The height of the peak of 5 5 0 n m represents the absorbance due to superoxide-dependent cytochrome c reduction. 02- production was calculated on the basis of the height of the peak at 550 nm using an extinction coefficient of 19.6 m M - ~ cm -t (Yonetani, 1965). The protein concentration was determined by the method of Smith et al. (1985), using the bicinchoninic acid protein assay reagent from Pierce Chemical Company 'Rockford,
IL),
RESULTS
AND
DISCUSSION
This new discontinuous assay is suitable for investigating t e m p e r a t u r e - d e p e n d e n t changes in N A D P H oxidase because of the use of a n i n c u b a t o r with thermometer. Figure 1 shows temperatured e p e n d e n t changes in activation of neutrophi] N A D P H oxidase evaluated using this new disc o n t i n u o u s microassay. Activation of N A D P H oxidase in a cell-free system was the highest at 24°C in the presence or absence o f 0.5 # M F A D . In addition to the presence o f an optimal t e m p e r a t u r e (24°C) in activation (reconstitution) o f the N A D P H oxidase in a cell-free system, activations of the oxidase at 3 T C , b o t h in the presence a n d absence of F A D , were very low; the same as those at 0°C. In order to further investigate reduced activation of" the oxidasc at a higher t e m p e r a t u r e (37°C), the stability o f solubilized m e m b r a n e s was evaluated. Figure 2 shows time-dependent changes in activation o f the oxidase after the p r e i n c u b a t i o n o f only solubilized m e m b r a n e s at 37°C, but when the reactions were carried o u t at 24~C in the presence (A) or absence (B) of F A D . Activation of the N A D P H oxidase at 24°C logarithmically decreased with increasing preincub a t i o n time o f the solubilized m e m b r a n e s at 37°C. In replotting the initial four or five data of each curve o n a log-log scale, linear regression analysis revealed lines with slopes , 0 . 7 6 a n d - 0 . 7 3 in the presence (A) and absence (B) o f F A D , respectively (data not
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