260

Biochimica et BiophysicaActa, 1040 (1990) 260-266 Elsevier

BBAPRO 33714

NADPH" nitroblue tetrazolium reductase found in plasma membrane of human neutrophil Yukio Nisimoto and Hidetsugu Otsuka-Murakami Department of Biochemistry, Aichi Medical University, Nagakute, Aichi (Japan) (Received 12 February 1990)

Key words: Tetrazolium reductase; Neutrophil; NADPH; (Human)

After phorbol 12-myristate 13-acetate (PMA) stimulation the increase of NADPH: nitroblue tetrazolium reductase activity in the plasma membrane almost corresponded with the stimulated activity of respiratory burst oxidase. Solubilization of plasma membranes from PMA-activated neutrophils with n-octyl glucoside resulted in high recoveries of the two enzymatic activities. When solubilized plasma membrane was subjected to non-denaturing polyacrylamide gel electrophoresis in the presence of 35 mM n-octyl glucoside, we could see three major bands stained with NADPH-dependent nitroblue reductase activity giving molecular masses of approx. 95, 45 and 40 kDa, respectively. Activity was specific for NADPH but not for NADH. These bands also stained weakly in the plasma membranes obtained from resting cells. The activities for NADPH oxidase and nitroblue tetrazolium reductase were found to elute as a very similar protein peak on an anion-exchange HPLC, at about 0.32 M KCI. This elution peak also contains 45 and 40 kDa proteins showing NADPH: nitroblue tetrazolium reductase activity.

Introduction Reliable information regarding the enzymatic reaction responsible for the respiratory burst and mechanism of activation is now of central interest to the understanding of physiological function in neutrophil. The key enzyme initiating the oxidase metabolism is the respiratory burst oxidase (or NADPH oxidase), which produces superoxide anion. However, this enzyme and its related regulatory mechanism are far more intricate than was previously thought. Because of the difficulty in its purification, the following possibilities were suggested: (1) the oxidase was considerably labile after solubilization: (2) dissociation constant of flavin as a cofactor is fairly high; and (3) the purified NADPH oxidase reported so far was only one component and not entirely sufficient to comprise a functional unit

Abbreviations: fMLP, formyl-methionyl-leucyl-phenylalanine;PMA, phorbol 12-myristate 13-acetate; SDS, sodium dodecyl sulfate; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; DFP, diisopropyl fluorophosphate; SOD, superoxide dismutase; NBT, nitroblue tetrazolium; HPLC, high-performance liquid chromatography. Correspondence: Y. Nisimoto, Department of Biochemistry, Aichi Medical University, Nagakute, Aichi 480-11, Japan.

producing superoxide anion. Thus, the respiratory burst oxidase has been considered to be a transmembraneelectron transport system consisting of two or more components as its catalytic center, for example, a flavoprotein [1-4], cytosolic NADPH-binding protein [5-8] and b-type cytochrome [9,10]. We have already reported that membrane-associated NADH dehydrogenase from bovine neutrophil catalyzes the reductions of ferricyanide, cytochrome b5 and nitroblue tetrazolium [11]. A menadione-stimulated pyridine nucleotide oxidase was also highly purified from bovine neutrophil membrane vesicles and characterized by comparing the properties of NADPH oxidase reported to date [12]. However, these enzymes were likely to be distinct from the NADPH oxidase in reference to their catalytic properties [12]. In a recent study, Kakinuma et al. demonstrated that whole membrane fraction separated from stimulated pig neutrophils gives a protein band staining with N A D P H : N B T reductase activity focused at pI 5.0 on isoelectric focusing gels, and the enzyme showed high specificity for NADPH and similar characteristics to the respiratory burst oxidase. The extracted NADPH: NBT reductase contained FAD, giving a high turnover number of O~--generating activity and its molecular mass was approximately 68 kDa [13]. In the present investigation, plasma membrane was isolated from human neutrophils, and the membrane-bound proteins

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261 were successfully separated on non-denaturing PAGE in the presence of n-octyl glucoside, and we identified three major bands stained with N A D P H : N B T reductase activity whose molecular masses were approx. 95, 45 and 40 kDa, respectively. Since HPLC on DEAE5PW also demonstrated 45 and 40 kDa proteins in a very similar protein peak possessing NADPH oxidase activity, the possibility that 45 and 40 kDa components are involved in respiratory burst oxidase system is also discussed.

Experimental procedures Materials Polyacrylamide gel electrophoresis standards, NADH, NADPH, deoxycholate, nitroblue tetrazolium, superoxide dismutase, ferricytochrome c, n-octyl glucoside, PMA, fMLP, fatty acid-free bovine serum albumin, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride were obtained from Sigma (St. Louis, MO). HESPAN (6% Hetastarch in 0.9% NaC1) and lymphocyte separation medium (LSM, 6.2% Ficoll plus 9.4% sodium diatrizoate) were obtained from American Critical Care Division of American Hospital Supply (McGaw Park, IL) and Biovetics (Kensington, MD), respectively. All other reagents were of the best grade commercially available.

Isolation of human neutrophils Normal human neutrophils were obtained from peripheral blood obtained by phlebotomy [14]. Erythrocytes were sedimented with HESPAN and the mononuclear cells were removed from the resulting supernatant by centrifugation through lymphocyte separation medium [15]. The resulting cells were greater than 95% neutrophilic granulocytes. Neutrophils were resuspended in phosphate buffer (pH 7.0) containing 0.6 mM CaC12, 1.5 mM KH2PO 4, 2.6 mM KC1, 0.5 mM MgC12, 136 mM NaC1, 8 mM Na2HPO 4 and 5.5 mM glucose.

Isolation of plasma membranes from human neutrophils Plasma membrane preparation was performed as described previously [16]. Cells (4.108) in 5 ml of ice-cold relaxation buffer (20 mM potassium phosphate buffer, pH 7.0, containing 0.13 M NaC1, 0.34 M sucrose, 1 mM EDTA, 0.1 mM PMSF and 10/~M DFP) were disrupted by sonication at 20 W for 45 s with constant cooling at 3°C. The sonicate was centrifuged (80 × g, 5 min, 4°C) to remove nuclei and unbroken cells. The supernatant was loaded onto 30-50% discontinuous sucrose gradient and centrifuged at 150000 ×g for 1 h. Plasma membrane fraction was separated, washed and resuspended in 25 mM phosphate buffer (pH 7.6) containing 35% glycerol and 20/~M FAD.

Solubilization of plasma membranes The plasma membranes were suspended at a final concentration of 1.2 mg protein per ml in solubilizing

mixture consisting of 35 mM octylglucoside, 35% glycerol, 20 #M FAD and 25 mM phosphate buffer (pH 7.6). The suspension was stirred continuously at 3°C for 6 h and then centrifuged at 105 000 x g for 1 h. The supernatant was used for the assay of NADPH oxidase and N A D P H : N B T reductase activities and for separation of proteins on polyacrylamide gel electrophoresis.

Determination of enzyme activity Superoxide anion production was determined by measuring the SOD-inhibitable reduction of cytochrome c, which was estimated from the difference between the amount of cytochrome c reduction in the absence and presence of 89/~g/ml SOD, as described previously [17]. NADPH-dependent cytochrome c reductase activity was quantitated at 36 °C by measuring the absorbance at 550 nm, using absorption coefficient of 18.5 mM -1 .cm -l (reduced-minus-oxidized cytochrome c). Nitroblue tetrazolium reduction was measured in 1.2 ml of a reaction medium consisting of 0.5 mM NBT, 0.2 mM NADPH, 25 mM phosphate buffer (pH 7.0), 8 mM Mg 2÷ and either cytosol or supernatant of the solubilized membrane (0.03-0.05 mg of protein). The absorbance change at 550 nm was measured at 36 o C for 10 min. NADPH : NBT reductase actNity was determined by using absorption coefficient of 28.6 mM -l • cm -1 at 550 nm [18].

Polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis was carried out according to the method of Rudolph and Krueger [19]. Non-denaturing electrophoresis was also performed in the presence of 35 mM octyl glucoside at 3°C according to the methods of Ornstein and Davis [20,21]. Following electrophoresis for 6 h, the gels were cut into two fragments, one of which was used for protein staining with Coomassie brilliant blue R-250 and the other was stained for N A D P H (or NADH):NBT reductase activity in the reaction mixture containing 50 mM phosphate buffer (pH 7.4), 0.4 mM NADPH (or 0.4 mM NADH), 0.5 mM NBT and 10% glycerol for 2 h at 25 o C.

High-performance liquid chromatography HPLC colunm of TSK gel DEAE-5PW (7.5 x 75 mm) from Tosoh was used. The column was equilibrated with 25 mM Tris-HC1 buffer (pH 7.6) containing 35 mM octyl glucoside and 10% glycerol. Approx. 1.2 ml (1.12 mg of protein) of solubilized plasma membranes (105 000 x g, supernatant), which was prefiltered using an HPLC nylon filter (3 mm diameter, 0.45 #m pore size), was injected. The column was eluted with equilibration buffer with or without KC1 linear gradient (0-0.5 M) at 1 ml/min, absorbance at 280 nm recorded and fractions of 1 ml were collected. The HPLC system

262 Mr(kDa)

M

1

2

3

4

of NADPH-dependent superoxide anion formation and NBT reduction.

Results

480 240 136

68

45 31

Fig. 1. Non-denaturing polyacrylamide gel electrophoresis of solubilized plasma membranes and NADH:NBT reductase activity. The supernatant (105000x g, 1 h) of solubilized plasma membranes (0.15 mg of protein) was subjected to electrophoresis (10% gel) in the presence of 35 mM octyl glucoside. After electrophoresis the gels were used for protein staining (lanes 1 and 2) with Coomassie brilliant blue and enzymatic activity staining for NADH:NBT reductase (lanes 3 and 4). The reductive reaction was performed in 50 mM sodium phosphate buffer (pH 7.4) containing 0.5 mM NBT, 0.4 mM NADH and 10% glycerol at 25 o C for 2 h. The arrow indicates the NBT-stained bands. Gel lanes 1 and 3 were the membranes from PMA-activated cells and lanes 2 and 4 were from resting ceils. Lane M contained marker proteins of known molecular mass. The following proteins were used as standards; urease tetramer (Mr, 480 kDa), urease dimer (240 kDa), bovine serum albumin dimer (136 kDa), bovine serum albumin monomer (68 kDa), egg albumin (45 kDa) and carbonic anhydrase (31 kDa). was at r o o m t e m p e r a t u r e , b u t i n d i v i d u a l fractions were transferred to 3 ° C i m m e d i a t e l y following collection. C o l u m n eluate fractions were a s s a y e d for the activities

H u m a n n e u t r o p h i l s were briefly i n c u b a t e d with a n d w i t h o u t 0.1 # M P M A (or 0.1 m M f M L P ) a n d were then f r a c t i o n a t e d to o b t a i n cytosol, p l a s m a m e m b r a n e s a n d granules with d i f f e r e n t N A D P H : N B T reductase and r e s p i r a t o r y b u r s t o x i d a s e activities. T r e a t m e n t of these m e m b r a n e fractions with o c t y l g l u c o s i d e p r o v i d e d soluble N A D P H : N B T r e d u c t a s e activity as well as N A D P H o x i d a s e activity. T h e two e n z y m a t i c activities of solubilized p l a s m a m e m b r a n e s f r o m d o r m a n t cells were low, but, r e s p i r a t o r y b u r s t o x i d a s e activity of the p l a s m a m e m b r a n e is g r e a t l y raised b y P M A - s t i m u l a t i o n of the cells w h i c h a p p e a r s to b e c o m p a r a b l e with the increase of N A D P H : N B T r e d u c t a s e activity ( T a b l e I). T h e s t i m u l a t o r y effects of s u p e r o x i d e a n i o n g e n e r a t i o n a n d N B T r e d u c t i o n were m o s t r e m a r k a b l y o b s e r v e d in p l a s m a m e m b r a n e , a n d the low N A D P H : N B T red u c t a s e activity was a l m o s t equally seen in cytosol f r o m b o t h resting a n d a c t i v a t e d n e u t r o p h i l s , whereas S O D insensitive N A D P H : c y t o c h r o m e c r e d u c t a s e of each fraction gave o n l y slight a c t i v a t i o n in s t i m u l a t e d cells. S e p a r a t e d soluble p l a s m a m e m b r a n e (0.05-0.15 mg of p r o t e i n ) was l o a d e d on p o l y a c r y l a m i d e gel electrop h o r e s i s in the p r e s e n c e o f 35 m M octyl glucoside a n d a f t e r e l e c t r o p h o r e s i s t h e gels w e r e s t a i n e d for NADH:NBT r e d u c t a s e activity (Fig. 1). O n l y one s t a i n e d b a n d w i t h N A D H : N B T r e d u c t a s e activity was seen with h i g h l y a g g r e g a t e d form whose a p p a r e n t m o l e c u l a r m a s s was m o r e t h a n 480 k D a , as d e s c r i b e d p r e v i o u s l y b y N i s i m o t o et al. [11]. T h e r e was no cons p i c u o u s d i f f e r e n c e in the intensities of the N A D H - d e p e n d e n t N B T s t a i n e d b a n d s b e t w e e n the solubilized m e m b r a n e s f r o m s t i m u l a t e d a n d u n s t i m u l a t e d neutrophils. I n contrast, the staining intensities of N A D P H - d e p e n d e n t N B T r e d u c t a s e activity in solubilized p l a s m a m e m b r a n e s f r o m a c t i v a t e d cells were clearly higher t h a n

TABLE I Respiratory burst oxidase and NADPH : N B T reductase activities of each fraction isolated from both resting and PMA-activated neutrophils

Separated plasma membranes and granules were solubilized with 35 mM n-octylglucoside, respectively, and their supernatant (105 000 x g, 1 h) were used for the assays of NADPH:NBT reduetase and NADPH:cytochrome c reductase activities at 36 °C for 10 rain. Cytosolic fraction (0.03-0.05 mg of protein) was also assayed for the activities. Fraction

NADPH : NBT reductase activity NADPH : cytochrome c reductase activity (nmol/min per nag protein) (nmol/min per mg protein) rest

Cytosol Solubilized granules Solubilized plasma membranes

2.1 6.6 4.9

PMA-activated 1.8 12 33

SOD-sensitive

SOD-insensitive

rest

PMA-activated

rest

PMA-activated

0.59 0.77 1.3

1.2 4.3 38

2.1 0.53 0.85

4.3 2.6 1.8

263 Mr" (kDo)

1

2

3

4

5

6

I 95 II~

45 , 40 II¢

Fig. 2. Gel electrophoresis of solubilized plasma membranes in the presence of octylglucoside and NADPH-dependent NBT reductase activity on gel. The supernatant (105000x g, 1 h) of solubilized plasma membranes was loaded onto 7.5% gel containing 35 mM of octylglucoside and then subjected to electrophoresis for 6 h at 3° C. After electrophoresis the gels were stained for NADPH:NBT reductase activity. The arrows on the left side denote the NBT-stained bands. Lanes 1, 2 and 3 are solubilized plasma membranes from PMA-activated neutrophils (0.12, 0.09 and 0.06 mg) and gel lanes 4, 5 and 6 are from unstimulated cells (0.05, 0.10 and 0.15 mg). The other conditions were the same as Fig. 1 except that 0.4 mM NADPH was added instead of NADH to the reaction mixture.

N A D P H : N B T reductase and respiratory burst oxidase (40 + 10 FM) are close, while those for N A D H of both N A D H : N B T reductase and N A D H dehydrogenase (6 ~M) appear to coincide [7]. Anion-exchange chromatography on DEAE-5PW shows the elution profile of plasma membrane-associated proteins in the presence of 35 m M octyl glucoside and 10% glycerol (Fig. 4). One poorly resolved protein peak appearing at high molarity of KC1 (0.32-0.35 M) gave both N A D P H oxidase and N B T reductase activities but no further peaks having these enzymatic activities were seen. On this H P L C column the two enzymatic activities were observed at very similar positions, corresponding to proteins with acidic isoelectric points. This peak fraction (retention times: 45, 46 and 47 min) possessing the two activities exhibited two distinct N A D P H : N B T staining bands with molecular masses of approx. 45 and 40 k D a (Fig. 4, inset). The average

1.00

7_~ 0.75

_c in those from resting cells (gel lanes 1 - 3 vs. 4 - 6 in Fig. 2). The distinct difference of N A D P H : N B T reducing activity between the resting and stimulated membranes found on the gel coincided with the results seen in Table I. Thus, we could see three major staining bands in extracts of plasma membranes from stimulated cells, whose molecular masses were approx. 95, 45 and 40 kDa, respectively. The two proteins with 45 and 40 k D a showed markedly stronger N B T reductase activity in the activated plasma membranes, although the 95 k D a protein presented almost the same intensity in the membranes from stimulated and unstimulated cells. In addition, both 45 and 40 kDa-stained bands were exclusively seen in the membrane rather than cytosolic fraction after stimulation (data not shown). The solubilized plasma membranes from PMAactivated neutrophils exhibited hyperbolic kinetics with respect to both N A D P H and N A D H concentrations in the N B T reductase assay. K m values for N A D P H and N A D H were determined from Lineweaver-Burk plots (Fig. 3) to be 78 and 16 FM, respectively, and the maximum N B T reductase activity (Vm~x) showed almost the same value as N A D H , when compared with N A D P H . The relative pyridine nucleotide specificity of the N B T reductase of stimulated plasma membrane suggests that the K m values for N A D P H of both

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NADPH: nitroblue tetrazolium reductase found in plasma membrane of human neutrophil.

After phorbol 12-myristate 13-acetate (PMA) stimulation the increase of NADPH:nitroblue tetrazolium reductase activity in the plasma membrane almost c...
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