Biochimica et Biopl~vsica Acta. 1074(1991) 12-18 ' 1991 ElsevierScience PublishersB.V.0304-4165/91/$03.50 A D O N I S 030441659100145Q
BBAGEN 23499
Inhibition of the oxidation of hydroxyl radical scavenging agents after alkaline phosphatase treatment of rat liver microsomes S u s a n a P u n t a r u l o 1 a n d A r t h u r I. C e d e r b a u m
2
i pto~lca I Chemisto ' Dicision. School of Pharmac.r and Biochemisto'. Unicersi(r of Buenos Aires. Buenos Aires (Argentina) and -" Department of Biochemistr)'. Mount Sinai School of Medicine. New York, N Y (U.S.A.)
(Received 13 July 1990) (Revised manuscriptreceived l0 December1990)
Key words: CytochromeP-450: NADPH-cytochrome-creductase: Phosphatase: Microsome;(Rat liver) Treatment of rat liver microsomes with alkaline phosphatase results in a loss in the F M N but not the FAD flavin prosthetic group of NAUPl-l-cytochrome P-450 reductase (Taniguchi, H. and Pyerin, W. (1987) Biochim. Biophys. Acta 912, 295-307). Experiments were carried out to evaluate the effect of preventing electron transfer from the FADH 2 to FMN component of the reductase, and subsequent mixed functioa oxidase activity, on reduction of te~ic chelates, production of H202, and the generation of "OH-like species by microsomes. Treatment with alkaline phosphatase was confirmed to decrease NADPH-cytochrome c, but not NADPH-ferricyanide, reductase activity by microsomes and by purified NADPH cytochrome P-450 reductase. The oxidation of hydroxyl radical scavenging agents by microsemes and reductase was decreased by the alkaline phosphatase treatment in accordance with the decline in cytochrome c reductase activity. This decrease in hydroxyl radical production occurred in the presence of various ferric chelate catalysts. Rates of microsomal reduction of the ferric chelates were also inhibited after alkaline pbosphatase treatment. Production of H202 was decreased in accordance to the fall in eytochrome c reductase activity and - O H production. Rates of H202 production appeared to be rate-limiting for the overall generation of . O H as the addition of an external H 2Oz-generating system stimulated "OH production as well as prevented the decline in , O H production caused by the alkaline phosphatase treatment. These results suggest that both the FAD and F M N flavin prosthetic groups of the reductase contribute towards the reduction of various ferric chelates. However, loss of the F M N component and activities dependent on electron transfer from this prosthetic group result in a decrease in H 2 0 z production, which appears to be responsible for the decline in the generation of -OH-like species by microsomes after treatment with alkaline phosphatase.
Introduction NADPH-cytochrome P-450 reductase contains 1 tool each of FMN and FAD per mole of enzyme [1-5]. The enzyme functions to transfer electrons from N A D P H to cytochrome P-450. F A D appears to be the site for accepting electrons from NADPH, followed by transfer to FMN and subsequently to cytochrome P-450 [6-9]. The enzyme can reduce other electron acceptors such as cytochrome c and ferricyanide. Dialysis of the purified reductase against potassium bromide depletes F M N but not FAD from the reductase. This depletion is associ-
Correspondence: A.I. Cederbaum, Department of Biochemistry.Box 11)20, Mount Sinai School of Medicine.One Ciustave L. Levy Place, New York. NY 10029.U.S.A.
ated with an almost complete loss of NADPH-cytochrome c reductase activity, whereas NADPH-ferricyanide reductase was reduced less than 30% [5,7,10]. It appears that F M N is necessary for the reduction of cytochrome c but not for ferricyanide reduction [7,8]. N A D P H can readily reduce the FMN-depleted enzyme, forming F A D H 2 [7,8,11]. Recently, Taniguchi and Pyerin [12,13] showed that incubation of rabbit liver microsomes with alkaline phosphaiase caused a decrease in NADPH-cytochrome c reductase, but not NADPH-ferricyanide reductase activity. This decrease could be correlated with a loss of NADPH-dependent monooxygenase activities. The alkaline phosphatase effect was relatively specific for N ADPH-cytochrome P-450 (c) reductase as the content of cytochromes P-450 and b s or activities of NADH-cytochrome b s (c) reductase were not affected [13]. Flavin
analysis showed a decrease of FMN without any effect on FAD and it was concluded that the alkaline phosphatase inactivated the reductase by interacting with and digesting the FMN component of the reductase [131. In the presence of iron, microsomes can oxidize a variety of hydroxyl radical (-OH) scavengers by a NADPH-dependent reaction [14]. The basic reaction appears to require reduction of ferric iron to the ferrous redox state and the production of H_,O, by the microsomal electron transfer chain, followed by a Fenton-type of reaction to yield -OH-like species [14]. The ability to allow electron reduction to the FAD but not the FMN flavin prosthetic group of NADPH-cytochrome P-450 reductase by alkaline phosphatase treatment, afforded an opportunity to carry out experiments to evaluate a role for each of these flavin components on the basi,: reaction scheme described above. Experiments were carried out to determine the effect of treating rat liver microsomes with alkaline phosphatase on the NADPHdependent reduction of ferric chelates, on the production of H20~, and on the generation of -OH-like species. Some of the results with microsomes, e.g., oxidation of -OH scavengers, were exttmded to the purified reductase.
Materials and Methods Liver microsomes were prepared from male Sprague-Dawley rats weighing about 150-200 g. The microsomes were prepared by differential centrifugation, washed twice with 125 mM KCI and stored at - 7 0 ° C until utilized. Alkaline phosphatase from Escherichia coli (Type 11I) was purchased from Sigma Chemicals and was used without further purification. NADPH-cytochrome P-450 reductase was purified by a slight modification of the method of Guengerich and Martin [15], as previously described [16]. Final preparations had specific activities ranging from 18 to 24 units per mg protein. Alkaline phosphatase treatment of the microsomes was carried out at 37°C essentially as described by Taniguchi and Pyerin [13]. Microsomes (about 2 mg protein) were incubated in 50 mM Tris-HCI buffer (pH 8.3) in the absence or presence of alkaline phosphatase (about 2 units per mg microsomal protein for most experiments) for varying time periods (30 min for most experiments) in a shaking water bath. At the indicated time, aliquots were removed and the control or the treated microsomes were added to the specific reaction system being studied, followed by the NADPH-generating system to initiate the reaction. NADPH-cytochrome c reductase activity was measured in a system containing 0.3 M potassium phosphate buffer (pH 7.7), 0.05 mM cytochrome c, and 0.1 mM NADPH [17]. NADPH-ferricyanide reductase activity was determined
in a system containing 0.1 M potassium phosphate buffer (pH 7.4), 1 mM potassium ferricyanide, and 0.1 mM NADPH [13] and activity determined using an extinction coefficient at 420 nm of 1.02 r a M ~ c m [181. The production of -OH or compounds with the oxidizing power of -OH by microsomes was assayed by measuring the generation of ethylene gas from 2-keto4-thiomethylbutyrate (KMB) or of formaldehyde from DMSO or t-butyl alcohol. Reactions were carried out essentiall 3' as previously described [19-21]. For most experiments, 50 /.tM ferric-EDTA was utilized as the iron catalyst since this is the most reactive iron complex for the promotion of -OH-like species Izy microsomes [22,231. The production of formaldehyde ~as determined by the Nash reaction [24] and the generation of ethylene was assayed by a head space gas chromatography procedure. All values were corrected for zerotime controls which contained acid prior to the addition of the NADPH-generating system. The production of HzO z was determined by measuring the generation of formaldehyde from the oxidation of methanol by the catalase-compound l complex [25]. The reduction of ferric chelates was monitored by the increase in absorbance at 520 nm when microsomes. 0.05 mM ferric chelate. 0.1 mM NADPH and 5 mM 2,2'-bipyridyl in 0.1 M phosphate buffer (pH 7.4) were mixed together [26]. Microsomal NADPH oxidation was determined from the decrease in absorbance at 340 nm of a reaction system containing 0.1 M phosphate buffer (pH 7.4), 0.05 mM ferric chelate, and 0.1 mM NADPH. The ferric complexes were prepared by dissolving ferric ammonium sulfate in 0.1 M HCI, and then diluting with the respective chelator to the appropriate stock concentration. Ferric-ATP was used as a 1 : 20 complex, whereas the other ferric complexes were utilized as a 1 : 2 complex. The phosphate buffer and the water utilized to prepare all solutions were passed through columns of chelex-100 resin to remove metals such as iron. All experiments were carried out in duplicate, with at least two different microsomal preparations: variability generally did not exceed 10%. Where indicated, results refer to mean + standard error.
Results Results in Fig. 1 confirm the observations of Taniguchi and Pyerin [13] that treatment of rat liver microsomes with alkaline phosphatase results in a decrease in NADPH-cytochrome c reductase activity whereas NADPH-ferricyanide reductase activity is not significantly affected. Different amounts of the NADPH-cytochrome c reductase were lost depending on the units of alkaline phosphatase utilized (Fig. IA) or depending on the length of incubation with a fixed amount of alkaline phosphatase (Fig. IB). In general, treatment with about
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30 60 Incubation time (rain)
90
Fig. l. (A) Effect of alkaline phosphatase treatment on NADPH-cytochrome c reductase (o) and NADPH-ferricyanide reductase (&) activity. Microsomes were treated with varying units of alkaline phosphatase or buffer for 60 rain and enzyme activities measured. (B) Microsomes were incubated in the absence of (o. 4) or presence (e, A) of 2 units per mg protein of alkaline phosphatase for 15, 30, 60, or 90 rain, followed by assays of NADPH-cytochrome c (O. e) or NADPH-ferrieyanide (n, A) reductase activity.
2 units of alkaline p h o s p h a t a s e p e r m g m i c r o s o m a l p r o t e i n for 30 rain p r o d u c e d a b o u t a 50% loss o f 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 activity, as c o m p a r e d to m i c r o s o m e s i n c u b a t e d with the ' I r i s b u f f e r alone. S o m e loss of 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 activity ( a b o u t 20%) was o b s e r v e d for s a m p l e s i n c u b a t e d in b u f f e r alone for 15 to 60 m i n as c o m p a r e d to m i c r o s o m e s kept o n ice. In all e x p e r i m e n t s d e s c r i b e d b e l o w , the effect of the alkaline p h o s p h a t a s e t r e a t m e n t w a s a l w a y s c o m p a r e d to s a m p l e s i n c u b a t e d with b u f f e r a l o n e for the s a m e time period. A c c o m p a n y i n g the loss o f 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 activity w a s a c o m -
p a r a b l e loss o f N A D P H o x i d a t i o n b y the m i c r o s o m e s , b o t h in the a b s e n c e o r p r e s e n c e o f f e r r i c - E D T A , as m e a s u r e d b y the d e c r e a s e in a b s o r b a n c e a t 340 n m . T h e p r o d u c t i o n o f e t h y l e n e f r o m K M B w a s u s e d to a s s a y for the g e n e r a t i o n o f . O H - l i k e species b y m i c r o s o m e s in the p r e s e n c e o f N A D P H a n d f e r r i c - E D T A . T r e a t m e n t o f the m i c r o s o m e s with a l k a l i n e p h o s p h a t a s e for 15, 30, o r 60 m i n resulted in a p r o g r e s s i v e loss o f e t h y l e n e g e n e r a t i o n as c o m p a r e d to c o n t r o l s i n c u b a t e d w i t h o u t a l k a l i n e p h o s p h a t a s e (Fig. 2A). T h e p e r c e n t loss o f m i c r o s o m a l o x i d a t i o n o f K M B to e t h y l e n e directly c o r r e l a t e d with the p e r c e n t loss o f N A D P H - c y t o -
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Fig. 2. (A) Effect of alkaline phosphatase treatment on microsomal oxidation of KMB to ethylene. Microsomes were treated with 2 units of alkaline phosphatase per mg protein for 15 (o), 30 (O), or 60 rain (zx). or with buffer (e), followed by assays for KMB oxidation for either 5 or 10 rain. Control values for microsomes incubated with buffer for 15, 30, or 60 min were similar ( + 87o)and were therefore grouped together. (B) Correlation between inhibition of NADPH-cytochrome c reductase activity by alkaline phosphatase and inhibition of KMB oxidation to ethylene. The various points were obtained for different incubation times with increasing units of alkaline phosphatase activity, followed by assays of NADPH-cytochrome c reductase activity and oxidation of KMB.
15 lOO
~
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"~ 60 E ~ 4O o_ 20
15
30
45
Incubation time (rnin)
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Fig. 3. Comparison of the effec~ of treatment with alkaline phosphatase on NADPH-cytochrome c (o) or NADPH-ferricyanide (O1 reductase activity and oxidation of the .OH scavengers DMSO (U) or t-butyl alcohol (~,). Microsomes were incubated with 2 units of alkaline phosphatase per mg protein for 15. 30. or 60 min, or with buffer, followed by assays of reductase activity or production of formaldehyde from the oxidation of 33 mM DMSO or 50 mM t-butyl alcohol in the presence of 50 t~xi f~,ric-EDTA. Control values were grouped together since they were ~imilar for the three incubation periods. Control values were about 165 and 450 nmol per min per mg protein for NADPH-cytochrome e and [erricyanide reductase activities. respectively, and about 4 to 5 nmol per rain per mg microsomal protein for DMSO and t-butyl alcohol oxidation to formaldehyde. c h r o m e e r e d u c t a s e activity (Fig. 2B). T o d e t e r m i n e if the effect o f alkaline p h o s p h a t a s e t r e a t m e n t o n m i c r o s o m a l " O H g e n e r a t i o n w a s u n i q u e for K M B as the c h e m i c a l scavenger, the o x i d a t i o n of o t h e r . O H s c a v e n g i n g a g e n t s w a s e v a l u a t e d . Previous studies h a v e s h o w n t h a t f o r m a l d e h y d e is a m a j o r p r o d u c t w h i c h arises f r o m the i r o n - d e p e n d e n t o x i d a t i o n of D M S O or t-butyl alcohol b y m i e r o s o m e s [20,21]. I n c u b a t i o n o f m i c r o s o m e s with 2 unit~ o f alkaline p h o s p h a t a s e p e r m g p r o t e i n for 15, 30, o r 60 m i n resulted in progressive loss o f f o r m a l d e h y d e o x i d a t i o n f r o m either D M S O o r t-butyl alcohol (Fig. 3). T h e decrease in o x i d a t i o n of these - O H s c a v e n g i n g a g e n t s a g a i n p a r a l l e l e d the d e c r e a s e in activity o f N A D P H - c y t o c h r o m e c reductase, whereas
N A D P H - ' f e r r i c y a n i d e r e d u c t a s e activity r e m a i n e d u n a f fected (Fig. 31. Similar e x p e r i m e n t s were carried o u t with the purified r c d u c t a s e to ascertain t h a t the results with microsomes treated with alkaline p h o s p h a t a s e were likely due to effects o n the reductase. T r e a t i n g the purified red u c t a s e for 20 o r 4 0 min with alkaline p h o s p h a t a s e c a u s e d decreases of 71 or 78%, respectively, of 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 activity, w h e r e a s N A D P H - f e r r i c y a n i d e r e d u c t a s e activity w a s not affected (Table 1). G l y c e r o l w a s present d u r i n g the i n c u b a t i o n at 3 7 ° C to help m a i n t a i n c o n t r o l r e d u c t a s e activity. T h e p r o d u c tion of ethylene f r o m K M B , as catalyzed b y ferricE D T A , w a s d e c r e a s e d b y the alkaline p h o s p h a t a s e t r e a t m e n t in direct r e l a t i o n s h i p to the fall in 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 activity (Table 1). Previous e x p e r i m e n t s h a d indicated t h a t certain ferric chelates s u c h as f e r r i c - E D T A a n d f e r r i c - D T P A were effective iron c a t a l y s t s for the g e n e r a t i o n of - O H - l i k e species by m i c r o s o m e s , w h e r e a s iron chelates s u c h as f e r r i c - A T P or ferric-citrate were m u c h less effective c a t a l y s t s [22,23]. T o e v a l u a t e w h e t h e r the inhibition of m i c r o s o m a l o x i d a t i o n o f - O H scavenger b y alkaline p h o s p h a t a s e t r e a t m e n t w a s specific o n l y for ferricE D T A as the iron catalyst, e x p e r i m e n t s with o t h e r ferric chelates were c a r r i e d out. T r e a t m e n t with alkaline p h o s p h a t a s e resulted in a similar degree of loss of K M B o x i d a t i o n r e g a r d l e s s o f the a d d e d ferric chelate (Table Ill. W h e n ferric i r o n is a d d e d , a n initial event in the u l t i m a t e g e n e r a t i o n o f " O H b y m i c r o s o m e s is r e d u c t i o n to the ferrous r e d o x state. T h e inhibition of m i c r o s o m a l - O H p r o d u c t i o n a f t e r t r e a t m e n t with alkaline p h o s p h a t a s e c o u l d reflect inhibition o f the r e d u c t i o n o f the a d d e d ferric chelate. In the p r e s e n c e o f N A D P H , micros o m e s c a t a l y z e d the r e d u c t i o n of various ferric chelates ( T a b l e 111). T r e a t m e n t with alkaline p h o s p h a t a s e resulted in a n i n h i b i t i o n o f the r e d u c t i o n o f the ferric chelates ( T a b l e 11I). T h e decrease in ferric chelate red u c t i o n a f t e r a l k a l i n e p h o s p h a t a s e t r e a t m e n t w a s less
TABLE I Effect of alkaline phosphatase treatment on NADPH cytochrome c and ferricvanide reduction and oxidation of KMB I~v purified reductase
NADPH-cytochrome P-450 reductase was incubated in a 50 mM Tris buffer (pH 8.1) containing 10t~ glycerol for either 20 or 40 rain at 37°C in the absence or presence of 2.6 units alkaline phosphatase (alk. phosph.) per/Jg reductase protein. At the indicated times, aliquots were removed and assays of cytochrome c or ferricyanide reduction or ethylene production from 10 mM KMB were conducted as described under Materials and Methods. Ferric-EDTA (50/AM iron/100 .~M EDTA) was present for the KMB assay which was carded out for 15. 30. 45 and 60 rain at 37°C. Incubation condition
NADPHcytochrome c reductase
NADPHferricyanide reductasc
Ethylene production time of reaction (min) 15 30
45
60
20 rain buffer 20 rain alk. phosph. 40 rain h ' ~¢~r 40 rain ~: . .~osph.
23.1 6,6 18.7 3.8
36.8 36.8 34.7 29.8
1.16 0.15 1.06 0.13
2.55 0.48 2.43 0.40
3.03 0.58 2.87 0.45
2.11 0.37 1.94 0.31
16 TABLE II
TABLE IV
t:ffect of alkuhne pho.v~hatase treatnlent on microsomal oxidation of KMB m the presence of t'arious ferric chelates
Effect of alkaline pho.sphatase treatment on H,O_, production t~r microsomes
The oxidation of KMB to ethylene by microsomes treated with 2 units per mg protein of alkaline phosphatase or with buffer for 30 min was a~saved in the presence of the indicated ferric chelates (50/~M final ferric concentration). The alkaline phosphatase treatment lowered the activities of NADPH-ferricyanide and cytochrome c reductase by 6 and 71q. respectively. Results are from two to three experiments.
The production of H202 by microsomes treated with 2 units per mg protein of alkaline phosphatase or with buffer for 30 rain was assayed as described in Materials and Methods in the absence of added iron. or in the presence of 50 aM ferric-EDTA or 50 #M ferric-ATP. Results are from three experiments. The alkaline phosphatase treatment lowered NADPH-ferricyanide reductase activity by 3% at~d NADPH-cytochrome c reductase activity by 49%.
Ferric chelate
Rate of KMB oxidation alkaline + alkaline phnsphatase phosphatase -
Effect of alkaline phosphatase (%)
Rate of H202 production alkaline phosphatase -
(nmol/min per mg protein)
Effect of alkaline phosphatase (%)
(nmol/min per mg protein)
Ferric ammonium sulfate Ferric-EDTA Ferric-DTPA Ferric-ATP Ferric-citrate
0.77+0.06 4.13+0.31 2.43 0.62 ± 0.07 0.92
0.19±0.05 1.31_+0.18 0.75 0.20 ± 0.02 0.14
-75 -68 - 69 - 67 - 84
t h a n the decrease in 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 . In addition, the residual rates o f ferric r e d u c t i o n (20 to 30 nmol per min per m g protein) were far in excess o f the rates of . O H p r o d u c t i o n w h i c h r e m a i n e d a f t e r t r e a t m e n t with alkaline p h o s p h a t a s e . F o r e x a m p l e , f e r r i c - E D T A c a t a l y z e d p r o d u c t i o n o f ethylene f r o m K M B or f o r m a l d e h y d e f r o m either D M S O o r t-butyl alcohol a f t e r t r e a t m e n t of the m i c r o s o m e s with a l k a l i n e p h o s p h a t a s e was a b o u t 1 to 3 n m o l p e r m i n p e r m g protein (Figs. 2 a n d 3; T a b l e Ii), w h e r e a s the rate o f f e r r i c - E D T A r e d u c t i o n was a b o u t 20 n m o l p e r rain p e r m g protein after alkaline p h o s p h a t a s e t r e a t m e n t . T h e s e results suggest t h a t r e d u c t i o n of the ferric c a t a l y s t w a s not rate-limiting for the overall p r o d u c t i o n of . O H b y the microsomes.
TABLE 111 Fffect of alkuline phosphatase treatment on NADPH.dependent reduction of ferric chelates
The reduction of the ferric chelates was determined as described in Materials and Methods with microsomes treated with 3 units per mg protein of alkaline phosphatase, or with barfer for 30 min. NADPHcytochrome c reductzse activity was lowered by 74+6~ by the alkaline phosphatase treatment, whereas there was no effect on the NADPH-ferricyanide reductase activity. Results are from three to four experiments. Rate of ferric reduction alkaline + alkaline phosphatase phosphatase -
Effect of alkaline phosphatase (%)
(nmol/min per mg protein) Ferric- EDTA Ferric-DTPA Fcrric-ATP Ferric-citrate
+ alkaline phosphatase
44 + 6 32 + 3 62 + 7 34±5
20 + 3 21 _+4 28 + 5 18±3
- 55 - 33 - 55 -47
None Ferric-EDTA Ferric-ATP
5,69 + 1.21 13.84 + 2.74 3.31 +0.75
2.56 + 0.43 6.94 + 0.29 1.74+0.18
- 55 - 50 -47
In view of the p o t e n t i n h i b i t i o n o f m i c r o s o m a l o x i d a tion of . O H s c a v e n g e r s b y c a t a l a s e o r g l u t a t h i o n e plus g l u t a t h i o n e p e r o x i d a s e [ 1 6 , 1 9 - 2 1 ] , H 2 0 2 is a r e q u i r e d p r e c u r s o r for the p r o d u c t i o n o f . O H . I n h i b i t i o n of H~O2 p r o d u c t i o n a f t e r a l k a l i n e p h o s p h a t a s e t r e a t m e n t c o u l d b e a c o n t r i b u t i n g f a c t o r for the d e c r e a s e in . O H p r o d u c t i o n . Result~ in T a b l e IV s h o w t h a t t r e a t m e n t o f the m i c r o s o m e s w i t h alkaline p h o s p h a t a s e resulted in a d e c r e a s e in H 2 0 2 p r o d u c t i o n , in the a b s e n c e o r prese n c e o f a d d e d iron, w h i c h w a s c o m p a r a b l e to the loss in 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 activity. R e s i d u a l r a t e s o f H 2 0 2 p r o d u c t i o n in the p r e s e n c e o f f e r r i c - E D T A (about 7 nmol per min per mg protein) were a closer reflection of residual r a t e s o f " O H p r o d u c t i o n (1 to 3 n m o l p e r m i n p e r m g p r o t e i n ) t h a n were the r e s i d u a l r a t e s o f f e r r i c - E D T A r e d u c t i o n (20 n m o l p e r m i n p e r m g protein). if p r o d u c t i o n o f H z O 2 is a r a t e - l i m i t i n g f a c t o r in the overall o x i d a t i o n o f • O H s c a v e n g e r s b y the m i c r o s o m e s , a d d i t i o n o f H 2 0 2 s h o u l d increase o x i d a t i o n o f the s c a v e n g e r s a n d p e r h a p s p r e v e n t the inhibition o f , O H p r o d u c t i o n p r o d u c e d b y the a l k a l i n e p h o s p h a t a s e treatment. The coupled oxidation of hypoxanthine by x a n t h i n e o x i d a s e w a s u s e d as a n e x t e r n a l l y a d d e d H z O 2 - g e n e r a t i n g system. R a t e s o f K M B o x i d a t i o n to e t h y l e n e in the a b s e n c e o f m i c r o s o m e s were s u b t r a c t e d f r o m total rates o f e t h y l e n e p r o d u c t i o n to a c c o u n t for • O H p r o d u c t i o n b y the x a n t h i n e o x i d a s e reaction, especially in the p r e s e n c e o f f e r r i c - E D T A [22,27,28]. T h e a d d i t i o n of the H 2 0 2 - g e n e r a t i n g s y s t e m p r o d u c e d a b o u t a 5070 increase in the r a t e of . O H g e n e r a t i o n for the c o n t r o l m i c r o s o m e s n o t t r e a t e d with alkaline p h o s p h a t a s e ( T a b l e V), s u g g e s t i n g t h a t H 2 0 2 is i n d e e d a r a t e - l i m i t i n g c o m p o n e n t for the overall o x i d a t i o n of • O H s c a v e n g e r s b y the m i c r o s o m e s . M o r e o v e r , the inhibition o f e t h y l e n e p r o d u c t i o n p r o d u c e d b y the alkaline p h o s p h a t a s e t r e a t m e n t c o u l d be p r e v e n t e d b y the
TABLE V Effect of an added H202-generating system on the inhibition of microsomal oxidation of KMB I~" treatment with Ikaline phosphata.~e
The oxidation of KMB to ethyleneby microsomestreated with 2 units per mg protein of alkaline phosphataseor with buffer for 30 rain was assayed as described in Materials and Methods. In someexperiments. an external H202-generating system consisting of 0.5 mM hypoxanthine plus 0.03 units of xanthine oxidase was added immediately before initiating the KMB oxidation experimentswith the NADPHgenerating system. Results are from two experiments. The alkaline phosphatase treatment lowered NADPH-cytochromec reductase activity by 56%, without any effect on NADPH-ferricyanide reductase activity. Ferric chelate Xanthine Rate of KMB oxidation Effectof oxidase -alkaline +alkaline alkaline phosphatase phosphatase phosphatasc None
+
(nmol/min per mg protein) 0.31 0.15 - 52 0.56 0.65 + 15
Ferric-EDTA +
3.15 4.89
1.48 3.91
-53 - 20
Ferric-ATP
0.35 0.55
0.18 0.64
- 5I + 16
+
addition of the H202-generating system (Table V), suggesting that the decrease in H202 production as a consequence of the alkaline phosphatase treatment is primarily responsible for the inhibition of -OH production. Under all conditions, production of . O H was insensitive to superoxide dismutase (43 units per ml) but was completely inhibited by added catalase (200 units per ml), indicating the requirement for H202, but not superoxide in the overall mechanism of -OH production (although superoxide may dismute to yield the H202). Discussion
These results demonstrate that treating rat liver microsomes or purified NADPH cytochrome P-450 reductase with alkaline phosphatase results in a decreased capacity to catalyze the oxidation of .OH scavenging agents such as KMB, DMSO, or t-butyl alcohol. Associated with this decline in -OH generation by microsomes was a corresponding decrease in the reduction of the ferric chelate catalyst and the generation of H202, the precursor of .OH. The alkaline phosphatase treatment was confirmed [12,13] to decrease the cytochrome c reductase activity, but not the ferricyanide reductase activity of rat liver microsomes and purified NADPHcytochrome P-450 reductase. These results point to a key role for the FMN component of the reductase in the overall pathway of -OH production. It appears that loss of the FMN prosthetic group of the reductase results in a decreased capacity to reduce various ferric
chelates, and more significantly, an eventual decrease in H202 production. The decrease in ferric reduction was observed for all the iron chelates evaluated, ranging from ferric-EDTA and ferric-DTPA, which are the most effective ferric complexes for catalysis of .OH generation by microsomes and reductase, to ferric-ATP and ferric-citrate, which are poorer catalysts of -OH production. Generation of -OH was inhibited after alkaline phosphatase treatment to compare.-." extents irrespective ol the ferric chelate complex, fhe decline in ferric chelate reduction did not strictly lollow the loss in reductase activity whereas the decline in -OH or H.,O 2 production was proportional to the loss in reductase a,,'tivity. Grover and Piette [29] have shown that the ability of the reductase to reduce oxygen to superoxide is equa!!y dependent upon the content of FMN and FAD aad that both flavin prosthetic groups have equal capacities for oxygen reduction to superoxide. Alexander et al. [10] have shown that the FMN-depleted reductase loses 40 to 50% of its NADPH oxidase activity. A similar situation may exist with the ferric EDTA reduction capacity, For example, the ferric/ferrous EDTA couple has a reduction potential of 0.12 V [30]. Since the redox potentials for F M N / F M N H 2 and F A D / FADH 2 are - 0 . 1 9 and -0.33 V, respectively [6,7], ferric-EDTA should be reduced by either flavin component of the reductase, analogous to oxygen reduction to superoxidc. Hence, the decline in ferric EDTA reduction is less than the fall in cytocbrome C reduction after alkaline phosphatase treatment. Previous experiments have shown that oxidation of KMB, DMSO, or t-butyl alcohol is inhibited by catalase or glutathione plus glutathione peroxidase, whereas addition of H202 or generation of additional H202 by the addition of redox cycling agents or hypoxanthine plus xanthine oxidase increases -OH generation [16,31,32]. The decrease in H202 production as a consequence of the alkaline phosphatase treatment appears to be responsible for the decline in -OH generation, as the latter can be fully prevented by the addition of a H202-generating system. In microsomes, H202 can arise either from the dismutation of superoxide which is generated by the reductase itself [29,33-35], or more likely from the decay of the oxy-P-450 complex [36-41], or from autooxidation of reduced ferrous chelates, and from the decay of the peroxy-P-450 complex [39-41]. All these reactions would be inhibited as a consequence of the alkaline phosphatase treatment, e.g., depletion of FMN causes a decrease of superoxide production from the reductase [29], and since cytochrome P-450-catalyzed activity is lost [12,13], H202 will not be generated from oxy- or peroxy-P-450. Inhibition of ferric reduction to ferrous, will also decrease H202 generation from autooxidation of ferrous chelates. The depletion of FMN by the alkaline phosphatase treatment with the subsequent decrease in H202 production results in the de-
18 c r e a s e d c a p a c i t y o f the m i c r o s o m e s to oxidize . O H s c a v e n g e r s . T h e s e s t u d i e s s u g g e s t t h a t b o t h flavin c o m p o n e n t s of N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e a r e r e q u i r e d for m a x i m a l rates o f g e n e r a t i o n o f - O H - l i k e species, a n d p r o v i d e s u p p o r t t h a t the r a t e of p r o d u c t i o n o f H 2 0 2 is a critical r a t e - l i m i t i n g f a c t o r for t h e o x i d a tion o f . O H s c a v e n g e r s b y m i c r o s o m e s . Acknowledgements These studies were supported by USPHS Grant AA 03312 from The National Institute on Alcohol Abuse and Alcoholism and INT-8901813 from The National Science F o u n d a t i o n . W e t h a n k Ms. Pilar Visco C e n i z a l for t y p i n g the m a n u s c r i p t , a n d D r . L i v i u C l e j a n for his generous provision of the NADPH cytochrome P-450 reductase. References 1 lya,~agi. T. and Mason. H.S. (19731 Biochemistry 12, 2297-2308. 2 Vermilion, J.L. and Coon, M.J. (1974) Biochem. Biophys. Res. Commun. 60,1315-1322. 3 Dignam, J.D. and Strobel, H.W. (19751 Biochem. Biophys. Res. Commun. 63. 845-852. 4 Yasukochi. Y. and Masters. B.S.S. (19761 J. Biol. Chem. 251, 5337-5344. 5 Vermilion. J.L. and Coon, M.J. (19781 J. Biol. Chem. 253, 26942704. 6 lyanagi, T., Makino. N. and Mason, H.S. (19741 Biochemistry 13, 1701-1710. 7 Vermilion, J.L. and Coon. M.J. (1978) J. Biol. Chem. 253, 88128819. 8 lyanagi, T., Makino, N, and Anan, F.K. (19811 Biochemistry 20, 1722-1730. 9 Kurzban, G.P. and Strobel, H.W. (19861 J. Biol. Chem. 261, 7824-7830. 10 Alexander, L.M., Hersh, L.B. and Masters, B.S.S. (1980) in Microsomes, Drug Oxidations and Chemical Carcinogenesis (Coon, M.J., Conney, A.H., Estabrook, R.W., Gelboin, H.V., Gillette, J.R. and O'Brien, P.J., eds.) pp. 285-288, Academic Press, New York. 11 Vermilion, J.L., Ballou, D.P. Massey, V. and Coon, M.J. (19811 J. Biol. Chem. 256. 266-277. 12 Pyerin. W., Jochum. Ch.. Taniguchi, H. and Wolf, C.R. (19861 Res. Commun. Chem. Pathol. Pharmacol. 53, 133-136. 13 Tanigucni. H. and Pyerin. W. (19871 Biochim. Biophys. Acta 912, 295-302.
14 Cederbaum, A.I. (19891 Free Radical Biol. Med. 7. 559-567. 15 Guengerich, G.P. and Martin. M.V. (19801 Arch. Biochem. Biophys. 205. 365-379. 16 Clejan. L. and Cederbaum. A.I. (19891 Biochem. Pharmacol. 38. 1779-1786. 17 Phillips. A.H. and Langdon. R.G. (1962) J. Biol. Chem. 237. 2652-2660. 18 Schellenberg. K.H. and Hellerman. L. (19581 J. Biol. Chem. 231. 547-556. i9 Cohen. G. and Cederbaum A.I. (19801 Arch. Biochem. Biophys. 199, 438-447. 20 Klein, S.M., Cohen, G. and Cederbaum. A.I. (1981) Biochemistry 20, 6006-6012. 21 Cederbaum. A.I.. Qureshi. A. and Cohen. G. (19831 Biochem. Pharmacol. 32, 3517-3524. 22 Winston. G.W.. Feierman, D.E. and Cederbaum, A.I. (1984) Arch. Biochem. Biophys. 232, 378-390. 23 Puntarulo, S. and Cederbaum, A.I. (1988) Arch. Biochem. Biephys. 264, 482-491. 24 Nash, T. (19531 Biochem. J. 55, 416-422. 25 Hildebrandt, A.G., Roots. I., Tjoe. M. and Heinemeyer, G. (19781 Methods Enzymol. 52, 342-350. 26 Egyed, A., May, A. and Jacobs, A. (19801 Biochim. Biophys. Acta 629, 391-398. 27 McCord. J.M. and Day, E.D. (19781 FEBS Lett. 86.139-142. 28 Halliwell, B. (19781 FEBS Len. 92, 321-326. 29 Grover. T.A. and Piette, L.H. (19811 Arch. Biochem. Biophys. 212, 105-114. 30 Schwa~enboch, G. and Heller. J. (1951) Heir. Chim. Acta 34, 576-591. 31 Cederbaum, A.I., Dicker, E. and Cohen, G. (19801 Biochemistry 19, 3695-3704. 32 Beloqui, O. and Cederbaum, A.I. (1985)Arch. Biochem. Biophys. 242, 187-196. 33 Aust, S.D., Roerig, D.L. and Pederson, T.C. (1972) Biochem. Biophys. Res. Commun. 47, 1133-1137. 34 Prough, R.A. and Masters, B.S.S. 0973) Ann. NY Aead. Sci. 212. 89-93. 35 Bosterling, B. and Trudell, J.R. 0981) Biochem. Biophys. Rcs. Commun. 98, 569-575. 36 Strobel, H.W. and Coon, M.J. (19/11 J. Biol. Chem. 246, 78267829. 37 Dybing, E., Nelson, S.D., Mitchell, J.R., Sasame, H.A. and Gillette, J.R. (19761 Mol. Pharmacol. 12, 911-920. 38 Gillette, J.R., Brodie, B.B. and LaDu, B.N. (1957) J. Pharm. Exp. Ther. 119, 532-541. 39 Kuthan, H., Tsuji, H., Graf, H., Ullrich, V., Werringloer, J. and Estabrook. R.W. (1978) FEBS Len. 91. 343-345. 40 Kuthan. H. and Ullrich V. (19821 Eur. J. Biochem. 126. 583-588. 41 Terelius, Y. and lngelman-Sundberg, M. (19881 Biochem. Pharmacol. 37, 1383-'1389.
BBAGEN 23499
Inhibition of the oxidation of hydroxyl radical scavenging agents after alkaline phosphatase treatment of rat liver microsomes S u s a n a P u n t a r u l o 1 a n d A r t h u r I. C e d e r b a u m
2
i pto~lca I Chemisto ' Dicision. School of Pharmac.r and Biochemisto'. Unicersi(r of Buenos Aires. Buenos Aires (Argentina) and -" Department of Biochemistr)'. Mount Sinai School of Medicine. New York, N Y (U.S.A.)
(Received 13 July 1990) (Revised manuscriptreceived l0 December1990)
Key words: CytochromeP-450: NADPH-cytochrome-creductase: Phosphatase: Microsome;(Rat liver) Treatment of rat liver microsomes with alkaline phosphatase results in a loss in the F M N but not the FAD flavin prosthetic group of NAUPl-l-cytochrome P-450 reductase (Taniguchi, H. and Pyerin, W. (1987) Biochim. Biophys. Acta 912, 295-307). Experiments were carried out to evaluate the effect of preventing electron transfer from the FADH 2 to FMN component of the reductase, and subsequent mixed functioa oxidase activity, on reduction of te~ic chelates, production of H202, and the generation of "OH-like species by microsomes. Treatment with alkaline phosphatase was confirmed to decrease NADPH-cytochrome c, but not NADPH-ferricyanide, reductase activity by microsomes and by purified NADPH cytochrome P-450 reductase. The oxidation of hydroxyl radical scavenging agents by microsemes and reductase was decreased by the alkaline phosphatase treatment in accordance with the decline in cytochrome c reductase activity. This decrease in hydroxyl radical production occurred in the presence of various ferric chelate catalysts. Rates of microsomal reduction of the ferric chelates were also inhibited after alkaline pbosphatase treatment. Production of H202 was decreased in accordance to the fall in eytochrome c reductase activity and - O H production. Rates of H202 production appeared to be rate-limiting for the overall generation of . O H as the addition of an external H 2Oz-generating system stimulated "OH production as well as prevented the decline in , O H production caused by the alkaline phosphatase treatment. These results suggest that both the FAD and F M N flavin prosthetic groups of the reductase contribute towards the reduction of various ferric chelates. However, loss of the F M N component and activities dependent on electron transfer from this prosthetic group result in a decrease in H 2 0 z production, which appears to be responsible for the decline in the generation of -OH-like species by microsomes after treatment with alkaline phosphatase.
Introduction NADPH-cytochrome P-450 reductase contains 1 tool each of FMN and FAD per mole of enzyme [1-5]. The enzyme functions to transfer electrons from N A D P H to cytochrome P-450. F A D appears to be the site for accepting electrons from NADPH, followed by transfer to FMN and subsequently to cytochrome P-450 [6-9]. The enzyme can reduce other electron acceptors such as cytochrome c and ferricyanide. Dialysis of the purified reductase against potassium bromide depletes F M N but not FAD from the reductase. This depletion is associ-
Correspondence: A.I. Cederbaum, Department of Biochemistry.Box 11)20, Mount Sinai School of Medicine.One Ciustave L. Levy Place, New York. NY 10029.U.S.A.
ated with an almost complete loss of NADPH-cytochrome c reductase activity, whereas NADPH-ferricyanide reductase was reduced less than 30% [5,7,10]. It appears that F M N is necessary for the reduction of cytochrome c but not for ferricyanide reduction [7,8]. N A D P H can readily reduce the FMN-depleted enzyme, forming F A D H 2 [7,8,11]. Recently, Taniguchi and Pyerin [12,13] showed that incubation of rabbit liver microsomes with alkaline phosphaiase caused a decrease in NADPH-cytochrome c reductase, but not NADPH-ferricyanide reductase activity. This decrease could be correlated with a loss of NADPH-dependent monooxygenase activities. The alkaline phosphatase effect was relatively specific for N ADPH-cytochrome P-450 (c) reductase as the content of cytochromes P-450 and b s or activities of NADH-cytochrome b s (c) reductase were not affected [13]. Flavin
analysis showed a decrease of FMN without any effect on FAD and it was concluded that the alkaline phosphatase inactivated the reductase by interacting with and digesting the FMN component of the reductase [131. In the presence of iron, microsomes can oxidize a variety of hydroxyl radical (-OH) scavengers by a NADPH-dependent reaction [14]. The basic reaction appears to require reduction of ferric iron to the ferrous redox state and the production of H_,O, by the microsomal electron transfer chain, followed by a Fenton-type of reaction to yield -OH-like species [14]. The ability to allow electron reduction to the FAD but not the FMN flavin prosthetic group of NADPH-cytochrome P-450 reductase by alkaline phosphatase treatment, afforded an opportunity to carry out experiments to evaluate a role for each of these flavin components on the basi,: reaction scheme described above. Experiments were carried out to determine the effect of treating rat liver microsomes with alkaline phosphatase on the NADPHdependent reduction of ferric chelates, on the production of H20~, and on the generation of -OH-like species. Some of the results with microsomes, e.g., oxidation of -OH scavengers, were exttmded to the purified reductase.
Materials and Methods Liver microsomes were prepared from male Sprague-Dawley rats weighing about 150-200 g. The microsomes were prepared by differential centrifugation, washed twice with 125 mM KCI and stored at - 7 0 ° C until utilized. Alkaline phosphatase from Escherichia coli (Type 11I) was purchased from Sigma Chemicals and was used without further purification. NADPH-cytochrome P-450 reductase was purified by a slight modification of the method of Guengerich and Martin [15], as previously described [16]. Final preparations had specific activities ranging from 18 to 24 units per mg protein. Alkaline phosphatase treatment of the microsomes was carried out at 37°C essentially as described by Taniguchi and Pyerin [13]. Microsomes (about 2 mg protein) were incubated in 50 mM Tris-HCI buffer (pH 8.3) in the absence or presence of alkaline phosphatase (about 2 units per mg microsomal protein for most experiments) for varying time periods (30 min for most experiments) in a shaking water bath. At the indicated time, aliquots were removed and the control or the treated microsomes were added to the specific reaction system being studied, followed by the NADPH-generating system to initiate the reaction. NADPH-cytochrome c reductase activity was measured in a system containing 0.3 M potassium phosphate buffer (pH 7.7), 0.05 mM cytochrome c, and 0.1 mM NADPH [17]. NADPH-ferricyanide reductase activity was determined
in a system containing 0.1 M potassium phosphate buffer (pH 7.4), 1 mM potassium ferricyanide, and 0.1 mM NADPH [13] and activity determined using an extinction coefficient at 420 nm of 1.02 r a M ~ c m [181. The production of -OH or compounds with the oxidizing power of -OH by microsomes was assayed by measuring the generation of ethylene gas from 2-keto4-thiomethylbutyrate (KMB) or of formaldehyde from DMSO or t-butyl alcohol. Reactions were carried out essentiall 3' as previously described [19-21]. For most experiments, 50 /.tM ferric-EDTA was utilized as the iron catalyst since this is the most reactive iron complex for the promotion of -OH-like species Izy microsomes [22,231. The production of formaldehyde ~as determined by the Nash reaction [24] and the generation of ethylene was assayed by a head space gas chromatography procedure. All values were corrected for zerotime controls which contained acid prior to the addition of the NADPH-generating system. The production of HzO z was determined by measuring the generation of formaldehyde from the oxidation of methanol by the catalase-compound l complex [25]. The reduction of ferric chelates was monitored by the increase in absorbance at 520 nm when microsomes. 0.05 mM ferric chelate. 0.1 mM NADPH and 5 mM 2,2'-bipyridyl in 0.1 M phosphate buffer (pH 7.4) were mixed together [26]. Microsomal NADPH oxidation was determined from the decrease in absorbance at 340 nm of a reaction system containing 0.1 M phosphate buffer (pH 7.4), 0.05 mM ferric chelate, and 0.1 mM NADPH. The ferric complexes were prepared by dissolving ferric ammonium sulfate in 0.1 M HCI, and then diluting with the respective chelator to the appropriate stock concentration. Ferric-ATP was used as a 1 : 20 complex, whereas the other ferric complexes were utilized as a 1 : 2 complex. The phosphate buffer and the water utilized to prepare all solutions were passed through columns of chelex-100 resin to remove metals such as iron. All experiments were carried out in duplicate, with at least two different microsomal preparations: variability generally did not exceed 10%. Where indicated, results refer to mean + standard error.
Results Results in Fig. 1 confirm the observations of Taniguchi and Pyerin [13] that treatment of rat liver microsomes with alkaline phosphatase results in a decrease in NADPH-cytochrome c reductase activity whereas NADPH-ferricyanide reductase activity is not significantly affected. Different amounts of the NADPH-cytochrome c reductase were lost depending on the units of alkaline phosphatase utilized (Fig. IA) or depending on the length of incubation with a fixed amount of alkaline phosphatase (Fig. IB). In general, treatment with about
5OO
~ ~
400A
400
300
,
~ 2oo4
200
~0.-----0
° ~
~
°
\ 0
,
t
I
0
2
0 ~ 0
1oo
o
o
3
0
Units of olkoline phosphotase
30 60 Incubation time (rain)
90
Fig. l. (A) Effect of alkaline phosphatase treatment on NADPH-cytochrome c reductase (o) and NADPH-ferricyanide reductase (&) activity. Microsomes were treated with varying units of alkaline phosphatase or buffer for 60 rain and enzyme activities measured. (B) Microsomes were incubated in the absence of (o. 4) or presence (e, A) of 2 units per mg protein of alkaline phosphatase for 15, 30, 60, or 90 rain, followed by assays of NADPH-cytochrome c (O. e) or NADPH-ferrieyanide (n, A) reductase activity.
2 units of alkaline p h o s p h a t a s e p e r m g m i c r o s o m a l p r o t e i n for 30 rain p r o d u c e d a b o u t a 50% loss o f 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 activity, as c o m p a r e d to m i c r o s o m e s i n c u b a t e d with the ' I r i s b u f f e r alone. S o m e loss of 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 activity ( a b o u t 20%) was o b s e r v e d for s a m p l e s i n c u b a t e d in b u f f e r alone for 15 to 60 m i n as c o m p a r e d to m i c r o s o m e s kept o n ice. In all e x p e r i m e n t s d e s c r i b e d b e l o w , the effect of the alkaline p h o s p h a t a s e t r e a t m e n t w a s a l w a y s c o m p a r e d to s a m p l e s i n c u b a t e d with b u f f e r a l o n e for the s a m e time period. A c c o m p a n y i n g the loss o f 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 activity w a s a c o m -
p a r a b l e loss o f N A D P H o x i d a t i o n b y the m i c r o s o m e s , b o t h in the a b s e n c e o r p r e s e n c e o f f e r r i c - E D T A , as m e a s u r e d b y the d e c r e a s e in a b s o r b a n c e a t 340 n m . T h e p r o d u c t i o n o f e t h y l e n e f r o m K M B w a s u s e d to a s s a y for the g e n e r a t i o n o f . O H - l i k e species b y m i c r o s o m e s in the p r e s e n c e o f N A D P H a n d f e r r i c - E D T A . T r e a t m e n t o f the m i c r o s o m e s with a l k a l i n e p h o s p h a t a s e for 15, 30, o r 60 m i n resulted in a p r o g r e s s i v e loss o f e t h y l e n e g e n e r a t i o n as c o m p a r e d to c o n t r o l s i n c u b a t e d w i t h o u t a l k a l i n e p h o s p h a t a s e (Fig. 2A). T h e p e r c e n t loss o f m i c r o s o m a l o x i d a t i o n o f K M B to e t h y l e n e directly c o r r e l a t e d with the p e r c e n t loss o f N A D P H - c y t o -
100 50
A
~ 4o 30
•
/
20
B
•
Z /
0
80
0
~ 60 @
/D
40
Z~
~ 20 O= ~
0
,
5 Reaction time (min)
,
10
0~ 0
20
4o
8'0
80
Percentinh;b;tionof cytochrome¢ reductose
Fig. 2. (A) Effect of alkaline phosphatase treatment on microsomal oxidation of KMB to ethylene. Microsomes were treated with 2 units of alkaline phosphatase per mg protein for 15 (o), 30 (O), or 60 rain (zx). or with buffer (e), followed by assays for KMB oxidation for either 5 or 10 rain. Control values for microsomes incubated with buffer for 15, 30, or 60 min were similar ( + 87o)and were therefore grouped together. (B) Correlation between inhibition of NADPH-cytochrome c reductase activity by alkaline phosphatase and inhibition of KMB oxidation to ethylene. The various points were obtained for different incubation times with increasing units of alkaline phosphatase activity, followed by assays of NADPH-cytochrome c reductase activity and oxidation of KMB.
15 lOO
~
8o
"~ 60 E ~ 4O o_ 20
15
30
45
Incubation time (rnin)
50
Fig. 3. Comparison of the effec~ of treatment with alkaline phosphatase on NADPH-cytochrome c (o) or NADPH-ferricyanide (O1 reductase activity and oxidation of the .OH scavengers DMSO (U) or t-butyl alcohol (~,). Microsomes were incubated with 2 units of alkaline phosphatase per mg protein for 15. 30. or 60 min, or with buffer, followed by assays of reductase activity or production of formaldehyde from the oxidation of 33 mM DMSO or 50 mM t-butyl alcohol in the presence of 50 t~xi f~,ric-EDTA. Control values were grouped together since they were ~imilar for the three incubation periods. Control values were about 165 and 450 nmol per min per mg protein for NADPH-cytochrome e and [erricyanide reductase activities. respectively, and about 4 to 5 nmol per rain per mg microsomal protein for DMSO and t-butyl alcohol oxidation to formaldehyde. c h r o m e e r e d u c t a s e activity (Fig. 2B). T o d e t e r m i n e if the effect o f alkaline p h o s p h a t a s e t r e a t m e n t o n m i c r o s o m a l " O H g e n e r a t i o n w a s u n i q u e for K M B as the c h e m i c a l scavenger, the o x i d a t i o n of o t h e r . O H s c a v e n g i n g a g e n t s w a s e v a l u a t e d . Previous studies h a v e s h o w n t h a t f o r m a l d e h y d e is a m a j o r p r o d u c t w h i c h arises f r o m the i r o n - d e p e n d e n t o x i d a t i o n of D M S O or t-butyl alcohol b y m i e r o s o m e s [20,21]. I n c u b a t i o n o f m i c r o s o m e s with 2 unit~ o f alkaline p h o s p h a t a s e p e r m g p r o t e i n for 15, 30, o r 60 m i n resulted in progressive loss o f f o r m a l d e h y d e o x i d a t i o n f r o m either D M S O o r t-butyl alcohol (Fig. 3). T h e decrease in o x i d a t i o n of these - O H s c a v e n g i n g a g e n t s a g a i n p a r a l l e l e d the d e c r e a s e in activity o f N A D P H - c y t o c h r o m e c reductase, whereas
N A D P H - ' f e r r i c y a n i d e r e d u c t a s e activity r e m a i n e d u n a f fected (Fig. 31. Similar e x p e r i m e n t s were carried o u t with the purified r c d u c t a s e to ascertain t h a t the results with microsomes treated with alkaline p h o s p h a t a s e were likely due to effects o n the reductase. T r e a t i n g the purified red u c t a s e for 20 o r 4 0 min with alkaline p h o s p h a t a s e c a u s e d decreases of 71 or 78%, respectively, of 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 activity, w h e r e a s N A D P H - f e r r i c y a n i d e r e d u c t a s e activity w a s not affected (Table 1). G l y c e r o l w a s present d u r i n g the i n c u b a t i o n at 3 7 ° C to help m a i n t a i n c o n t r o l r e d u c t a s e activity. T h e p r o d u c tion of ethylene f r o m K M B , as catalyzed b y ferricE D T A , w a s d e c r e a s e d b y the alkaline p h o s p h a t a s e t r e a t m e n t in direct r e l a t i o n s h i p to the fall in 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 activity (Table 1). Previous e x p e r i m e n t s h a d indicated t h a t certain ferric chelates s u c h as f e r r i c - E D T A a n d f e r r i c - D T P A were effective iron c a t a l y s t s for the g e n e r a t i o n of - O H - l i k e species by m i c r o s o m e s , w h e r e a s iron chelates s u c h as f e r r i c - A T P or ferric-citrate were m u c h less effective c a t a l y s t s [22,23]. T o e v a l u a t e w h e t h e r the inhibition of m i c r o s o m a l o x i d a t i o n o f - O H scavenger b y alkaline p h o s p h a t a s e t r e a t m e n t w a s specific o n l y for ferricE D T A as the iron catalyst, e x p e r i m e n t s with o t h e r ferric chelates were c a r r i e d out. T r e a t m e n t with alkaline p h o s p h a t a s e resulted in a similar degree of loss of K M B o x i d a t i o n r e g a r d l e s s o f the a d d e d ferric chelate (Table Ill. W h e n ferric i r o n is a d d e d , a n initial event in the u l t i m a t e g e n e r a t i o n o f " O H b y m i c r o s o m e s is r e d u c t i o n to the ferrous r e d o x state. T h e inhibition of m i c r o s o m a l - O H p r o d u c t i o n a f t e r t r e a t m e n t with alkaline p h o s p h a t a s e c o u l d reflect inhibition o f the r e d u c t i o n o f the a d d e d ferric chelate. In the p r e s e n c e o f N A D P H , micros o m e s c a t a l y z e d the r e d u c t i o n of various ferric chelates ( T a b l e 111). T r e a t m e n t with alkaline p h o s p h a t a s e resulted in a n i n h i b i t i o n o f the r e d u c t i o n o f the ferric chelates ( T a b l e 11I). T h e decrease in ferric chelate red u c t i o n a f t e r a l k a l i n e p h o s p h a t a s e t r e a t m e n t w a s less
TABLE I Effect of alkaline phosphatase treatment on NADPH cytochrome c and ferricvanide reduction and oxidation of KMB I~v purified reductase
NADPH-cytochrome P-450 reductase was incubated in a 50 mM Tris buffer (pH 8.1) containing 10t~ glycerol for either 20 or 40 rain at 37°C in the absence or presence of 2.6 units alkaline phosphatase (alk. phosph.) per/Jg reductase protein. At the indicated times, aliquots were removed and assays of cytochrome c or ferricyanide reduction or ethylene production from 10 mM KMB were conducted as described under Materials and Methods. Ferric-EDTA (50/AM iron/100 .~M EDTA) was present for the KMB assay which was carded out for 15. 30. 45 and 60 rain at 37°C. Incubation condition
NADPHcytochrome c reductase
NADPHferricyanide reductasc
Ethylene production time of reaction (min) 15 30
45
60
20 rain buffer 20 rain alk. phosph. 40 rain h ' ~¢~r 40 rain ~: . .~osph.
23.1 6,6 18.7 3.8
36.8 36.8 34.7 29.8
1.16 0.15 1.06 0.13
2.55 0.48 2.43 0.40
3.03 0.58 2.87 0.45
2.11 0.37 1.94 0.31
16 TABLE II
TABLE IV
t:ffect of alkuhne pho.v~hatase treatnlent on microsomal oxidation of KMB m the presence of t'arious ferric chelates
Effect of alkaline pho.sphatase treatment on H,O_, production t~r microsomes
The oxidation of KMB to ethylene by microsomes treated with 2 units per mg protein of alkaline phosphatase or with buffer for 30 min was a~saved in the presence of the indicated ferric chelates (50/~M final ferric concentration). The alkaline phosphatase treatment lowered the activities of NADPH-ferricyanide and cytochrome c reductase by 6 and 71q. respectively. Results are from two to three experiments.
The production of H202 by microsomes treated with 2 units per mg protein of alkaline phosphatase or with buffer for 30 rain was assayed as described in Materials and Methods in the absence of added iron. or in the presence of 50 aM ferric-EDTA or 50 #M ferric-ATP. Results are from three experiments. The alkaline phosphatase treatment lowered NADPH-ferricyanide reductase activity by 3% at~d NADPH-cytochrome c reductase activity by 49%.
Ferric chelate
Rate of KMB oxidation alkaline + alkaline phnsphatase phosphatase -
Effect of alkaline phosphatase (%)
Rate of H202 production alkaline phosphatase -
(nmol/min per mg protein)
Effect of alkaline phosphatase (%)
(nmol/min per mg protein)
Ferric ammonium sulfate Ferric-EDTA Ferric-DTPA Ferric-ATP Ferric-citrate
0.77+0.06 4.13+0.31 2.43 0.62 ± 0.07 0.92
0.19±0.05 1.31_+0.18 0.75 0.20 ± 0.02 0.14
-75 -68 - 69 - 67 - 84
t h a n the decrease in 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 . In addition, the residual rates o f ferric r e d u c t i o n (20 to 30 nmol per min per m g protein) were far in excess o f the rates of . O H p r o d u c t i o n w h i c h r e m a i n e d a f t e r t r e a t m e n t with alkaline p h o s p h a t a s e . F o r e x a m p l e , f e r r i c - E D T A c a t a l y z e d p r o d u c t i o n o f ethylene f r o m K M B or f o r m a l d e h y d e f r o m either D M S O o r t-butyl alcohol a f t e r t r e a t m e n t of the m i c r o s o m e s with a l k a l i n e p h o s p h a t a s e was a b o u t 1 to 3 n m o l p e r m i n p e r m g protein (Figs. 2 a n d 3; T a b l e Ii), w h e r e a s the rate o f f e r r i c - E D T A r e d u c t i o n was a b o u t 20 n m o l p e r rain p e r m g protein after alkaline p h o s p h a t a s e t r e a t m e n t . T h e s e results suggest t h a t r e d u c t i o n of the ferric c a t a l y s t w a s not rate-limiting for the overall p r o d u c t i o n of . O H b y the microsomes.
TABLE 111 Fffect of alkuline phosphatase treatment on NADPH.dependent reduction of ferric chelates
The reduction of the ferric chelates was determined as described in Materials and Methods with microsomes treated with 3 units per mg protein of alkaline phosphatase, or with barfer for 30 min. NADPHcytochrome c reductzse activity was lowered by 74+6~ by the alkaline phosphatase treatment, whereas there was no effect on the NADPH-ferricyanide reductase activity. Results are from three to four experiments. Rate of ferric reduction alkaline + alkaline phosphatase phosphatase -
Effect of alkaline phosphatase (%)
(nmol/min per mg protein) Ferric- EDTA Ferric-DTPA Fcrric-ATP Ferric-citrate
+ alkaline phosphatase
44 + 6 32 + 3 62 + 7 34±5
20 + 3 21 _+4 28 + 5 18±3
- 55 - 33 - 55 -47
None Ferric-EDTA Ferric-ATP
5,69 + 1.21 13.84 + 2.74 3.31 +0.75
2.56 + 0.43 6.94 + 0.29 1.74+0.18
- 55 - 50 -47
In view of the p o t e n t i n h i b i t i o n o f m i c r o s o m a l o x i d a tion of . O H s c a v e n g e r s b y c a t a l a s e o r g l u t a t h i o n e plus g l u t a t h i o n e p e r o x i d a s e [ 1 6 , 1 9 - 2 1 ] , H 2 0 2 is a r e q u i r e d p r e c u r s o r for the p r o d u c t i o n o f . O H . I n h i b i t i o n of H~O2 p r o d u c t i o n a f t e r a l k a l i n e p h o s p h a t a s e t r e a t m e n t c o u l d b e a c o n t r i b u t i n g f a c t o r for the d e c r e a s e in . O H p r o d u c t i o n . Result~ in T a b l e IV s h o w t h a t t r e a t m e n t o f the m i c r o s o m e s w i t h alkaline p h o s p h a t a s e resulted in a d e c r e a s e in H 2 0 2 p r o d u c t i o n , in the a b s e n c e o r prese n c e o f a d d e d iron, w h i c h w a s c o m p a r a b l e to the loss in 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 activity. R e s i d u a l r a t e s o f H 2 0 2 p r o d u c t i o n in the p r e s e n c e o f f e r r i c - E D T A (about 7 nmol per min per mg protein) were a closer reflection of residual r a t e s o f " O H p r o d u c t i o n (1 to 3 n m o l p e r m i n p e r m g p r o t e i n ) t h a n were the r e s i d u a l r a t e s o f f e r r i c - E D T A r e d u c t i o n (20 n m o l p e r m i n p e r m g protein). if p r o d u c t i o n o f H z O 2 is a r a t e - l i m i t i n g f a c t o r in the overall o x i d a t i o n o f • O H s c a v e n g e r s b y the m i c r o s o m e s , a d d i t i o n o f H 2 0 2 s h o u l d increase o x i d a t i o n o f the s c a v e n g e r s a n d p e r h a p s p r e v e n t the inhibition o f , O H p r o d u c t i o n p r o d u c e d b y the a l k a l i n e p h o s p h a t a s e treatment. The coupled oxidation of hypoxanthine by x a n t h i n e o x i d a s e w a s u s e d as a n e x t e r n a l l y a d d e d H z O 2 - g e n e r a t i n g system. R a t e s o f K M B o x i d a t i o n to e t h y l e n e in the a b s e n c e o f m i c r o s o m e s were s u b t r a c t e d f r o m total rates o f e t h y l e n e p r o d u c t i o n to a c c o u n t for • O H p r o d u c t i o n b y the x a n t h i n e o x i d a s e reaction, especially in the p r e s e n c e o f f e r r i c - E D T A [22,27,28]. T h e a d d i t i o n of the H 2 0 2 - g e n e r a t i n g s y s t e m p r o d u c e d a b o u t a 5070 increase in the r a t e of . O H g e n e r a t i o n for the c o n t r o l m i c r o s o m e s n o t t r e a t e d with alkaline p h o s p h a t a s e ( T a b l e V), s u g g e s t i n g t h a t H 2 0 2 is i n d e e d a r a t e - l i m i t i n g c o m p o n e n t for the overall o x i d a t i o n of • O H s c a v e n g e r s b y the m i c r o s o m e s . M o r e o v e r , the inhibition o f e t h y l e n e p r o d u c t i o n p r o d u c e d b y the alkaline p h o s p h a t a s e t r e a t m e n t c o u l d be p r e v e n t e d b y the
TABLE V Effect of an added H202-generating system on the inhibition of microsomal oxidation of KMB I~" treatment with Ikaline phosphata.~e
The oxidation of KMB to ethyleneby microsomestreated with 2 units per mg protein of alkaline phosphataseor with buffer for 30 rain was assayed as described in Materials and Methods. In someexperiments. an external H202-generating system consisting of 0.5 mM hypoxanthine plus 0.03 units of xanthine oxidase was added immediately before initiating the KMB oxidation experimentswith the NADPHgenerating system. Results are from two experiments. The alkaline phosphatase treatment lowered NADPH-cytochromec reductase activity by 56%, without any effect on NADPH-ferricyanide reductase activity. Ferric chelate Xanthine Rate of KMB oxidation Effectof oxidase -alkaline +alkaline alkaline phosphatase phosphatase phosphatasc None
+
(nmol/min per mg protein) 0.31 0.15 - 52 0.56 0.65 + 15
Ferric-EDTA +
3.15 4.89
1.48 3.91
-53 - 20
Ferric-ATP
0.35 0.55
0.18 0.64
- 5I + 16
+
addition of the H202-generating system (Table V), suggesting that the decrease in H202 production as a consequence of the alkaline phosphatase treatment is primarily responsible for the inhibition of -OH production. Under all conditions, production of . O H was insensitive to superoxide dismutase (43 units per ml) but was completely inhibited by added catalase (200 units per ml), indicating the requirement for H202, but not superoxide in the overall mechanism of -OH production (although superoxide may dismute to yield the H202). Discussion
These results demonstrate that treating rat liver microsomes or purified NADPH cytochrome P-450 reductase with alkaline phosphatase results in a decreased capacity to catalyze the oxidation of .OH scavenging agents such as KMB, DMSO, or t-butyl alcohol. Associated with this decline in -OH generation by microsomes was a corresponding decrease in the reduction of the ferric chelate catalyst and the generation of H202, the precursor of .OH. The alkaline phosphatase treatment was confirmed [12,13] to decrease the cytochrome c reductase activity, but not the ferricyanide reductase activity of rat liver microsomes and purified NADPHcytochrome P-450 reductase. These results point to a key role for the FMN component of the reductase in the overall pathway of -OH production. It appears that loss of the FMN prosthetic group of the reductase results in a decreased capacity to reduce various ferric
chelates, and more significantly, an eventual decrease in H202 production. The decrease in ferric reduction was observed for all the iron chelates evaluated, ranging from ferric-EDTA and ferric-DTPA, which are the most effective ferric complexes for catalysis of .OH generation by microsomes and reductase, to ferric-ATP and ferric-citrate, which are poorer catalysts of -OH production. Generation of -OH was inhibited after alkaline phosphatase treatment to compare.-." extents irrespective ol the ferric chelate complex, fhe decline in ferric chelate reduction did not strictly lollow the loss in reductase activity whereas the decline in -OH or H.,O 2 production was proportional to the loss in reductase a,,'tivity. Grover and Piette [29] have shown that the ability of the reductase to reduce oxygen to superoxide is equa!!y dependent upon the content of FMN and FAD aad that both flavin prosthetic groups have equal capacities for oxygen reduction to superoxide. Alexander et al. [10] have shown that the FMN-depleted reductase loses 40 to 50% of its NADPH oxidase activity. A similar situation may exist with the ferric EDTA reduction capacity, For example, the ferric/ferrous EDTA couple has a reduction potential of 0.12 V [30]. Since the redox potentials for F M N / F M N H 2 and F A D / FADH 2 are - 0 . 1 9 and -0.33 V, respectively [6,7], ferric-EDTA should be reduced by either flavin component of the reductase, analogous to oxygen reduction to superoxidc. Hence, the decline in ferric EDTA reduction is less than the fall in cytocbrome C reduction after alkaline phosphatase treatment. Previous experiments have shown that oxidation of KMB, DMSO, or t-butyl alcohol is inhibited by catalase or glutathione plus glutathione peroxidase, whereas addition of H202 or generation of additional H202 by the addition of redox cycling agents or hypoxanthine plus xanthine oxidase increases -OH generation [16,31,32]. The decrease in H202 production as a consequence of the alkaline phosphatase treatment appears to be responsible for the decline in -OH generation, as the latter can be fully prevented by the addition of a H202-generating system. In microsomes, H202 can arise either from the dismutation of superoxide which is generated by the reductase itself [29,33-35], or more likely from the decay of the oxy-P-450 complex [36-41], or from autooxidation of reduced ferrous chelates, and from the decay of the peroxy-P-450 complex [39-41]. All these reactions would be inhibited as a consequence of the alkaline phosphatase treatment, e.g., depletion of FMN causes a decrease of superoxide production from the reductase [29], and since cytochrome P-450-catalyzed activity is lost [12,13], H202 will not be generated from oxy- or peroxy-P-450. Inhibition of ferric reduction to ferrous, will also decrease H202 generation from autooxidation of ferrous chelates. The depletion of FMN by the alkaline phosphatase treatment with the subsequent decrease in H202 production results in the de-
18 c r e a s e d c a p a c i t y o f the m i c r o s o m e s to oxidize . O H s c a v e n g e r s . T h e s e s t u d i e s s u g g e s t t h a t b o t h flavin c o m p o n e n t s of N A D P H - c y t o c h r o m e P - 4 5 0 r e d u c t a s e a r e r e q u i r e d for m a x i m a l rates o f g e n e r a t i o n o f - O H - l i k e species, a n d p r o v i d e s u p p o r t t h a t the r a t e of p r o d u c t i o n o f H 2 0 2 is a critical r a t e - l i m i t i n g f a c t o r for t h e o x i d a tion o f . O H s c a v e n g e r s b y m i c r o s o m e s . Acknowledgements These studies were supported by USPHS Grant AA 03312 from The National Institute on Alcohol Abuse and Alcoholism and INT-8901813 from The National Science F o u n d a t i o n . W e t h a n k Ms. Pilar Visco C e n i z a l for t y p i n g the m a n u s c r i p t , a n d D r . L i v i u C l e j a n for his generous provision of the NADPH cytochrome P-450 reductase. References 1 lya,~agi. T. and Mason. H.S. (19731 Biochemistry 12, 2297-2308. 2 Vermilion, J.L. and Coon, M.J. (1974) Biochem. Biophys. Res. Commun. 60,1315-1322. 3 Dignam, J.D. and Strobel, H.W. (19751 Biochem. Biophys. Res. Commun. 63. 845-852. 4 Yasukochi. Y. and Masters. B.S.S. (19761 J. Biol. Chem. 251, 5337-5344. 5 Vermilion. J.L. and Coon, M.J. (19781 J. Biol. Chem. 253, 26942704. 6 lyanagi, T., Makino. N. and Mason, H.S. (19741 Biochemistry 13, 1701-1710. 7 Vermilion, J.L. and Coon. M.J. (1978) J. Biol. Chem. 253, 88128819. 8 lyanagi, T., Makino, N, and Anan, F.K. (19811 Biochemistry 20, 1722-1730. 9 Kurzban, G.P. and Strobel, H.W. (19861 J. Biol. Chem. 261, 7824-7830. 10 Alexander, L.M., Hersh, L.B. and Masters, B.S.S. (1980) in Microsomes, Drug Oxidations and Chemical Carcinogenesis (Coon, M.J., Conney, A.H., Estabrook, R.W., Gelboin, H.V., Gillette, J.R. and O'Brien, P.J., eds.) pp. 285-288, Academic Press, New York. 11 Vermilion, J.L., Ballou, D.P. Massey, V. and Coon, M.J. (19811 J. Biol. Chem. 256. 266-277. 12 Pyerin. W., Jochum. Ch.. Taniguchi, H. and Wolf, C.R. (19861 Res. Commun. Chem. Pathol. Pharmacol. 53, 133-136. 13 Tanigucni. H. and Pyerin. W. (19871 Biochim. Biophys. Acta 912, 295-302.
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