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Vo1.153, No. 3,1988
June 30,1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 973-978
A NEW AND SUITABLE RECONSTRUCTED SYSTEM
FOR NADPH-DEPENDENT MICROSOMAL LIPID PEROXIDATION
Hisanori Minakami a, Hiroyuki Arai b, Minoru Nakano b~,
Katsuaki Sugioka b, Shingo Suzuki c, and Akemi Sotomatsu d
aDepartment of Obstetrics and Gynecology, Jichi Medical School, Tochigi, Japan
bCollege of Medical Care and Technology, Gunma University, Maebashi, Gunma,
371 Japan,
CDepartmentof Medicine, Hamamatsu Medical School, Shizuoka, Japan
dDivision of Neurology, Gunma University Hospital, Gunma, Japan
Received March 8, 1988
In order to evaluate the 02 participation in NADPH-dependent microsomal lipid peroxidation, we used reconstructed system which contained detergent-solubil- ized NADPH-dependent cytochrome P-450 reductase, cytochrome P-450, phospho- lipid liposomes, NADPH and F~+-ADP. Lipid peroxidation, monitored by the formation of thiobarbituric acid-reactive substance, was increased with in- creasing concentration of detergent-solubilized NADPH cytochrome P-450 reduc- tase, cytochrome P-450 or F_~+-ADP. Cytochrome P-450-dependent lipid per- oxidation was parallel to 09 generation monitored by chemiluminescence probe with 2-methyl-6-(p-methoxyp~enol)-3,7-dihydroimidazo~l,2-a]pyrazin-3-one. Lipid peroxidation was significantly inhibited by superoxide dismutase, but not by catalase or sodium benzoate. The reconstructed system herein describ- ed is considered to be very close to NADPH-dependent microsomal lipid per- oxidation system. © 1988 Acade~= Press, Inc.
It has been known that free iron or loosely bound iron is required for
NADPH-dependent microsomal lipid peroxidation (1,2). In order to prove this
mechanism, several investigators used reconstructed systems, which contained
NADPH, protease-solubilized NADPH-dependent cytochrome P-450 reductase, Fe 3+-
ADP or Fe3+-ADP-EDTA and phospholipid liposomes (3 - 5). Such a reconstruct-
ed system is different from NADPH-dependent microsomal lipid peroxidation
system in that the former requires iron at higher concentration and consumes
To whom correspondence should be addressed.
Abbreviations used are:
MCLA, 2-methyl-6-(methoxyphenol)-3,7-dihydroimidazo[l,2-~ pyrazin-3-one; SOD,
superoxide dismutase.
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0006-291)[/88 $1.50 Copyright © 1988 by Academic Press, Inc,
All rights of reproduction in any form reserved.
Vol. 153, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NADPH to a lesser extent (4), compared with the latter (2,6). Mitomycin, a
quinoid anticancer drug, which consumes NADPH rapidly and produces O; to a
great extent in the presence of protease-solubilized enzyme, strongly enhanc-
ed the lipid peroxidation in the reconstructed system (7). It seems likely,
therefore, that microsomes contain another system elevating O; generation in
addition to NADPH-cytochrome P-450 reductase-O2-system. A reconstructed
system containing detergent-solubilized NADPH-cytochrome P-450 reductase,
cytochrome P-450 and liposomes has already been reported to involve a O~ gen-
eration in intact microsomes (8,9). In the present paper, we report that a
suitable reconstructed system for NADPH-dependent microsomal lipid peroxida-
tion is a system containing microsomal phospholipid liposomes, cytochrome P-
450, detergent-solubilized NADPB-dependent cytochrome P-450 reductase (but
not protease-solubilized enzyme) and Fe3+-ADP.
MATERIALS AND METHODS
Reagents All chemicals were reagent grade, 2-methyl-6-(p-methoxyphenol)-3,7- dihydroimidazoLI,2-aJpyrazin-3-one (MCLA) was kindly donated by Prof. T.Goto. Fe3+-ADP complex was prepared by the method as previously described (i0). Fe3+(3mM)-ADP(5OmM) complex, a stock solution, was diluted to adequate con- centration with water before use. Preparation ofliposomes Microsomal phospholipid was obtained from rat liver microsomes and freed from cholesterol and free fatty acids (i0). Liposomes were then prepared by the method described previously (i0). Enzyme sources Bovine erythrocyte superoxide dismutase (SOD) and bovine liver catalase were obtained from Toyobo,Co. and Sigma Chemical Co.~respect- ively. NADPH-cytochrome P-450 reductase, a detergent-solubilized enzyme (specific activity ffi 60 nnits/mg of protein), and cytochrome P-450 (5.6 nmol/ mgof protein) were prepared from liver microsomes of phenobarbital-treated rats by the method of Van Der Hoeven and Coon (Ii). NADPH-cytochrome P-450 reductase, protease-solubilized enzyme (63 units/mg of protein), was prepared from rat liver microsomes by the method of Omura and Takasue (12). Commercial catalase, NADPH-cytochrome P-450 reductase or cytochrome P-450 was dialyzed against i0 mMTris-HCl buffer at pH 7.5 before use. Specific activ- ity of cytochrome P-450 reductase was determined according to the method described by Vermilion and Coon (13). Incubation experiments The incubation mixture for lipid peroxidation contain- ed liposomes (0.85 ~mol of lipid phosphorus/ml), 1.0 mM NADPB, Fe3+-ADP com- plex, cytochrome P-450, NADPH-cytochrome P-450 reductase (protease-solubili- zed or detergent-solubilized enzyme) and 0.I M Tris-HCl buffer at pH 7.5, in a total volume of 1.0 ml. The incubation mixture for chemiluminescence contained all components described above, except for Fe3+-ADP. Ten~MMCLA was used as chemiluminescence probe. In some case, O.I~M SOD, O.l~Mheat- treated SOD (autoclave-treated enzyme, 40 min at 120°C), 20 ~g of catalase/ml, 20 mM sodium benzoate or 50~M desferrioxamine methansulfonate was added to the reaction mixture. The reaction was initiated by the addition of NADPH and carried out at 37°C. Assay Lipid peroxidation was determined by the formation of thiobarbituric acid-reactive substance during I0 min and expressed as a concentration of malondialdehyde (14). MCLA-dependent luminescence, originated by O; + MCLA reaction, was determined by chemiluminescence intensity in a luminescence reader (Aloka,Co.) and expressed as integrated light intensity corrected for control as previously described with 2-methyl-6-phenyl-3,7-dihydroimidazo [iz2-a~pyrazin-3-one (a luminescence probe) and activated leukocyte system (02 generationg system)(15).
974
Vo l . 153, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS
Lipid peroxidation in reconstructed system containing four components
(Fe3+-ADP, NADPH, cytochrome P-450 and liposomes) at fixed concentrations and
detergent-solubilized enzyme at a variety of concentrations increased with
increasing enzyme concentration. Omission of cytochrome P-450 from the re-
constructed system greatly suppressed the lipid peroxidation. However, a
substantial lipid peroxidation, over control (reconstructed system including
desferrioxamine or excluding Fe3+-ADP), occurred in the reconstructed system
excluding cytochrome P-450, but did not significantly increase with increas-
ing enzyme concentration. These results are shown in Fig IA.
With fixed concentrations of four components, the lipid peroxidation in the
reconstructed system increased with increasing cytochrome P-450 concentration
40
.S.¢
A jL_...._.....- o
/ o ~ f ° i /°
~o __,3-------- ~ ~ - - - ' E - - " - - - ' ~
= ~5,4 .6, .as .i,
=: ENZYME, UNIT/ml Lid
B
. ,8
5 .10 .15 .20 . .50 CYTOCHROME P450,nmolAnl
80
7o
60
5o
4o
50
~o
IO
C
/ 0
/
I I I I I
20 40 6O 8O IOO ( F ~ , pM
Fig 1 Cytochrome P-450 and NADPH-cytochrome P-450 reductase-dependent lipid peroxidation in the presence of Fe3+-ADP complex
(A), Comglete system contained 1.0 mM NADPH, 0.3 nmol of cytochrome P-450; 20 pH Fdh+-ADP, 0.85 jamol of phospholipid liposomes, 0.1 M Tris-HC1 buffer at pH 7.5, and various concentrations of NADPH-cytochrome P-450 reductase in a total volume of 1.0 ml (Curve 1). The following exceptions from or additions to the complete system were made: minus cytochrome P-450, Curve 2; minus Fe3+-ADP, Curve 3; plus 50 pM desferrioxamine methansulfonate, Curve 4. (B), Complete system cogtained 1.0 mM NADPH, 0.1 unit of NADPH-cytochrome P- 450 reductase, 20Ad~ Fe3+-ADP, 0.85jamol of phospholipid liposomes, 0.1 M Tris-HC1 buffer at pH 7.5 and various concentrations of cytochrome P-450, in a total volume of 1.0 ml (Curve i). The following exceptions from or addi- tions to the complete system were made: plus 0.i ~M SOD, Curve 2; plus 0.I heat-treated SOD, Curve 3; plus 20 ~g of catalase, Curve 4; plus 20 mM sodium benzoate, Curve 5; minus NADPH-cytochrome P-450 reductase, curve 6; plus 0.I unit of protease-solubilized NADPH-cytochrome P-450 reductase instead of detergent-solubilized NADPH-cytochrome P-450 reductase, Curve 7; and minus Fe3+-ADP, Curve 8. (C), Complete system contained 1.0 mM NADPH, 0.3 nmol of cytochrome P-450, 0.I unit of NADPH-cytochrome P-450 reductase, 0.85 ~mol of phospholipidlipo- somes, 0.i M Tris-HCl buffer at pH 7.5 and various concentrations of FeS~-ADP in a total volume of 1.0 ml (o o). SOD (O.ijaM) was added to the complete system (r---R).
975
V o l . 1 5 3 , N o . 3 , 1 9 8 8 B I O C H E M I C A L A N D BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S
up to 0.15 nmol/ml. Such a lipid peroxidation was significantly and slightly
inhibited by intact SOD and heat-treated SOD, respectively, but not influenc-
ed by catalase or sodium benzoate, indicating that only 0;, bu£ no "OH, is
involved in the lipid peroxidation. Either omission of detergent-solubilized
enzyme or replacement of detergent-solubilized enzyme with protease-solubili-
zed enzyme significantly suppressed the lipid peroxidation in the recons-
tructed system. A little or no lipid peroxidation occurred when Fe3+-ADP was
excluded from the reconstructed system. These results are shown in Fig lB.
To know the effect of Fe 3+ concentration in the reconstructed system, four
components at fixed concentrations were incubated with Fe 3+ at a variety of
concentrations and lipid peroxidation was detected. As shown in Fig IC,
increasing concentration of Fe 3+ in iron-ADP complex increased lipid per-
oxidation in the reconstructed system, which was significantly inhibited by
indicates that both 02 and Fe 3+ are highly required for promoting SOD. This
phospholipid peroxidation.
O; generation in biological systems is usually measured by cytochrome c
reduction. However, cytochrome c is directly reduced by NADPH-cytochrome P-
450 reductase in the presence of NADPH. Thus, we applied achemiluminescence
probe with MCLA for the determination of O; in our systems. As Shown in
Fig 2~ O; generation in the reconstructed system excluding Fe3+-ADP increased
with increasing cytochrome P-450 concentration, and was parallel to lipid
peroxidation in the reconstructed system, when experimental values were
I . E .6 0 - i
IEn" o @ I I I I I I I i
o .o4 .o8 .ta Je .2o .z4 .2a .~ C'Y'FOCHROME ~450, nmolhnl
Fi~ 2 Correlation between integrated light intensity (0; generation) and lipid peroxidation during i0 min-incubation
Experimental reaction mixture contained 1.0 ~MNADPB, 0.i unit of NADPH-cyto- chrome P-450 reductase, 0.3 nmol of cytochrome P-450, 0.85 ~mol of phospho- lipid liposomes, additive, and 0.I M Tris-HCl buffer at pB 7.5 in a total volume of 1.0 ml. Ten~M MCLA was added to the above reaction mixture for chemiluminescence (o o), while Fe3+(20 ~M)-ADP(0.33 mM) was added to the above system for lipid peroxidation (6 6). Both reactions were performed simultaneously. The experimental value was expressed relative to the value obtained with 0.3 nmol of cytochrome P-450. Each value was corrected for control (excluding cytochrome P-450).
976
Vol. 153, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fi R 3
LIPID PEROXIDATION
02 ~ Fe2~.ADP
NADP'/\2P4 (Fe 202 Possible mechanism of phospholipid peroxidation in the reconstructed system. Abbreviations used are: UPL, unsaturated phospholipid; P-450, cytochrome P-450.
corrected for their corresponding controls (experimental systems excluding
cytochrome P-450).
DISCUSSION
It has been reported that aminopyrine, a substrate of cytochrome P-450-
mediated hydroxylation, inhibits NADPH-dependent microsomal lipid peroxida-
tion~probably by competing for reducing equivalents (16,17) or by interacting
with cytochrome P-450 (5). On the other hand, cytochrome P-450 in oxidized
form is proposed to act as a propagator of lipid peroxidation in the recons-
tructed system containing protease-solubilized cytochrome P-450 reductase,
Fe3+-ADP and liposomes (5). However, there is no direct evidence for the
function of this cytochrome on NADPH-dependent microsomal lipid peroxidation.
The present work clearly indicates that cytochrome P-450 links with its
reductase (detergent-solubilized enzyme) and produces O; at an appreciable
amount, which probably reduces Fe3+-ADP to promote lipid peroxidation (Fig 3).
In Fig 3, the generation of O; was based on the mechanism proposed by
Ingelman-Sundberg and Johansson (9). Fe3+-ADP can be directly reduced by
NADPH-cytochrome P-450 reductase (3). However, in the presence of both Fe 3+-
ADP and cytochrome P-450, electron flux from NADPH to cytochrome P-450 via
reduced enzyme is considered to be much faster than that to Fe3+-ADP. Thus
iron-ADP is mainly reduced by O; released from cytochrome P-450-02 complex
(perferryl ion type compound), in keeping with the case of mitomycin-induced
phospholipid peroxidation (7). Even though further study with highly purifi-
ed cytochrome P-450 or its specific fraction should be done, our reconstruct-
ed system would be very close to NADPH-dependent microsomal lipid peroxida-
tion system.
REFERENCES
I. Hochstein,P.,Nordenbrand,K.,and Ernster,L.(1964) Biochem.Biophys.Res.Commun. 14,323-328.
2. Poyer,J.L.,and McCay,P.B.(1971) J.Biol.Chem. 246,263-269.
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Vol. 153, No. 3, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3. Noguchi,T.,and Nakano,M.(1974) Biochim.Biophys.Acta 368,446-455. 4. Sugioka,K.,and Nakano,M.(1976) Biochim.Biophys.Acta 423,203-216. 5. Svingen,B.A.,Buege,J.A.,O'Neal,F.O.,and Aust,S.D.(1979)
J.Biol.Chem. 254,5892-5899. 6. May,H.E.,and McCay,P.B.(1968) J.Biol.Chem. 243,2296-2305. 7. Nakano,H.,Sugioka,K.,Nakano,M.,Mizukami,M.,Kimura,H.,Tero-Kubota,S.,and
Ikegami,Y.(1984) Biochim.Biophys.Acta 796,285-293. 8. Kuthan,H.,and Ullrich,V.(1982) Eur.J.Biochem. 126,583-588. 9. Ingelman-Sundberg,M.,and Johansson,I.(1984) J.Biol.Chem. 259,6447-6458. i0. Sugioka,K.,and Nakano,M.(1982) Biochim.Biophys.Acta 713,333-343. Ii. Van Der Hoeven,T.A.,and Coon,M.J.(1974) J.Biol.Chem. 249,6302-6310. 12. Omura,T.,and Takasue,S.(1970) J.Biochem.(Tokyo) 67,249-257. 13. Vermilion,J.L.,and Coon,M.J.(1978) J.Biol.Chem. 253,2694-2704. 14. Ohkawa,B.,Ohnishi,N.,and Yagi,K.(1979) Anal.Biochem. 95,351-358. 15. Nakano,M.,Sugioka,K.,Ushijima,Y.,and Goto,T.(1986)
Anal.Biochem. 159,363-369. 16. Wi1Is,E.D.(1969) Biochem.J. 113,333-341. 17. Orrenius,S.,Dallner,G.,and Ernster,L.(1964)
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