THE JOURNAI, OF IDEOLOGICAL CHEMISTRY

Vol. 248, No. 20, Issue of Octolscr 25, pp. 7131-7141, 19i3

J’rinted in U.S.A.

Microsomal Electron Transport

THE ROLE OF REDUCED NICOTINAMIDE ADENINE DIIWCLEOTIDE PHOSPHATE-CYTOCHROME c REDUCTASE IN LIVER MCROSOlIAL LIPID PEROXIDATION*

(Received for publication, January 29, 1973)

THOMAS C. PEDERSON, JOHN A. BUEGE, AXD STEVEX D. AUST

From the Department of Biochemistry, Michigan State lj’~liversity, East Laming, Xichigau 4882s

SUMMARY

NADPH-cytochrome c reductase in rat liver microsomes was solubilized by bromelain digestion and purified to homo- geneity. An antibody preparation obtained by immuniza- tion with this enzyme was found to inhibit the NADPH- cytochrome c reductase activity of both the purified enzyme and intact microsomes by more than 90%. This antibody also inhibited the NADPH-dependent peroxidation of micro- somal lipid which occurs in the presence of ferric ion chelated by ADP (ADP-Fe). The bromelain-solubilized reductase will also promote NADPH-dependent peroxidation of ex- tracted microsomal lipid, but only if ferric ion chelated by ethylenediaminetetraaacetate (EDTA-Fe) is included in the reaction mixture. The optimal conditions for this reaction are those required for the optimal reduction of EDTA-Fe by the purified reductase. Other ferric ion chelators, regardless of whether or not they promoted the reduction of ferric ion, would not replace EDTA in promoting lipid peroxidation. A purified preparation of the microsomal enzyme, NADH- cytochrome bS reductase, which reduces EDTA-Fe, will also promote the peroxidation of extracted microsomal lipid. Intact microsomes, in the presence of ADP-Fe, are specific for NADPH instead of NADH in promoting the peroxidation of microsomal lipids; however, in the presence of both EDTA-Fe and ADP-Fe, both NADH and NADPH promote lipid peroxidation. These results indicate that the NADPH- dependent peroxidation of microsomal lipid involves the ac- tivity of NADPH-cytochrome c reductase, and suggest that an additional microsomal electron transport component is involved. The function of this additional component in the lipid peroxidation reaction can apparently be replaced by EDTA-Fe.

The endoplasmic reticulum, isolated from mammalian liver as a microsomal fraction, is characterized by an NADPII-de-

* Thcsc st~~tlies were supported in part by General Itesearch Support C;r:tnt JCIL05F56 from the (:enerxl Research Support Branch, Division of Research Facilities on l~cso~~rccs, National Institutes of Ilealth, I~Cnvironmentnl Protection Administration Grant 15PA 00801, and ?;ational Institrltes of Health Training Grant GM 1001 from the National Institllte of (ieneral Medical Srienccs.

pendent electron transport system associated with the mixed function oxidase activity responsible for the hydroxylation of a large variety of drugs, steroids, carcinogens, and other lipid- soluble compounds (1, 2). The microsomes will also catalyze an SADPII-dependent peroxidat,ion of endogenous lipid if ferric ions and a chelator such as ADP or pyrophosphate are present (3). This reaction, which involves the transient formation of phospholipid peroxides, destroys the polyunsaturated fatty acid moieties and produces a variety of degradation products in- cluding malondialdehyde, which is measured as an assay of peroxidation activity (4-7). It has been shown that drug metabolism involves the activity of the microsomal flavoprotein, NADPH-cytochrome c reductase (8), and it has bren suggested that this enzyme is also involved in SADPH-dependent lipid peroxidation (1, 9).

The observation that the presence of drug metabolism sub- strates undergoing hydroxylation inhibits SADPI-I-dependent lipid peroxidation in microsomes led to the conclusion that drug metabolism and lipid peroxidation compete for reducing equiva- lents from a common electron transport component (10, 11). However, we found that many of the drug metabolism substrates were good antioxidants and were capable of inhibiting the peroxidation reaction directly. Other substrates, which would inhibit lipid peroxidation only if they mere metabolized! produced hydroxylation products which inhibited the peroxidation reac- tion. This was confirmed by showing that the production of such hydroxylation products also inhibited ascorbate-promoted peroxidation of microsomal lipids.’ This again raised the ques- tion of whether microsomal lipid pcroridation and drug metab- olism share any common elect,ron transport components. We have continued this investigation of the microsomal electron transport system by concentrating our efforts on the lipid peroxi- dation activity to determine \T-hich microsomal components are involved and by what mechanism these components promote XADPEI-dependent pcroxidation of microsomal lipid.

In this paper we present evidence which shows that the NADPH-dependent lipid peroxidation activity in intact micro- somes depends on the activity of NADPH-cytochrome c reduc- tase. We previously reported in a preliminary communication that the purified reductase would ako promote XADPH-de- pendent peroxidation of extracted microsomal lipid under appro-

1 T. C. Pederson, J. A. Brlege, and 8. I). Alwt, lmpltblished obsrrvation.

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priate reaction conditions (12). We will further describe this system and show that, in addition to ferric ion chelated by ADP, the peroxidation activity requires the presence of an additional artificial electron transfer component, ferric ion chelated by cthylcnediaminetctraacetate.

KXPERIME1L‘TAL PROCEDURE

Xefhods

Enzpe Purificatiolz-The m&hod employed to isolate SADI’II-cxytochrome c rcductasc, using protcolytic digestion followvcd by gel filtration and DEAE-cellulose affinity chromatog- raphy, is similar to that of Omura and Takcsue (13). Micro- somcs from the livers of male rats (250 ,::), fed water containing O.l%, phenobarbital 10 days prior to being killed, were isolated by a method previously &scribed (14). The microsomes were then nashetl with 0.05 RI ‘I’ris-HCl (~1-1 8.0 at O-4“) containing 1 m&l ED’I’A. About 2 to 3 g: of washed microsomes were re- suspcndetl ill the same buffer at a concentration of 10 mg per ml and incubated anaerobically with 0.1 mg of bromelain per ml for 3 hours at O-4”. ‘The mixture was centrifuged at 105,000 x g for 60 min and the pellet was discarded. l’hr supcrnatant fraction was concentrated by ultrafiltration on a Diaflo PJI-30 mcmbranc, and this concentrate was applied to a Sephadex G-100 column (2’7 >( 825 mm) and eluted n-ith the same buffer. The fractions containing the XADPI-I-cytochrome c reductase RC- tivity were adsorbed onto a DEAE-cellulose c>olumn (12 x 160 mm) and cluted with a linear 0 to 0.5 M KC1 gradient (total vol- ume, 100 ml) csontaining 0.05 M Tris-HCl (pH 8.0 at O-4”) and 1 rnir EDTA. The peak fractions containing retluctase activity wcrc combined and diluted about 3-fold with 0.05 a~ Tris-I-ICI (pH 8.0 at O-4”) containing 20% glycerol and adsorbed onto a second DEAE-cellulose column (10 x 130 mm) pre-equilibrated n-ith the above buffer. The enzyme was elutctl with a linear 0.15 to 0.35 JI KC1 gradient (total volume, 30 ml) containing 0.05 JI Tris-IICl (pII 8.0 at. O-4”), 20% ,&~rol, and 0.1 m&r EDT& The enzyme in the combined peak fractions from this column could be stored at O-4” for scvnal months with only motlriatc loss of activity.

N;\l)I-r-c~~toc,llrome b5 reductasc was purified by the method of Takesue alit1 Omura (15)) which involved lysosomal digestion to solubilizc> the enzyme. The only change made in the purifica- tion method was the addit)ion of 0.1 maI dithiothreitol to all buffc1.s used after (NH&S04 fractionation to prevent loss of activity as a result of sulfhydryl oxidation (16).

-.lssa,y of C~JfOCkrO7~7e c Reductase Act&U--‘She assays for the reduction of cytochrome c by SADPH were all made with 75 FM cgtochromc c and 0.1 111~1 NADPH in 0.3 JI phosphate buffer, p1-I 7.5, at 25”. The reduction of cytochrome c was measured by following the increase in absorbance at 550 11111 on a Pcrkin-Elmer model 124 spoctrophotometer. The rate is espresed as micro- equivalents of cytochrome c reduced per min Ilsing- an extinction coefficient of 2.10 X IO4 M-l cm-l (17).

Preparation of Antibody to NADI’II-Cutochrowe c Reductase- An adult malt rabbit was immunized with the purified reductase by three weekly, cutaneous injections (abdomen and toe pads). Each injection contained 0.85 mg of the purified reductasc ad- ministered in 6, 2, and 1 ml of 50’/, Frcund’s complete adjuvant, in the lst, 2114 and 3rd weeks, respectively. -1 booster injection (1.0 m$ was given 1 month after the third injection, and 10 days later blood was collected from the car vein and the serum was separated from the whole blood. The y-globulin fraction from both immune and preimmune serum was prepared by the method of Masters et al. (8).

Lipid Peroxidation and Drug Metabolism ASS~,IJS in Nicro- somes-The NADI’H-dependent peroxidation of microsomal lipid was assayed by incubating microsomcs in a Dubnoff shaker at 37” in reaction mistures (usually of 5 ml total volume) con- taining 0.05 Al Tris-HCl (pl-I ‘7.5 at 37”), 2 m3I ADP, 0.12 rnlr Fe(XOJ3, and an JXADI’IIgenerating system containing 7 mAI MgC&, 2 ml1 m-isocitrate, 0.1 m&l SADP+, and 0.05 unit of SADP-isocitrate dehydrogenaee (which had been passed through a Sephades G-50 column) per ml. The reaction was initiated by the addition of the isocitrate and SADI’+, and the extent of lipid peroxidation at various time intervals was determined by removing 0.5-ml aliquots from the reaction misturc and meas- uring the malondialdehyde present by the thiobarbituric acid reaction (18). To prevent any additional peroxidation of lipid during the color development with the thiobarbituric acid rea- gent, 0.01 volume of 2%;; butylated hydroxytoluene in ethanol was added to the thiobarbituric acid reagent just prior to use, producing a finely divided suspension of the antioxidant. Drug metabolism was measured as aminopyrinc demethylasc activity by methods previously published (14).

IWraction of JIicrosomal Lipid and PreparatiojL 0J” Liposomes- Total lipid was extracted from washed microsomes by the method of Folch et al. (19) with the added care of keeping all solvents under nitrogen and performing all operations under nitrogen at O-4” to minimize the auto-oxidation of unsaturated lipids. Lipid was measured as the amount of total lipid phosphorous by the method of Isartlctt (20). The extracted lipid could be kept for scvcral weeks in chloroform-methanol, 2:1, stored at, -2O”, under nitrogen.

Aqueous suspensions of microaomal lipid n‘rrc prepared by,- sonication under anaerobic conditions. This was accompli&cd by transferring an aliquot of the stock lipid solution to a thin walled plastic tube, removing the chloroform-methanol under a stream of nitrogen, adding nitrog‘en-saturated buffer, and then capping the tube under a stream of nitrogen. l‘hc scaled tube was placed in a small glass vessel filled with watcxr kept at ice bath tcmpcrature. The probe of a I~ranson model S 125 sonifier was placed in the outer vessel above the end of the plastic tube and a sonication current of 10 amps was applied for approxi- mately 5 min. The final concentration of lipid in the: suspension was 2.5 pmolcs of lipid phosphorus per ml, and the buffer rou- tinely used was 0.25 M Tris-HCl (pI1 G.8 at 3T”) containing 0.25 31 XaCl.

Lipid Peroxidation of Eriracted Microsown Lipid-‘l’he assays for the pcrosidation of extracted microsomal lipid promoted by the purified cnzgme lvere performed in the following manner. Cnless specified otherwise, reaction mixtures (uar~~dl~ of 5 ml total volume) containing 0.25 JI Tris-IICl (pII 6.8 at 37”), 0.25 11 SaCI, 0.5 pmole of lipid phosphorus per ml, 2 m>r A\DP, 0.22 m>r Fc(N03)~, and 0.1 rnhI EDTA were incubated at 37” under an atmosphere of air in a Dubnoff shaker. The i’orination of malon- dialdehyde was assayed by the method described for the perosi- dation of lipid in intact microsomcs. The rate of oxidation prior to the addition of the enzyme was determined after 11~ addition of 0.2 mhf -1;ADPH to incubation mixtures which had lxxn pre- incubated for 2 min at 37”. The rate of malolldialtl(,llS-de pro-

duction measured after the adclition of enzyme was adjusted for this nonenzymatic rat,e.

I?rgthrocyte IIemolysis-Radical-like intermediate< produced during the lipid peroxidation reaction were detected lvith the use of an erythrocyte hemolysis assay (21). Erytllrocytes, ob- tained from the blood of a young male goat, were washed and packed. The incubation mixtures for erythrocyte hemolysis

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+ FIG. 1. Sodium dodecyl sulfate polyacrylamide disc gel elec-

trophoresis of 40 pg of the purified NADPH-cytochrome c reduc- tase. The sample (in 1% sodium dodecyl sulfate) was heated in a boiling water bath in the presence of dithiothreitol prior to electrophoresis. Above the gel is a densitometer tracing of the gel stained with Coomassie blue.

contained 0.1 ml of packed cells per ml in a reaction mixture containing 0.1 M Tris-HCl (pH 6.8 at 37”), 0.1 M NaCI, 2.0 mM ADP, 0.12 mM Fe(N03)3, 0.05 mM EDTA, and 1 unit of iso- citrate dehydrogenase per ml. The incubation mixtures were preincubated for 2 min at 37” in a Dubnoff shaker, and the reac- tion was initiated by the addition of the substrates necessary for the generation of NADPH: 2 InM sodium isocitrate, 7 mM MgCl,, and 0.1 mu NADPf. The reaction mixture with intact micro- somes contained approximately 1 mg of microsomal protein per ml. The reaction mixture with the purified reductase contained. 2.4 pg of the reductase and 1.0 pmole of lipid phosphorus per ml. The percentage of hemolysis was assayed as described by Pfeifer and McCay (21) after 10 min of incubation in the presence of NADPH.

Other Methods-Protein was determined by the method of Lowry et al. (22) and standardized with bovine serum albumin (Pentex) using e:?&, at 280 and nm equal to 6.6 (23). The ab-

sorption spectrum of the purified flavoprotein was measured on a Cary model 15 spectrophotometer, and the extinction values at 276 nm and 455 nm of the oxidized enzyme were verified on a Gilford spectrophotometer. Sodium dodecyl sulfate polyacryl- amide gel electrophoresis was performed by the method of Fair- banks et al. (24), and the gels were scanned in a Gilford spectro- photometer wibh a scanning attachment.

Materials

Bromelain was obtained as a gift from the Dole Company. Cytochrome c (Sigma type VI), ADP, isocitrate dehydrogenase (Sigma type IV), sodium isocitrate, NADP+, NADPH, thio- barbituric acid, and butylated hydroxytoluene were obtained from Sigma Chemical Company, St. Louis, Missouri. o-Phen- anthroline was obtained from K & K Laboratories, Plainview, New York. All other reagents used were analytical grade.

RESULTS

Puri$cation of NADPH-Cytochrome c Reductase-The purified NADPH-cytochrome c reductase solubilized by bromelain had a specific activity of 56 peq per min per mg as measured by the reduction of cytochrome c. This value is considerably higher than the specific activity reported by Omura and Takesue for their trypsin-solubilized preparation of this reductase (13). The

difference is largely due to their use of lower ionic strength buffer

2.0

3bo 460 5bo 660

WAVELENGTH (nm)

FIG. 2. Absorption spectrum of the purified NADPH-cyto- chrome c reductase (1.61 mg of protein per ml) in 0.05 M Tris-HCl, pH 7.7, 20% glycerol, 0.1 mM EDTA, and about 0.25 M KC1 at ambient temperature. -, oxidized; - - -, reduced by addition of 0.1 mM NADH; a. .*, reduced by Na&Od.

in the cytochrome c reductase assay. The reduction of cyto- chrome c by this enzyme requires high ionic strength for optimal activity (25, 26). The purity of the enzyme was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results, shown in Fig. 1, indicate a single band with an apparent minimal molecular weight of 71,000. The absorption spectrum of the purified enzyme, shown in Fig. 2, has a characteristic flavin absorption spectrum which disappears upon reduction. Furthermore, when the enzyme was aerobically reduced by NADH instead of NADPH, a half-reduced flavin spectrum was observed. The ratio of the extinction at 276 nm to that at 455 nm of the oxidized enzyme was 6.7. Passage of the enzyme through a short Sephadex G-25 column did not change any of its spectral properties. These spectral characteristics are similar to those reported by Omura and Takesue (13).

Involvement of NADPH-Cytochrome c Reductase in Microsomal Lipid Proxidation-The y-globulin from the serum of a rabbit immunized with the purified reductase had a high antibody titer against the reductase, as evidenced by the formation of a single immunoprecipitin line in double diffusion agar plates using both the purified reductase and detergent-solubilized microsomes.2

2 A. F. Welton, T. C. Pederson, J. A. Buege, and S. D. Aust, manuscript in preparation.

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0, 00 0.1 02 03 04 05 06 07 08

FIG. 3. Inhibition of NADPH-q%ochrome c (C?/lI.-c) reductase activity by antibod:- to the enzyme. ---, r-globulin from im- mune serum; -----, T-globulin from preirnm~me serum. Assays n-ith purified reductase ( l ) contained 0.1 pg of the enzyme per ml; assays n-ith intact microsomes (0) contained 10 pg of microsomal protein per ml. .Lssays n-ere performed as described under “Methods.”

TABLE I

Comparison of inhibition by antibody of activity of A’ADPH-cyto- ch,ott~e c reductase with either cytochrome c or ferricyanitle

as terminal acceptor

All assay mixt\wcs contained 0.2 .ug of the purified enzyme per ml and, nhcrr indicated, 0.1 ml1 K,Fe(CN)s. In the assays con- taining K$e(CN)G, the activit.y was assayed as XAl>PH oxida- tion by observing the change in absorbance at 340 nm. All other conditions were the same as those described in the legend to Fig. 3.

Reaction mixture

Colltrol. Pl11s r-globulin front immune

scr1m1

Reductase activity

51 37 4%

67

The antibody was also a wry good inhibitor of the SADPII-cyto- chrome c rcductasc activity of the enzyme. ils shown in Fig. 3, the activities of both the purified reductasc and the intact microsomes wcrc inhibited more than 9Oc/; by y-globulin from immune serum, and no inhibition was observed with y-globulin from prcimmunc serum. The specificity of this inhibition was further sl~owr~ by the fact that the microsomal NADH-cyto- chronic c reductase activity, assayed under the conditions de- scribed in the legend to Fig. 3, m-as unaffected by concentrations up to 0.89 mg of y-globulin from immune swum per ml. The degree of inhibition was found to depend on the terminal electron acceptor. As shown in Table I, the antibody IT-as only about onc- half as effectirc at inhibiting the activity in the presence of fer- ricyanide as in the presence of cytocliromc c. The involvement of S~~DPII-cytochrome c reductase in microsomal KADPH- dependent reactions was investigated using the antibody to the purified enzyme in the microsomal reaction mixtures. As shown

1 molt of 1,11)1’.1; ~11 )I’-Fe, ferric ion chelatrd 1)~ :I 17.fold molar excess ot’ ;\1)1’; iY Is:;\l, ~~‘-eth~lnlal(~imid~~.

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in Fig. 4, both lipid pcroxidation and aminopyrinc tlcrnethylase activity wcrc significantly inhibited by y-globulin from immune serum. In contrast, the y-globulin from preimmune serum had no effect on aminopyrine demethylat.ion and caused only slight inhibition of lipid pcroxidation activity.

Peroridaiion of Extracted ,q,! icrosowal Lipid ~JJ Purified

~rADPM-C1lt~~Ilro/,le c Reductase--The purified rcductase will promote SXDPII-dependent peroxidation of an aqueous dis- persion of extracted microsomal lipid as assayed by measuring the formation of malondialdehyde. An illustrative example of the time course oi’ this reaction, demonstrating the measurement of the peroxidation activity prior to and following the addition of enzyme to the reaction mixture, has been published previously (12). The basic requirements for the reaction arc listed in Table II, showing that the peroxidation promoted by the purified re- ductase does require SADPH, and that heating the enzyme at 100” destroys the peroxidation actirity. The reaction also re- quires that two forms of ferric ion be present in the reaction mixture. EDT&Fe3 must be included, in addition to the ferric ion added as hDl’-Fe. The presence of ADP is not required for peroxidation activity in this system, but it was included since it is required in the reaction mixture for the lipid peroxidation activity promoted by intact microsomes (3). This lipid peroxi- dation reaction was also inhibited by the y-globulin fraction from the antiserum to SADPII-cytochrome c rcductasc (Table III). However, the degree of inhibition by the antibody in this peroxi- dation system, which includes ADP-Fe and EDTA-Fe, was significantly less than that observed in the perosidation pro- moted by intact microsomes in the presence of ADP-Fe.

The correlation between the rate of malondialdchyde produe- tion and the amount of reductase is shown in Fig. 58. A similar relationship bctn-ccn activity and the amount of lipid used is shown in Fig. 5B. 1Zoth show relatively linear correlations at the lowc~ concentrations used. The rate of malondialdehyde production as a function of pH is shown in Fig. 5C. As can be seen, the optimal activity occurs at about p1-I 6.8, but the activit? decreases quite rapidly above pH 7 in contrast, to lipid perosi- dation in ~~hole microsomes, which still proceeds readily at pH 8 (27). The lipid per oxidation reaction catalyzed by the purified reductasc rcquircs high ionic strength, wit,h the optimal concen- tration of SnCl bciug 0.4 M or greater, as shown in Fig. 50. In the prrsc’ncc of 0.25 M Tris-HCl, 0.25 M KaC1 will allow op- timal activity. ‘I‘hc relationship between the ionic strength and the pcroxitlation activity in this system, which albo contains EDTLFc, is similar to the relationship betwtlcll the ionic strength aucl the IXU’A-Fe reductase activity of the purified reductasc described by Iiamin and Masters (26).

In addition to SADPH-dependent productiol~ of malondi- aldrhydc, further gcncral similarity between the lipid peroxida- tion activit,y in whole microsomes and the reaction (Bntalyzed by the purified reductase was shown with the erythrocytc hcmolgsis assay used by Plcifcl, and &Cay (21) to dcmonstrat v the produc- tion of a transient, radical-like species during the SADPH- dependent peroxidation of lipid in whole microsomc,s. After 10 min of incubation in the presence of KSDI’II, the pc,roxidation reaction mirturc containing extracted microsomal lipid, pur- ified reductase, -IDI’-Fe, and EDTA-Fc cnn~d 81(>1 hemolysis of the ergthrocytcq included, whereas the pcroxidation reaction mixture containing intact microsomes and alDP-Fc caused 90y0

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FIG. 4 (rrpper). Inhibition of mi- crosomal NAI>PH-dependent lipid peroxidation and aminopyrine de- methylation by antibody to NADPH- cytochrome c reductase. --, y- globulin from immune serum; ----- r-globulin from preimmune serum: All reaction mixtures contained 0.125 mg of microsomal protein per ml. The lipid pcroxidation activity in the absence of r-globulin promoted the formation of 1.2 nmoles of malondial- dehyde per min per ml. The amino- pyrine demethylation activity under the same cwnditions produced 1.5 nmoles of formaldehyde per min per ml. Assays v\-ere performed as de- scribed under “Methods.”

FIG. 5 (m,idcl/e). Effects of various reaction conditions on the lipid per- oxidation activity promoted by puri- fied NAI)PH-~ytochrome c reductase. .4ssays were performed as described under “Methods.” -4, activity as a function of enzyme concentration; B, activity as :L ftmction of the micro- somal lipid conc’entration; C, activity as a funct,ion of pH. All assay mix- tures in I{ and C contained, per ml, 0.35 ,~g of a preparation of the reduc- tasr which had lost about one-half of its enzymatic activity. D, activit! as a fllnctiori of salt concentration. l , \vit,h 0.05 M Tris-HCl; 0, with 0.25 nr Tris-lIC,‘l. All reaction mix- tures contained 0.06 ~g of the pllrified redllctase prr ml.

FIG. 6 (locr~r). 1,ipid peroxidation as a flmctiorr of I,:I)T.4-Fe concentra- tion. All wac~tion mistllres cow tained 0.32pg of the plu’ified redactase per ml and 0. I2 mu Fe(N03)~ chelated by 2.0 m,w Al)l’, in addition to the concentration of I<:I)TA-Fr indicated. i\ssnys WPW pc,rl’ormcd as described under L‘Methodr ”

60

>I .Z .>

,” 40

3

20

I 0, O(

‘Wd P.~..,.&,i.n 4 1 011 0’2 013 oi4

$-Globulin, mg ml-’

1 2.0 i

C

%

:

0.0

- 1 100 2bo

Enzyme(ng/ml)

B

I l.O( ./’

--e----4

i i

I 0.25 0.50

Lipid(pMoles P/ml)

5 0.04

0.0 0.2 04

PH [NaCfl M

hemolysis. Neither reaction system caused erythrocyte hemoly- sis in the absence of SbDPI-I.

Role 01 BDTI-Fe in Lipid Pero.ridnlion-In addition to ADP-

Fe, normally required for microsomnl NADPII-dependent lipid perosidatiotl, the> peroxidation reaction catalyzed by the purified rcdurtase rcquirctd the l)resence of EDTA (12). However, as the concent~ration of ICUT. approached 0.12 mM (the concentration of ferric ion), inhibition occurred, suggesting that chelating all

the ferric ion with EDTA inhibited the reaction. Therefore, the optimal level of EDTA was determined under conditions in which the concentration of ADP-Fe remained constant by adding ferric ion chelated by a molar equivalent of EDTA (EDTA-Fe). Under these conditions, no inhibition was observed at higher concentrations of EDTh (Fig. 6). The concentration of EDTA- Fe routinely used in the lipid perosidation system was 0.1 m;\r, al- though there is still some increase in the peroxidation activity

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Reqzcirements for peroxidation of lipid catalyzed by purified NADPH-cytochrome c reductase

The complete system contained 0.13 pg of the purified reductase per ml. ilssays were performed as described under “Methods.”

Reaction mixture Malondialdehyde formed

nnzoles/miw/ml

TABLE II TABLE IV

Lipid peroxidation catalyzed by puriJied ATADPH-cytochrome c reductase with other ferric ion chelators used in place

of EDTA

All chelates other than KzFe(CN)e were prepared with FeC13 and the indicated chelator in a I:1 molar ratio. The concentra- tion of each chelate in the reaction mixture was 0.1 mM. Each reaction mixture also contained 0.12 mM Fe(N03)3 and 2 mM ADP. The activity in parentheses is the rate observed 5 min after the reaction had been initiated as compared to the initial rate. Assays were performed as described under “Methods.”

Complete............................. 2.40 Boiled enzyme. I 0.00 Minus NSDPH . 0.04 Minus ADP-Fe. 0.00 Minus EDTA-Fe. 0.10

Chelate NADPH oxidase Malo;i&;lddhyde

Comparison of inhibition by antibody of lipid peroxidation promoted by either puri$ed reductase or intact microsomes

The reactions promoted by the purified reductase contain 0.2pg of the enzyme per ml, and the reaction promoted by intact micro- somes contains 0.13 mg of microsomal protein per ml. Assays were performed as described under “Methods.”

None ........................... EDTA-Fe. ..................... Citrate-Fe ...................... o-Phenanthroline-Fe. ........... K,Fe(CN)G .....................

Ireq/mwmg nmoles/min/ml

0.50 0.11 22.6 2.46

0.21 0.23 1.10 0.15 (1.65)

78.0 0.09 I

TABLE V

Reaction mixture

Control. Plus r-globulin from immune

serum 0.1 mg per ml. 0.4 mg per ml.

Plus r-globulin from preim- mime serum

O.lmgperml_.,_.__.._._. 0.4 mg per ml.

- - Malondialdehyde formed

- With purified reductase and extracted lipid

?moles/ n~in/nzl

2.10

1.12 0.59

2.00 l.iG

Y" GO?kl701

53 28

95 84

With intact microsomes

nmoles/ miw//ml

1.20

0.19 0.05

1.10 0.95

L

% control

16 4

92 80

observed at higher concentrations. This concentration is similar to that required for optimal EDTA-Fe reductase activity

catalyzed by the purified reductase (26). Several other ferric ion chelators were used in place of EDTA, and t,he ability of these chelates to promote XdD1’I-I oxidation as well as lipid peroxidat,ion was investigated. As sholvn in Table IV, none of the substitute ferric chelates would replace EDTB-Fe in the lipid peroxidation system even though some stimulated the SADPH- oxidase act,ivity of the reductasc.

Since the microsomal flavoprotrin NADH-cytochrome bS

reductase will reduce fcrricyanide (15) aud has been suggested to reduce EDTAL1-Fe (28), its ability to reduce E:DTA-Fe was in-

vestigated. When the activity of a purified preparation of Nnl)H~cytochrorue b5 rcductase was assayed under the conditions

used in the lipid peroxidation assay, the presence of EDTA in- creased the rate ol ?;ADII oxidation by 13 pcq per min per mg, compared with an increase of 25 peq per min per mg in the NRDI’H osidase activity of NBDPH-cytochrome c reductase. When pu&ed NADH-cytochrome bj reduct,ase was used in place

of S=lDI’I~-c~tochrorne c reductase, NADH readily promoted the peroxidation of cxtrnctetl microsornal lipid (Table V). As has been previously reported (27, 29), n’,lDH will not replace N;\DPII in promoting the rapid peroxidation of lipid in intact

microsomrs in the prescuce of ADP-Fe. HoJvever, in the prcs- encc of both EDT&Fe and ADP-Fe, RADII is just as effective

7139

NADH-dependent lipid peroxidation in presence of EDTA-Fe

The reaction mixtures for the peroxidation of extracted micro- somal lipid contained either 0.22 mg of purified NADPH-cyto- chrome c reductase or 0.38 mg of purified NADH-cytochrome 65 reductase per ml. All other conditions were the same as those described under “Methods.” The reaction mixtures for lipid peroxidation in intact microsomes contained 0.2 mg of microsomal protein per ml. The assays were performed as described under “Methods,” except that NADH (0.5 mM), NADPH (0.5 mM), and EDTA-Fe (0.1 mb) were added as indicated.

Reaction mixture

Extracted microsomal lipid in presence of ADP-Fe and EDTA-Fe plus

NADPH & NADPH-cytochrome c reductase NADH & NADH-cytochrome bj reductase

Intact microsomes in presence of ADP-Fe plus NADPH NAl1H.........................................

Intact microsomes in presence of ADP-Fe and E:DTA-Fe plus

NADPH........................................ NADH .

Malondialde- hyde formed

2.19 2.00

2.38 0.48

4.14 4.24

as NADPH in promoting microsomal lipid peroxidation. This

can be correlated with the observation that, under the conditions used in the lipid peroxidation reaction, EDTA-Fe increased the N1ZDH oxidase activit,y of microsomes by 0.067 peq per min per

mg and the IY’BDPH oxidase activity by 0.060 peq per min per mg.

DISCUSSIOS

Antibodies to the rnicrosomal flavoprotcin, NADI’I-I-cyto- chrome c reductase, which also inhibit’ the activity of the enzyme,

have been used by other investigators to show that this enzyme is the initial component in the XADPH-dependent electron trans- port systcrn associated with the cytochrome P-450-catalyzed hy- droxylation of drugs and other compounds (8, 30). In this paper we have shown that antibodies to this enzyme mill inhibit the

S,4DI’I-I-dependent perosidatiou of endogenous microsomal

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TABLE VI

The NI:1Ltrc~:1trtl microsomes were prepared by incubating microsomcs (-2 mg of protein per ml) with 10 mlf Kl*XL\I in 0.05 M

Tris-IICl, pII 7.7, at 25” for GO min anti then passing them through a ,Seph:nlc~s G50 c01un111. The untreated microsomes were also passed throrigll the Sephndex colunm. The X‘\;AI)l’H-cytochrome c redrrctasc activity in the untreated microsomcs was 0.52 peq per min per rrrg; that in the ?;J,AI-treated microsomes was 0.02 ~eq per min per nrg. The lipid peroxidation reaction mixtures con- tained O-4 mg of protein of the microsomal preparation indicated per ml. The rcac%ion mixtures including the purified XADPH- cytochronrc c redrrctase contained 5 ~g of the redrrctase per ml, and the .I<:1 )TiZ-Fc was added at a concentration of 0.1 rnM where indic:Ltotl. All other conditions are those described under “nleth- ods” for the lipid pcroxidntion assay in microsomes.

Reaction system Malondialde- hyde formed

Untrcatcvl microsonres ?;lClI-l.lc:rtcd microsomcs n’l~:Xtreatctl nricrosomcs plus pruifictl rcduct:rse P\‘I*:lI-trratcd niicrosomes plrrs purified redlrctnsc

and 1~:1)‘1’.2-Fc 4.90

lipid a11(1 aminolryrinc demcthylation, iudicating that the activity of N~11>1’11-c~~toclirorllr c reductase is also involved in pro- moting the lipid pcroxidation renrtion. A bromelain-solubilized preparation of this enzyme will also promote the peroxidation of extracted microsomal lipid if EDT&Fe is also included in the reaction mixture. IKWA-Fe is required in addition to the ADP- Fe required for the S;~1-)PH-tl~pendent peroxidation promoted by intact microsomes (3, 31).

‘l’hc S-\l)I-‘ll-del)entlent peroxidation of extracted microsomal lipid can be promoted by a trypsinsolubilized preparation of the enzyme’ or, as prcviouslg reported (IL?), by t,hc KADPH-cyto- chrome I’-450 rcductase fraction isolated with the use of deoxy- cholatc by Strobcl et al. (32). In each case, the lipid peroxidation activity i,equircls thcl presence of EDTL4-Fc.1 Therefore, the re- quiremc,nt for JWl’h-Fe iii the reaction mixture is not due to proteolytic 2alternt,ion of the rcductase. ‘l‘hc purified reductase prepared by bromclain solubilization has an apparent minimal molecular weight, of 71,000, as determined by sodium dodecyl sulfate polyncryl:rmitlc ~;rl electrophoresis. 11-c hacc found that the rcchictase iii intact nricrosomes, prior to protease treatment, has ali apparent mininnd molecular weight of 79,000.2 Or- renius et nl. (9) demonstrated that as soon as the reductase is released from the micro,somnl mcmbrauc by tryptic digestion the rcductasf: will apparently no longer couplf with the microso- ma1 electron transport system, since both drug hydroxylation and lipid pcrositlat)ion activities are lost in parallel with the solubilization of 11~~ reductasc. 11-c also attempted to recon- st,itutc lipid pci,ositlation using L~~T-etl~ylmnleimirie treatment to iuactivatc the microsomal reductasc and theii ntldiilg back an equivalent amount of purified reductasc. AR shown in Table VI, the SEAI-trcatcd microsoniei, 1vliicli have lost nearly all their S~1D1’TI-c~tocl~ron~e c reducta,qc activity, do not become susccptiblc to S;\I)I’II-clcl,eiideilt lipid pcrositlation in the presence of .-Krl’-Fr when purified retluctase is added back. Yet the lipitl in the SI~LCtreated microsomcs is just as susceptible to pcroxitlation as is extracted microsomnl lipid, since in the presence of both ADI’-Fe and EDT&Fe the purified reductase

will readily promote the peroxidation of cntlogenous lipid in the SERI-trcatcd microsomrs.

EDl’&Fc iH wadilg reduced by the purified rctluct,asc in the

presence of high ionic strength (26), and lipid pcroxidation ac- tivity promoted by the purified reductasc also requires high ionic strength, suggesting that it is the reduction of EDTA-Fe that is responsible for promoting the peroxidation activity. This is supported by the observation that the microsomal enzyme RADII-cytochromc b, rcductase will reduce ED’l’d-Fe and will also promote S,~I~I~I-dcl~er~de~~t peroxidation of extracted mi- crosomal lipid in the presence of both ED’I’A-Fe and ADP-Fe. Furthermore, the presrnce of EDT&Fe in a peroxitlation re- action mixture containing: intact microsomcs enables KADH to promote lipid pcrosidntion just as readily as NADPH, which can be related to the observation that NADH reduced EDTA-Fe just as readily as KADPH in intact microsomes. However, several other ferric ion chelates, including those which arc reduced by the enzyme, would not replace EDTA-Fc in the pcrosidation reaction, indicating that EDTAFe has additional properties rrhich account for its role in the lipid peroxidation reaction. It is these properties which are likely responsible for the shift to a more acidic pH optimum for the lipid pcrosidation activity promoted by the purified reductase. A similar change to an acidic ~1-1 optimum has been observed in the pcroxidation of microsomal lipid promoted by ascorbic acid arid ferric ion (27).

In intact microsomes, in the presence of ADPFc, the peroxi- dation of endogenous lipid is specifically promoted by KL4DPH. HoIvever, in the presence of both EDT&Fe and 11DP-Fe, both NADH and NADPII readily promote lilpid peroxidation. This suggests that an additional microsomal electron transport com- poncnt, specifically reduced by ?;ADl’lI-cvtochromc c rcductase, is involved in promoting the SXDI’I-I-depelltleIlt peroxidation of microsomal lipid. In the perosidation of extracted microsomal lipid promoted by the purified rrductase, the function of this microsomal component is replaced by EDTAFe. The exist,cncc of such a microsomal component is also supported by the observed effect of the antibody on peroxidation activity in the two systems. The antibody is much more effcctivc at inhibiting NADPII-dependent peroxidation activity in intact microsomes than the pcroxidation activity promoted by the pu- rified rcductase in the presence of EDTA-Fc. This is analogous to the observation that the antibody is much more effective at inhibiting the reduction of cytochrome c, a macromoleculr, than ferricyaiiidc reduction. If there is a microsomal electron trans- port component in additioii to S-‘lDPH-cytochrolle c reductase involved in the SADI’H-dcpendcnt, lipid peroxidation activity, its identity remains unkiiown, but it may likely bc inrolvcd in other microsomal activities.

The prcsc~ce of ferric ion not chelated by I*:DTA is required for malondialtl~~hyde formation in all the reaction sy+ms for the peroxidation of polyunsaturated lipid (5, 31, 33)) including those which employ obviously different means of promoting peroxidation (34). Tt is likely that the role of this ferrir ion is in the pcrosidxtirc generation of free radicals which initiate radical chain oxidation and the formation of &gradation prod- ucts, including malondialdehyde. The gcncration of such a reactive radical species during SADPI-I-depcndcut lipid perosi- dation in intact microsomes, as detected by the hcmolysis of erythrocytcs (al), has also been shown to occur during the peros- idation of extracted microsomal lipid promoted by the purified reductasc. All the results presented in this paper were obtained under reaction conditions in Tvhich all ferric ion, other than that’ chelated by EDTA, was chelated by ADP, because ADP is

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7141

normally required for optimal pcroxidation activity in intact 15.

microsomes (3). ADP is not required as a chelator of ferric ion in other peroxiation reactions (34)) including the peroxidation

16.

of extracted lipid by the purified reductase. The role of ADP :A: may be to prevent precipitation of ferric ion at neutral pH or chelation of ferric ion by the other nonspecific chelators in the 19. reaction system. This will be a subject of subsequent publica- tions. 20.

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Thomas C. Pederson, John A. Buege and Steven D. AustLIVER MICROSOMAL LIPID PEROXIDATION

INADENINE DINUCLEOTIDE PHOSPHATE-CYTOCHROME c REDUCTASE Microsomal Electron Transport: THE ROLE OF REDUCED NICOTINAMIDE

1973, 248:7134-7141.J. Biol. Chem. 

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