May 6, 2018 - lyzed protein oxidation, which is accelerated by AH, and inhibited by ..... 20 1.1~ AH,; 4, NADPH + 100 unit of SOD; 5, NADPH + 50 m~ GSH; 6,.
T m JOURNAL OF BIOUXICAL CHEMISTRY 0 1994 by The American soeiety for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 18, Issue of May 6, pp. 13390-13397, 1994 Printed in U S A .
NADPH-initiated Cytochrome P450-mediated Free Metal Ion-independent Oxidative Damageof Microsomal Proteins EXCLUSIVE PREVENTION BY ASCORBIC ACID* (Received for publication, October 25, 1993, and in revised form, January 21, 1994)
Chinmay K. Mukhopadhyay and InduB. ChatterjeeS From the Department of Biochemistry, University College of Science, Calcutta 700 019, Zndia
In this paper we demonstrate that NADPH-initiated cally relevant of these is the NADPWcytochrome P450 (cyt oxidative damage of microsomal proteins occurs in the P450)reductase/cytochrome P450/Fe(III)/02 system. Ascorbate absence of free metal ions and that this protein oxida- (AH,) has often been added to the MCO system to accelerate Oxi- protein oxidation. In fact, AHJOJFe(II1) has been a favorite tion is mediated by cytochrome P450 (cyt P450). dized proteins are rapidly degraded by proteases. As- nonenzymatic MCO system. However, these i n vitro studies corbate (AH,) specifically inhibits free metal ionwith added iron salts may have less relevance to the i n vivo independent cyt P45O-mediated protein oxidation and situation where in the normal physiological condition most of thereby prevents subsequent proteolytic degradation. the metals are not freeand remain tightly boundwith proteins. Other scavengers of reactive oxygen species including In this paper we demonstrate that NADPWcyt P450 reductase/ superoxide dismutase, catalase, and glutathione are in-cyt P450 system produces oxidative modifications of microsoeffective. This is in variance with free metal ion-catamal proteins in the absence of free metal ions and that the lyzed protein oxidation, which is accelerated by AH,and inhibited by catalase. Oxidative damage of proteins hasoxidized proteinsare rapidly hydrolyzed by different proteases. been assessedby the production of carbonyl groups, bi-We further demonstratethat whereas in the presence of ADPFe3+oxidation of proteins is accelerated by AH,, cyt P450-metyrosineformation,andtryptophanloss.Themechanism of protein oxidation has been studied using a re- diated protein oxidation in the absence of free metal ions is exclusively preventedby AH,. Other scavengers of reactive oxyconstituted system comprised of purified NADPH-cyt P450 reductase, cyt P450, and isolated microsomal pro-gen species including superoxide dismutase (SOD), catalase, teins as well as model polypeptides, e.g. poly-L-proline and glutathione are ineffective. Using a reconstituted system and poly-L-lysine. Cyt P450 FeS+ is reduced by NADPH- comprised of purified NADPH-cyt P450 reductaselcyt P450 and isolated microsomal proteins as well as poly-L-proline and polycyt P450 reductase to cyt P450 Fe2+, which consumes L-lysine, we have studied the mechanisms of oxidative damage oxygen in a stoichiometric proportion to produce cyt P450 Fe2+0,, the resonance form of which aisperferryl of the microsomal proteins or the model polypeptides and the moiety, cyt P450 FeS+-O,. It is proposed that cyt P450 inhibition by AH,. FeS+.O; abstracts hydrogen from amino acid side chains MATERIALSANDMETHODS leading to the production of carbonyl derivatives. TenNADPH, fluorescamine, glutathione (GSH),bovine erythrocyte SOD, tatively, AH, prevents protein oxidation by interacting AH,, mannitol, poly-L-proline(M,30,000),poly-L-lysine(M,30,000with cytP450 FeS+-O,. 70,000), and ADP were purchased from Sigma. Catalase (free of SOD) was obtained from the Centre for Biochemicals,New Delhi, India. a"& copherol was a gift from E. Merck (India). PhenylmethylsulfonylfluoReactive oxygen species can oxidatively modify enzymes and ride (PMSF), 1,lO-phenanthroline, and 2,4-dinitrophenyl hydrazine (DNPH) were obtained from Merck Darmstadt, Germany. Di-ethyltriproteins in intact cellsand intracellular organellesas well as in amine pentaacetic acid (DTPA)was obtained from Kock-Light Laborathe in vitro system (1, 2). The oxidatively modified proteins tories Ltd., United Kingdom. Desfemioxamine was a gift from Cibabecome highly susceptible to proteolytic degradation by various Geigy, Basel, Switzerland. Antibody to cytochrome P450 LM2 was a proteases (3-7). The reactive oxygen species (O,, H,O,, 'OH) generous gift from Prof. G. Padmanaban, Indian Institute of Science, are produced by a variety of enzymatic and nonenzymatic sys- Bangalore, India. Preparation of Microsomes-Microsomal fractions were prepared tems, generally referred to as metal ion-catalyzed oxidation from liver homogenates (1:4, w/v) of young male (350-450 g) guinea pigs (MCO)' systems (1, 2). Irrespective of whether the system is in 1.15%KC1 by centrifuging at 9,000 x g for 20 min for two successive enzymatic or nonenzymatic, all MCO systems, thus far detimes, followed bycentrifugation at 105,000x g for 1h. The microsomes scribed, appear to require the presence of free transition metal were washed three times with 1.15%KC1 and suspended in 0.1 M potassium phosphate buffer, pH 7.4, at a concentration of approximately ions in the incubation medium. Perhaps the most physiologi10 mgproteidml of suspension. The microsomes were kept in aliquots at -20 "C and used within 48 h of preparation. Heat-denatured micro* This work was supported in part by a grant from the Council of somes wereprepared by heating themicrosomal suspension for 10 min Scientific and Industrial Research. The costs of publication of this ar- at 100 "C followed by homogenization to a uniform suspension. tide were defrayed in part by the payment of page charges. This article Preparation of Delipidized Microsomal Protein-Microsomal protein must therefore be hereby marked "advertisement"in accordance with 18 (5 ml = 50 mg of protein) was delipidized by homogenizing freshly U.S.C. Section 1734 solely to indicate this fact. prepared microsomal suspension with two volumes of a mixture of $ To whom correspondence should be addressed: Dept. of Biochemistry, University College of Science, 35 Ballygunge Circular Rd., Calcutta dichloromethane/methanol (l:l, v/v) in thepresence of desfemioxamine in nitrogen atmosphere, followed by centrifugation at 1,000 x g for 5 700 019 India. The abbreviations used are: MCO, metal ion-catalyzed oxidation; min. The upper methanol layer was collected.The process was repeated AH , ascorbic acid; SOD, superoxide dismutase; cyt P450 or cyt P450 twice tofree the microsomes fromany contaminating lipid. The methaFe3', cytochrome P450;DTPA, diethyltriamine pentaacetic acid; PMSF, nol layer was then centrifuged at 18,000 x g for 15 min to form a pellet. phenylmethylsulfonylfluoride; PAGE, polyacrylamide gelelectrophore- The pellet was washed with ethyl ether, vortexed, and centrifuged two times, dried under nitrogen, and finally suspended by homogenization sis; DNPH, 2,4-dinitrophenyl hydrazine. ~~~~
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Ascorbic Acid Prevents Oxidative Damage in 50 m~ potassium phosphate buffer, pH 7.4. Estimation of Protein-Proteins were estimated according to the method of Lowry et al. (8). Incubation System-Incubation mixture using microsomal suspension contained 1mg of microsomal protein in a final volume of 250 pl of 50 nm potassium phosphate buffer, pH 7.4. NADPH (0.24 nm or a s indicated) was added to initiate the reaction. For NADPH-initiated protein oxidation in the reconstituted system, 300 pl (final volume) of the incubation mixture contained 0.55 nmol of cyt P450 and 96 units of NADPH-cyt P450 reductase and 1 mg of protein in 50 m~ potassium phosphate buffer, pH 7.4. Reaction was initiated with NADPH (0.24 mM). Sodium Dodecyl Sulfate-Polyacylamide Gel Electrophoresis (SDSPAGE)-10% polyacrylamide gel electrophoresis in thepresence of SDS was performed according to the method of Laemmli (9).The gels were stained with Coomassie Brilliant Blue R-250. Degradation of proteins was quantitated wtih a LKB 2202 ultroscan laser densitometer. The staining intensities of protein bands were converted to electrophoretic peaks for analysis. Protein concentration was calculated from the integration of peak areas. Fluorescence Measurement-Fluorescamine reactivity (lo), bityrosine production, and tryptophan destruction (11)were measured with a Hitachi fluorescence spectrophotometer model F 3010. Assay of Carbonyl Content-Protein carbonyls were measured by reaction with DNPH as described by Levine et al. (12).After incubation with NADPH for required time in the presence of PMSF and EDTA to minimize proteolytic degradation and loss of protein carbonyl, the protein was precipitated with 20% trichloroacetic acid and the carbonyl content was measured in theprecipitate. A blank preparationin 2 M HCI was kept. The difference in absorbance between the DNPH-treated sample versus the HCl control was determined at 390 nm. The results are expressed as nanomoles of carbonyl groupdmilligram protein using a molar coefficient of 22,000 for the DNPH derivatives. Purification of Cyt P450 Reductase and Cyt P450-Both the enzymes were purified from liver microsomes of phenobarbital-treated rabbit according to the method of van der Hoven and Coon ( 13).The preparation contained 6.5nmol of cyt P45O/mg protein as assayed by the method of Omura and Sato (14) and 63 units of cyt P450 reductase/mg protein as assayed by the method of Masters et al. (15). Measurement ofoxygen Consumption-For the measurement of oxygen consumption during NADPH-initiated protein oxidation in the reconstituted system, the incubation mixture was the same a s described before under Incubation Systems except that thevolume of the incubation mixture was 2 ml instead of 300 pl and theamounts of protein, cyt P450 reductase, and cyt P450 were increased in the same proportion. Oxygen consumption was estimated by oxygraph using Clark electrodes.
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kD
U
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FIG.1. NADPH-initiated time-dependent degradation of microsomal proteins asevidenced by SDS-PAGE. One mg (-100 pl) of microsomal suspension was incubated at 37 “C with 0.24 mM NADPH in a final volume of 250 pl as described under “Materials andMethods.” m e r required time of incubation, 25 1.11of the incubation mixture were withdrawn and subjected to polyacrylamide gel electrophoresis under denaturing condition. Lune 1, unincubated; lane 2, after 15 min of incubation; lane 3, after 30 min of incubation; lane 4, after 45 min of incubation; lane 5, after 60 min of incubation; lune 6, after 60 min of incubation without addition of NADPH.
IS
RESULTS
NADPH-initiated Degradation of Microsomal ProteinsAlmost all the invitro systems thus fardescribed for oxidative degradation of proteins depend upon the presence of free transition metal ions in the medium. However, we have observed that oxidative degradation of microsomal proteins occurs independently of added metal ions. Fig. 1 shows that addition of NADPH to the microsomal suspension in theabsence of added metal salts results in rapid degradation of the microsomal proteins ina time-dependent manner, as evidenced by SDS-PAGE. No degradation was obtained in the absence of NADPH. In addition, boiled microsomes were ineffective. Analysis of the SDS-PAGE by densitometric scanning (Fig. 2) indicated progressive loss of protein bands to the extentof about 92% after 1 h of incubation. In separate experiments using CO-binding assay (14), we observed a progressive loss of cyt P450 during the incubation. About 64% ofthe cyt P450 was lostafter 30 min and 80% after 1 h. A similar observation was made with cyt P450 reductase activity (15).About 67% of the activity was lost after 30 min and93% after 1h of incubation. Earlier pioneering studies indicate that MCO the system comprised of NADPWcyt P450 reductasekyt P450/Fe(III)/02 catalyzes oxidative modification of proteins (1, 16). In the MCO system, the electron donor was needed to catalyze the reduction of 0, to H,O, and of Fe(II1) to Fe(I1). It has been proposed that Fe(I1) and H,O,
2
13391
30 Time (min
.
45
60
)
FIG.2. Analysis of the SDS-PAGE by densitometric scanning. Scanning densitometry (see “Materials and Methods”) was used to quantify the loss of (Coomassie Blue stained) protein bands following SDS-PAGE (Fig. 1).Values are the means of three independent determinations for which standard deviations are less than 10%.
generate in situa n activated oxygen species which reacts with the side chain amino acid residues producing oxidative modifications. The oxidatively modified proteins become highly susceptible to proteolytic degradation. The MCO system for protein oxidation is stimulated by which reduces Fe(II1) to Fe(I1) and is inhibited by catalase which removes H,O,. In order to understand the natureof the reactive oxygen species involved in theNADPH-initiated oxidative damage of the microsomal proteins in theabsence of added iron salts, we have investigated the effects of different scavengers of reactive oxygen species. Fig. 3 shows that NADPH-initiated microsomal protein degradation is exclusively prevented by low concentration of (20 w).Higher concentrations of A H,up to 100 PM produced similar inhibition. It has been mentioned above that incubation of microsomes in thepresence of NADPH results in marked loss of c y t P450 and cyt P450 reductase activity. It has (20 p ~ in) the been further observed that thepresence of incubation medium completely prevents theselosses. However,
AH,
AH,
AH,
Ascorbic Acid Prevents Oxidative Damage
13392 1
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20 40 Time (min.)
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FIG.4. Time-dependent release of fluorescamine-reactivematerials after incubation of microsomes with NADPH. Incubation mixture (250pl) contained 1 mg of microsomal protein in 50 m~ potassium phosphate buffer, pH 7.4. Reaction was initiated by adding 0.24 14.2 m~ NADPH and incubated at 37 "C withshaking.Reactionswere stopped with additionsof 250 pl of 20% trichloroacetic acid followed by centrifugation a t 12,000 x g for 10 min. Neutralized supernatants were FIG.3. Exclusive prevention of NADPH-initiated microsomal used to determine the fluorescamine reactivity by using an excitation emission wavelengthof 475 nm (10). Data protein degradation by AH, as evidenced by SDS-PAGE.Incuba- wavelength of 390 nm and an represent means of four estimations, S.D. < 10%. tionsandotherconditionsweresame as in Fig. 1 except that the incubations were carried outfor 60 min. Lane I , after incubation without additionof NADPH; lune 2, after incubation withNADPH; lune 3, after incubation with NADPH in thepresence of 20 ~MAH,; lune 4, after incubation with NADPH in the presence of 40 pg of catalase; lune 5, after incubation with NADPH in the presence of 50 m~ GSH; lune 6, after incubationNADPH in thepresence of a-tocopherol (20 p). a-Tocopherol was used as a dispersion in sodium deoxycholate (final concentration 0.02%).
-2 0 . 1 -
other scavengers including GSH, catalase, and a-tocopherol failed to protect the microsomal proteins from oxidative degradation. These resultsindicate that H,O, is not involved in the free metal ion-independent NADPH-initiated oxidative degradation of microsomal proteins. Although it is known that microsomal NADPWcyt P450 reductasekyt P450 system generates O,, SOD has been ineffective. This isprobably because we have observed in a separate experiment that the amount of 0, produced in the incubation system is too small (approximately 1.5 nmol) to produce any appreciable amount of protein oxidation. It will be shown later in this paper thatincreased an amount of 0, (15 nmol) generated by the xanthine/xanthine oxidase system, produces oxidative damage of the microsomal proteins which is inhibited by SOD. Besides demonstrating oxidative degradation of microsomal proteins bySDS-PAGE, the degradation has alsobeen evidenced by the formation of fluorescamine-reactive materials. Fluorescamine reacts with NH, groups. Production of fluorescamine reactive materials indicates production of new NH, terminus peptides after proteolytic cleavage of the oxidized proteins. Fig. 4 shows that incubation of microsomes with NADPH results in the progressive release of fluorescaminereactive material in the trichloroacetic acid extract of the microsomal suspension. As observed in Fig. 3, Fig. 5 shows that the production of fluorescamine-reactive material isexclusively prevented by AH,. SOD, catalase, GSH, a-tocopherol, mannitol, and histidine areineffective. The possibility that adventitious free iron present in the incubation mixture wasinvolved in the NADPH-initiatedprotein oxidation waseliminated because desfenioxamine (10-50 p ~ )a, strong chelatorof Fe(III), did not inhibit oxidative degradation of microsomal proteins (Fig. 5). Effect of ADP-Fe3+on the NADPH-initiated Microsomal Protein Degradation-It has been shown above that NADPH-initiated microsomal protein degradation in theabsence of added metal salts is completely prevented by (Figs. 3 and 5). In
AH,
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FIG.5. Effects of scavengers of reactive oxygen species and desferrioxamine on the NADPH-initiatedoxidative degradation of microsomal proteins as evidenced by fluorescamine reactivity. Incubations (as under "Materials Methods") and were camed outat 37 "C for 60 min with shahng in thepresence of different scavengers and desfemoxamine. After incubation trichloroacetic acid-precipitated supernatants were testedto determine the fluorescamine sensitivity as in Fig. 4. 1,incubated without NADPH; 2, with NADPH; 3, NADPH + 20 1.1~AH,; 4, NADPH + 100 unitof SOD; 5 , NADPH + 50 m~ GSH; 6, NADPH + 40 pg catalase; 7, NADPH + 20 p~ a-tocopherol; 8,NADPH + 10 m~ histidine; 9, NADPH + 10 p~ desferrioxamine. Results are means of four independent experiments, S.D. < 10%. Basal value of fluorescamine reactivity (54.2 f 3.8)obtained with unincubated microsomes was deductedfrom each of the above data.
contrast to this, when ADP-Fe3+(1.7 mM ADP and 50 p~ FeCl,) is added to the incubationmixture,proteindegradation is stimulated 48% which is further stimulated (63%)by AH,. This is apparently because in the presence of ADP-Fe3+,the microsomal NADPH-cyt P450 reductasekyt P450 becomes an enzymatic MCO system as described by others (1, 17) where the electrons from cyt P450 reduceFe(II1) to Fe(I1) and theprotein oxidation is catalyzed by Fe(I1) and H,O, (1).It is, therefore, expected that should stimulate the ADP-Fe3+-mediated protein oxidation by reducing Fe(II1) to Fe(I1) and that this oxidation should be inhibited by catalase. We have observed that ADP-Fe3+-mediated microsomal proteindegradation is 91% inhibited by catalase (40 pg) and 89% by GSH (50 mM). Mannitol (20 mM) also inhibited partially(27%) indicating that in the presence of ADP-Fe3+'OH is probably involved to some extent in the protein degradation. These results indicate that the mechanism of free metal ion-independent NADPH-initiated microsomal proteindegradation is distinctlydifferent from that catalyzed by ADP-Fe3+. Involvement of Proteases in the NADPH-initiated Oxidative Degradation of Microsomal Proteins-Results obtained with
AH,
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Ascorbic Acid Prevents Oxidative Damage TABLEI Effects of protease inhibitors on NADPH-initiated microsomal protein degradation
40
7
Conditions of incubation and estimation of fluorescamine reactivity are as in Fig. 4 except that the incubation time was 60 min. Concentrations of protease inhibitors used were: EDTA, 2 nm; DTPA, 2 m; o-phenanthroline, 1 m; PMSF 100 pg/ml. Data represent means of four estimations, &.D. Addition
% inhibition of fluorescarnine reactivity
None EDTA DTPA o-Phenanthroline PMSF PMSF + EDTA
0 68 2 3 62 f 2 71 f 3 80 f 4 91 f 4 Time ( m i n . )
FIG.6.NADPH-initiated modification of microsomal proteins as evidenced by bityrosine formation. Conditions of incubation are described under “Materials and Methods.” Results are given as fluorescence measurements at 325 nm excitation and 410-420 nm emission. Bityrosine fluorescence was measured in 20 nm HEPES buffer, pH 7.0. Values are means of three independent determinations for which stanwithout NADPH; A, with NADPH; dard deviation are less than 10%. 0, 0, NADPH plus 20 w AH,.
both enzymatic and nonenzymatic MCO systems indicate that there is a direct relationship between protein oxidation and increased proteolytic susceptibility (1, 11,16, 18-20). In other words, oxidative degradation of proteins is a two-step process, namely, (i) oxidative modification and (ii) proteolytic degradation of the oxidatively modified proteins. Table I shows that proion-independteolytic cleavageis also involved in the free metal ent NADPH-initiated microsomal protein degradation because this degradationis markedly inhibitedby PMSF, an inhibitor of trypsin and chymotrypsin,as well as by metalloproteinase inhibitors, e.g. EDTA, DTPA, and 0-phenanthroline. In the absence of NADPH, protein degradationdoes not occur, indicating that oxidative modification is a priorrequirement.Inthe 3 SO 350NADPH-initiated oxidative degradation of proteins, thedifference between the mechanismof the MCO system and that in the absence of free iron ions apparently lies in the stage of oxidative modification, proteolytic degradation being common 2 SOSO in both thecases. Oxidative Modification as Evidenced by Bityrosine Formation and Zkyptophan Loss-Davies et al. (11)have shown that exposure of bovine serum albumin to .OH generated by 6oCobalt radiation resulted in bityrosine formation and tryptophan I50 150 loss. Apparently tyrosil radicals are produced as a result of c’ hydrogen abstraction by .OH, and thetyrosil radicals may then 1 I I react with other tyrosil radicalsor with tyrosine molecules to 0 I O 20 30 form several biphenolic compounds of which bityrosineappears Time ( m i n . ) to be the majorproduct. It has also been observedthat NADPHFIG. 7. Loss of tryptophan following incubation of microsomes initiated oxidative modification of microsomal proteins leads to with NADPH. Conditions of incubation are given under “Materials and bityrosine formation at a n early stage, which is completely Methods.”Results are given as loss of tryptophan fluorescence intensity (Fig. 6). No bityrosine is produced in the measured a t 280 nm excitation and 340-350 nm emission. Fluorescence prevented by absence of NADPH. Fig. 7 shows that NADPH-initiated oxida- studies were performedwith 20 nm HEPES, pH 7.0. Values are means without NADPH; tive modification of microsomal protein in the absenceof free of three independent determinations, S.D. < 10%. 0, 0, with NADPH; 0, with NADPH + 20 p AH,. iron ions also results in the loss of tryptophan, asevidenced by the loss of fluorescence emission at 340-350 nm (280 nm excitation). The loss of tryptophan iscompletely prevented by AH, 0.05 nmoVmg protein, n = 6).There is no increase in the car(Fig. 7). bonyl content after incubationof microsomes in the absenceof Oxidative Modificationof Microsomal Proteins as Evidenced NADPH. Fig. 9 shows that carbonyl formation occurs at a n by the Introduction of Carbonyl Groups-Studies on homopoly- early stage of the oxidative modification of microsomal promers of amino acids indicate that carbonyl derivatives are teins. Maximum carbonyl formationtakes place around 15 min formed in the metal ion-catalyzed oxidation of Pro, Arg, Lys, of the incubationperiod. Table I1 shows that NADPH-initiated and His residues (2,7,18,21). In fact, measurement of “protein carbonyl formation in the microsomal proteins is exclusively carbonyls” has been used as a sensitive assay for oxidative prevented by AH,. SOD, GSH, catalase,a-tocopherol, mannitol, damage to proteins (1, 22, 23). We have estimated carbonyl and histidine areineffective. groups by reactionwith 2,4-&nitrophenyl hydrazine (12). Involvement of Cyt P450 in the NADPH-initiated Microsomal PMSF plus EDTA were added to the incubation mixture to studies (1, 17) on the enzymatic ProteinOxidation-Earlier prevent proteolytic degradation of the oxidized proteins and MCO system comprised of NADPWcyt P450reductasekyt thereby loss of carbonyl in the soluble peptides. Fig. 8 shows P450 indicate the requirement of a metal ion, e.g. Fe(III), which that when NADPH is added to the microsomal suspension, is reduced by the electron donor system to Fe(I1) that in turn carbonyl formation increases almost linearly with increased catalyzes protein oxidation. It has been considered that the NADPH concentration up to 0.24 mM studied. Unincubated protein must containa metal ion-binding site. In this paper it microsomes give only low values of protein carbonyl (0.44 f has been shown earlier that addition of desferrioxamine to the
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Ascorbic AcidOxidative Prevents
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Damage TABLEI1 NADPH-initiated carbonyl formation in microsomes and effects of antioxidants Conditions of incubations and estimation of carbonyl are described
under “Materials and Methods.” Incubation time30was min. PMSF(25 pg) and 2.0 mM EDTA were present during incubation to minimize the loss of peptides in thesupernatant. Carbonyl groups wereestimated in 20% trichloroaceticacid-precipitatedmicrosomalproteins.Additions were: AH,, 20 p;SOD, 100 units; GSH, 50 m; catalase, 40 pg; a-tocopherol, 20 p;mannitol, 20 m;histidine, 10 m. Results are means of four independent experiments, &.D. System
0
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FIG.8. Introduction of carbonyl groups in microsomal proteins as a functionof NADPH concentration. Conditions of incubation and estimation of carbonyl groupare described under “Materials and Methods.” Incubations were carried out for 30 min. Unincubated control value (0.44f 0.05 nmoVmg protein) was subtracted from the respective experimental data. Values are means of six independent determinations for which standard deviations are lessthan 10%.
Carbonyl formed
nmol hydrazonelmg protein 0.44f 0.05
Control +NADPH +NADPH + +NADPH + SOD +NADPH + catalase +NADPH + GSH +NADPH + a-tocopherol +NADPH + mannitol +NADPH + histidine
1.03f 0.09 0.44 f 0.05 1.04f 0.10 1.03 t 0.11 1.02t 0.11 1.01t 0.09 1.04f 0.10 1.04t 0.10
AH,
TABLEI11 Inhibitory effect of antibody to cytochrome P450 on NADPH-initiated carbonyl formation in microsomal proteins Conditions of incubation and estimation of carbonyl are described under “Materials and Methods.” The amount of antibody or y-globulin from preimmuneserum used was0.1 mg of protein each. Data represent means of four independent experiments, 2S.D. Carbonyl formed nmol hydrazonelmgprotein
Control +NADPH +y-Globulin from immuned +y-Globulin from preimmune
serum serum
0.44f 0.05 1.03 0.09 0.44 f 0.05 1.02f 0.1
this system. I t has also been observed that desferrioxamine does not inhibit protein oxidation in the reconsti(10-50 J~M) Time ( m i n . ) tuted system indicating that contaminating Fe(II1) is not inFIG.9. Time-dependent introduction of carbonyl groups in volved. Fig. 10 shows that in the reconstituted system using NADPH-initiated microsomal protein oxidation.Conditions of in- isolated microsomal proteins, carbonyl formation increases lincubation and determination of carbonyl groups are described under early with increased cyt P450 concentrationup to1 nmol stud“Materials and Methods.” Acontrol value of 0.44 f 0.05 nmoVmg protein ied. No protein oxidation was obtained in the absence of cyt for unincubated microsomes wassubtracted from the respective experimental data. Values are the meansof four independent determinations, P450. In addition, boiled c y t P450 was ineffective. Moreover, when the incubation was carried out using carbon monoxideS.D. < 10%. saturated cyt P450 or in the anaerobic condition, no carbonyl incubation system containing NADPH plus microsomes does formation could be detected. Similar resultswere obtained usand poly-L-Pro in place of isolated microsomal not inhibit proteinoxidation indicating that free Fe(111)is not ing PO~Y-L-LYS needed for protein involved in this oxidation. I t is considered that in the absence proteins (Table IV). Cyt P450-reductase was of Fe(III), probably c y t P450 carries out thefunction of redox oxidation, but increase of cyt P450 reductase with a constant iron. This is substantiatedby the observation that antibody to amount of cyt P450 (0.5 nmol) didnot increasecarbonyl formation (Table IV). Fig. 10 and Table IV show that protein oxidacyt P450 completely prevents NADPH-initiated microsomal completely prevented by protein oxidation (Table 111).To understand the mechanismof tion in the reconstituted system is is exclusive because other scavenaction of c y t P450 in theNADPH-initiated oxidative modifica- AH,. The prevention by tions of proteins, we have used a reconstituted system com- gers of reactive oxygen species including SOD, catalase, GSH, a-tocopherol, thiourea, mannitol, and histidine areineffective prised of purified cyt P450, NADPH-cyt P450 reductase, and isolateddelipidized microsomal protein as well as model (data not shown). Proteolytic Susceptibility of Cyt P450-mediated Oxidized polypeptides, e.g. poly-L-Pro and PO~Y-L-LYS. Proteins-Earlier works (3-7, 10, 16) have provided evidence Cyt P450-mediated Protein Oxidation Using Reconstituted System-Previous reports indicate that the enzymatic MCO that MCO marks proteinsfor proteolytic degradation. Roseman system of NADPWcyt P450 reductasekyt P450/Fe(III)/02 re- and Levine (6)have shown that proteinases catalyze rapid quires free metal ions for catalyzing the oxidation of proteins degradation of the oxidized forms of glutamine synthetase but and homopolymers (1, 17). We have observed that in a recon- have little or no ability to degrade the unoxidized enzymes. stituted system comprised of purified NADPWcyt P450 Davies and co-workers (7, 11, 24-27) have demonstrated that reductasdcyt P45Oholated delipidized microsomal proteins or prior exposure of highly purified proteins to oxygen radical model polypeptides, e.g. poly-L-Pro and PO~Y-L-LYS, protein oxi- generation system in vitro makes them highly susceptible to dation as evidenced by carbonyl formationis a direct functionof degradation by proteases. Since carbonyl formation provides a means of assessing the extentof oxidative damage in proteins, c y t P450 concentration. Addition of iron salts is not needed in 0
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Ascorbic Damage Oxidative Prevents Acid I .2
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FIG.10.NADPH-initiated formation of carbonyl groups in the
reconstituted system as a function of cytochromeP450 concentration. Conditions of incubation and determination are described under "Materials and Methods." Incubations were carried out for 30 min. Data represent means of four independent estimations; S.D. < 10%.0, with NADPH; x, with NADPH + 20 p AH,.
Carbonyl (n moles)
FIG. 11.Correlation between carbonyl content and proteolytic degradation of isolated microsomal proteins. NADPH-initiated formation of carbonyl groupsin the reconstituted system are described in Fig. 10. Afterincubation for 10 min, trypsin (20 pg) was added to all of the systems and release of fluorescamine-reactivepeptides was measured as described in Fig. 4. A basal fluorescence value of 15 was obtained by incubation of isolated microsomal proteins with trypsin in the absence of NADPH. Data represent means of four independent estimations, S.D. < 10%.
TABLE IV Oxidation ofmodel polypeptides as a function ofcytochrome (Cyt) P450 concentration and protection by AH, in the reconstituted system Conditions of incubation and estimation of carbonyls are described under "Materials and Methods." The amount of poly-L-Lys or poly-L-Pro used was 1mg each. Data represent means of four independent experiments, S . D .
Fe3+ and cyt P450 reductase, addition of NADPH led to the reduction of cyt P450Fe3+as evidenced by the shiftof the soret band from 417 to 414 nm and appearanceof single band at 542 nm in place of two separate bands at 534 and 568 nm, respectively (data not shown). It isknown that cyt P450 Fez+readily combines with molecular 0, (28). We have observed that when Carbonyl formed Addition NADPH is added to a mixture of purified cyt P450 reductase PO~Y-L-LYS Poly-~-Pm and various amountsof cyt P450, 0, is consumed in a stoichinmol hydmzonelmg protein ometric ratio. One nmol of 0, is consumed per nmol of cyt P450 Nil Nil Cyt P450 reductase (96 units) indicating theformation of c y t P450 Fez+0, which can be writNil Nil Cyt P450 (0.5 nmol) ten as theresonance form of cyt P450 Fe3+.0,. When microso0.39 i: 0.03 Cyt P450 reductase (96 units) + 0.53 2 0.04 mal protein (1mg) or poly-L-Pro (1 mg) is added to the reconcyt P450 (0.5 nmol) stituted system containing cyt P450 as mentioned above, the Cyt P450 reductase (192 units) + 0.41 i: 0.03 0.54 f 0.04 cyt P450 (0.5 nmol) protein is oxidized but no further 0, consumption takes place. 0.76 i: 0.05 1.05 2 0.09 Cyt P450 reductase (96 units) + This indicates that oxidation of protein is carried out by a n cyt P450 (1.0 nmol) active oxygen species produced from cyt P450. Although the Nil + Nil Cyt P450 reductase (96 units) nature of the active oxygen species responsible for protein oxicyt P450 (1.0 m o l ) + AH, (20 p) Nil + Nil Cyt P450 reductase (96 units) dation is not clear, we consider that cyt P450 Fe3+.0,, which is boiled cyt P450 (1.0 nmol) a perfenyl moiety, oxidizes amino acid side chains of proteins producing carbonyl groups. It is known that 0, can reduce Fe3+to Fez+(29).In a separate one would expect a relationship between carbonyl formation and proteolytic susceptibility. "his has been substantiated by experiment we have observed that 0; can also reducecyt P450 subjecting the oxidatively modified microsomal proteins to Fe3+to cyt P450 Fez+ (datanot shown).' This indicates thatfor trypsin. Oxidative modification, as evidenced by carbonyl for- oxidative modification of proteins, NADPH-cyt P450 reductase mation, has been done by incubating isolated delipidized mi- may be replaced by 0;. This has actually been observed by a reconstitutedsystem containing using 0, generated by the xanthine/xanthine oxidase system crosomal proteinswith NADPWcyt P450 reductasekyt P450 in the absence of added (30). In anincubation mixture (300 pl) containing 1nmol of cyt Fe(II1). Fig. 11shows that when oxidized microsomal protein is P450 in 50 mM potassium phosphate buffer, pH 7.4, 1 mg of treated with trypsin, theproduction of fluorescamine-reactive isolated microsomal protein, 10 p~ desferrioxamine, and 0.5 material increases linearly with increase in the carbonyl con- mM xanthine, addition of 0.1 nmol of xanthine oxidase produced tent. Although the microsomal proteinsused for oxidative 15 nmol of O z , and this was accompanied by the formation of with tryp- 1.06 nmol of protein carbonyl after an incubation period of 15 modification was a denatured protein, but treatment sin produced only a small amount of fluorescamine-reactive min at 37 "C. In this system the formation of protein carbonyl (20 p ~ but ) also by SOD (100 material within the shortperiod (1 h) of incubation (Fig. 11). was inhibited not only by AH, (20 p~ to 1mM) failed to prevent proteolytic degradation of units). Mechanism of Inhibition of Cyt P450-mediated Protein Oxioxidatively modified microsomal proteins, indicating thatonce was ineffective to prevent the dation by AH,--NADPH-initiated cyt P450-mediated protein the protein was oxidized, degradation. Mechanism of Cyt P450-mediatedProtein Oxidation-In the When 0.5 nmol of XOD was added to a spectrophotometriccuvette MCO system, theelectron donor reduces Fe(II1) to Fe(I1) which containing 20 m~ acetaldehyde and 5 nmol of cyt P450in 1ml of 20 m~ in conjunction with H,O, produces an active oxygen species for Tris acetate buffer, pH 7.4, containing 20% glycerol, there was generation of 100 nmolof O,, and thiswas accompaniedby a shift of the soret the oxidation of proteins. It is known that NADPH-cyt P450 band of cyt P450Fe3+from 417 to 414 nmwith a decrease in absorbance. reductase reduces cyt P450 Fe3+to cyt P450 Fez+(28). We also Also a single banda t 542 nm replacedthe two bands of cyt P450Fe3+at observed that in the reconstituted system containing cyt P450 534 and 568 nm.
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Ascorbic Acid Prevents Oxidative Damage
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oxidation involves three separate steps,namely (i) reduction of site on the protein after which protein Fe(1I) complex reacts cyt P450 Fe3+to cyt P450 Fez+by NADPH-cyt P450 reductase, with H,O, to generatein situan activatedoxygen species (.OH, (ii) oxygen uptake by cyt P450Fez+ to produce the active oxygen ferry1 ion), which in turn reacts with side chain amino acid species, tentatively theperferryl moiety, cyt P450 Fe3+.02,and residues at the metal binding site (1).In this process, some (iii) oxidation of amino acid side chainsof proteins by cyt P450 amino acid residues particularly Pro, His, Lys, and Arg are Fe3+.0Z.We have observed that AH, neither reduces cyt P450 converted to carbonyl derivatives. Among the enzymatic MCO Fe3+nor interferes with the NADPH cyt P450 reductase-cata- systems, perhaps NADPH-cyt P450 reductasekyt P4501 lyzed reduction of cyt P450 Fe3+to cyt P450 Fez+.When AH, (20 Fe(III)/O, is themost physiologically relevant one. Nonenzymic nmol) was added to a spectrophotometric cuvette containing a MCO system comprised of AHJFe/O, has also been considered mixture (1 ml) of NADPH (240 nmol)/cyt P450-reductase (96 to mimic the characteristicof the enzymatic MCO systems (18, units)/cyt P450 ( 5 nmol), the shift of the soret band form 417 321, and for convenience it has been used by many researchers nm or the appearance of a single band at 542 nm characteris- working in this field. Although the MCO system has provided tics of cyt P450 Fez+(13)were notaltered. Additionally, preven- valuable information about elucidating themechanism of oxiwas not accompanied by the dative damageof proteins, the free metal ion-catalyzed protein tion of protein oxidation by inhibition of 0, uptake by cyt P450 Fe". When AH, (40 nmol) oxidation system appears to haveless relevance to the in vivo was added to a mixture (2 ml) containing cyt P450 reductase situation, where in the normalphysiological condition most of (192 units), cyt P450 (1nmol), microsomal proteins (1mg), or the metal ions are not free and remain tightly bound with poly-L-Pro (1mg), the oxidation of protein was completely pre- proteins. The present study on cyt P450-mediated protein oxivented, but 0, uptake (1 nmol) was not inhibited. Lack of in- dation in theabsence of free metal ions is more relevant to the hibition of 0, uptake by cyt P450 Fez+ in thepresence of AH, in vivo condition and may represent an in vitro model for oxiindicates that does not prevent the formation of the pro- dative damage in vivo. The mechanism of cyt P450-mediated posed cyt P450 Fe3+. O;(cyt P450 Fe2+.0, s cyt P450 Fe3+. free metal ion-independent protein oxidation is distinctly dif0;). It therefore appears that AH, prevents protein oxidation ferent from that catalyzed by the MCO system involving Fe(I1) by interacting with cyt P450 Fe3+.0,. During thisprocess, AH, and H,O,. We consider that in the cyt P450-mediated protein is oxidized. Dehydroascorbate is ineffective. We have observed oxidation the active oxygen species involved is the preferryl that during the initial period of 10 min of incubation of the moiety, cyt P450 Fe3+.0,. The initial step of the formation of cyt reconstituted system mentioned above for 0, consumption, 1 P450 Fe3+.0, is the reduction of cyt P450Fe3+to cyt P450 Fe". nmol ofAH, is oxidized per nmol of cyt P450. When microsomal The most physiological electron donor system for cyt P450 Fe3+ protein (1mg) or poly-L-Pro(1mg) is added to the reconstituted is the NADPH-cyt P450 reductase. Cyt P450 Fe3+may also be system containing AH,, protein oxidation is completely pre- directly reducedby 0:. Subsequently cyt P450 Fez+ reactswith H, oxidation do not molecular 0, to produce cyt P450 Fe2+.0,, the resonance form of vented but the profile of 0, uptake and A prevents protein which is cyt P450 Fe3+.0,. Although the mechanism of oxidachange, supporting our proposition that oxidation by interacting withcyt P450 Fe3+.0, and not with thetion of the side chain amino acid by cyt P450 Fe3+.0, to the oxidized protein. corresponding carbonyl derivatives is not clear, yet a plausible mechanism may be similar to thatproposed by Stadtman and DISCUSSION Oliver (1)except that Fe(I1) and H,O, are replaced by cyt P450 Results presented in this paperclearly show that NADPH- Fe3+.02.Using lysyl residue as a model, the proposed mechainitiated oxidative damage of microsomal proteins occurs inde- nism is shown below: pendently of added metal salts. Theprotein oxidation is mediated by cyt P450 and is exclusively inhibited by AH,. Other RCH,NH, + Cyt P450 Fe3+.0, + H+ RCH-NH, + Cyt P450 Fe3++ H,O, scavengers of reactive oxygen species such as SOD, catalase, RCH NH, + RCH = NH mannitol, GSH, a-tocopherol, and histidine fail to prevent it. The failure of SOD and catalase to inhibit protein oxidation RCH = NH + H,O RCHO + NH, rules out the involvement of 0: and H,O, in the NADPH-cyt P450 reductasekyt P450-mediated protein oxidation. Mannitol MECHANISM 1 a 'OH trapping agent, does not inhibit indicating that 'OH is In this mechanism cyt P450 Fe3+.0z abstracts a hydrogen not involved in the cyt P450-mediated protein oxidation. The ineffectiveness of GSH and a-tocopherol can be ascribed by the from the carbon atom bearing the €-amino group producing a fact that GSH and a-tocopherol inhibit metalion-catalyzed pro- carbon-centered radical followed by its conversion to an imino tein oxidation (31). These results are at variance with that derivative, which then undergoes spontaneous hydrolysis proobtained by the in vitro oxidative modifications of proteins ducing an aldehydederivative of the lysyl residue. In thecase using enzymatic or non-enzymatic MCO systems. All the MCO of proline residue, hydrogen can be abstracted from carbon-5 systems thus far described depend upon the presence of free producing the corresponding imino group,followed by hydrolysis producing glutamyl semialdehyde (1). transition metal ions in the incubation medium. The metal The exclusive prevention of the cyt P450-mediated freemetal ion-catalyzed protein oxidation is accelerated by AH,. To supmay be explained by port the occurrence of MCO systems in vitro,postulations have ion-independent protein oxidation by been made for the existence of a mobile pool of iron ina micro- the consideration that AH, provides an easily donatable hydrocould act as a physi- gen for abstraction by cyt P450 Fe3+.0,, as proposed in the environment within thecells, where ological reducing agent. Recently, the mechanism of free metal following reactions ion-catalyzed protein oxidation has been extensively reviewed Cyt P450 Fe3+.0, + AH, + Cyt P450 Fe3'.0; + + H' (1,16). The current view of the free metal ion-catalyzed protein oxidation is that the electron donor system is needed only to AH'+AH'+A+AH, catalyze the reduction of Fe(II1) to Fe(I1). The reduction of Fe(II1) to Fe(I1) can also be mediated by 02,which can directly REACTION 1 react with Fe(II1) to form Fe(I1) and 0,. It hasbeen considered In this process, is oxidized to ascorbate free radical that the protein undergoing oxidation must contain a metal binding site. It is believed that Fe(I1) binds to the metal binding (AH'), which probably decays by disproportionation to AH, and
AH,
AH,
AH,
-
4
AH,
AH,
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Ascorbic Acid Prevents Oxidative Damage
AH,
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dehydroascorbate (A) (33). Thus will prevent the reaction aging and other inflammatory diseases supposed to be associbetween cyt P450 Fe3+.0, and the side chain amino acid resi- ated with protein oxidation. dues. These results indicate that if the cyt P450Fe3+. 0,-mediated protein oxidation occurs in vivo, then a physiREFERENCES ological function of A H,would be to protect the proteins from 1. Stadtman, E. R., and Oliver, C. N. (1991) J. Biol. Chem. 266,2005-2008 2. Amici, A,, Levine, R. L., Tsai, L., and Stadtman, E., R. (1989) J. Biol. Chem. oxidative damage in the body. 426,33414346 Although free metal ion-catalyzed protein oxidation appears 3. Farber, J. M., and Levine, R. L. (1982) Fed. P r o c . 41,865 to have less relevance to the in vivo situation, yet the MCO 4. Rivett, A. J. (1985) J. Biol. Chem. 260, 300-305 5. Rivett, A. J. (1985) J. Biol. Chem. 260, 1260fk12606 system has made a pioneering contribution in that the oxida6. Roseman, J. E., and Levine, R. L. (1987) J . Biol. Chem. 260,12600-12606 tively modified proteins become highly sensitive to proteolytic 7. Davies, K. J. A,, and Goldberg, A. L. (1987) J. Biol. Chem. 260, 8227-8234 8. Lowry, 0.H., Rosenbergh, N. J., Fan; A. L., and Randall, R. J. (1951)J . Biol. degradation. We have also observed that irrespective of the Chem. 193,265-275 difference between the mechanism of cyt P450-mediated pro9. Laemmli, U. K. (1970)Nature 227,680485 tein oxidation and that catalyzed by the MCO system, the sus- 10. Pacifici, R. E., and Davies, K. J. A. (1990) Methods Enzymol. 186,485-502 ceptibility of the oxidized protein to rapid proteolytic degrada- 11. Davies, K. J. A,, Delsignore, M. E., and Lin, S. W. (1987) J. Biol. Chem. 262, 9902-9907 tion is common in both the cases. However, in the MCO 12. Levine, R. L., Garland, D., Oliver, C. N., Amici, A,, Climent, I., Lenz, A,, Ahn, system AH, stimulates protein oxidation and thereby accelerB., Shaltiel, S., and Stadtman,E., R. (1990)Methods Enzymol. 186.464478 13. Van Der Hoeven, T., and Cwn, M. J. (1974) J. Biol. Chem. 249,63024310 ates subsequent proteolytic degradation. On the other hand, 14. Omura, T.,and Sato, R. (1964) J. Biol. Chem. 239, 237fk2378 prevents protein oxida- 15. Masters. B. S. S., Williams, C. H., Jr., and Kamin, H. (1967) Methods Enzymol. in the cyt P450-mediated system, 10,565-573 tion and thereby protects the protein from subsequent degra16. Stadtman, E. R. (1990) J. Biol. Chem. 29,6323-6331 dation. 17. Fucci, L., Oliver, C. N., Coon, M. J., and Stadtman, E. R. (1983) Proc. Natl. The prevention of the oxidative damage of microsomal proAcad. Sei. U. S. A. 80, 1521-1525 teins by is supported by our results obtained in vivo (34, 18. Levine, R. L., Oliver, C. N., Fulks, R.M., and Stadtman, E. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2120-2124 H, 19. Rivett, 35). We have shown in this paper that in the absence of A A. J. (1986) Curr. 7bp. Cell. Regal. 28, 291-337 incubation of microsomes with NADPH results in drastic oxi- 20. Stadtman, E. R. (1986) %rids Biochem. Sci. 11, 11-12 21. Stadtman, E. R., and Wittenberger, M. E. (1985)Arch. Biochem. Biophys. 239, dative degradation of almost all the microsomal proteins in379-387 cluding cyt P450and cyt P450 reductase. After a brief periodof 22. Murphy, M. E., and Kehrer, J. P. (1989) Biochem. J. 260,359-364 1 h incubation, about 80% of the cyt P450 and 93% cyt P450 23. Stadtman, E. R. (1990) Free Radical Biol. Med. 9, 315-325 24. Davies, K.J. A., and Goldberg, A. L. (1987) J. Biol. Chem. 262,8820-8826 reductase activity are lost. If these results occur in vivo, then 25. Davies, K.J. A. (1987)J. Biol. Chem. 262,9895-9901 deficiency there would bemarked 26. Davies, K.J. A., and Delsignore, M.E. (1987)J. Biol. Chem. 262,990S9913 one would expectthat in loss of the microsomal proteins including cyt P450and cyt P450 27. Davies, K. J. A., Lin, S. W., and Pacifici, R. E. (1987) J. Biol. Chem. 262, 9914-9920 reductase. In fact, it hasbeen shown in theguinea pig (36,37) 28. Porter, T.D, and Coon, M. J. (1991) J . Biol. Chem. 266, 13469-13472 that in deficiency (21 days) there is about 60% lossof the 29. Minotti, G . (1992) Arch. Biochem. Biophys. 297, 189-198 total microsomal proteins accompanied by a marked decrease 30. McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 604M055 31. Palamanda, J. R., and Kehrer, J. P. (1992) Arch. Biochem. Biophys. 293, 103of the liver microsomal enzymes including cyt P450 (40-45%), 109 cyt P450 reductase activity (80-85%), and the activities of the 32. Levine, R. L. (1983) J. Biol. Chem. 258, 11823-11827 B. H. J., Richter, H. W., and Chau, P. C. (1975) Ann. N. Y. Acad. Sci. drug-metabolizing enzymes in general (50450%).The decrease 33. Bielski, 258,231-237 was not due to lack of protein synthesis (36). Recently, we have 34. Chakrabarty, S., Nandi, A,, Mukhopadhyay, C. K., and Chatterjee, I. B. (1992) Mol. Cell. Biochem. 111, 41-47 deficiency, modified proshown that even in subclinical S., Nandi, A., Mukhopadhyay, C. K., and Chatterjee, I. B. (1993) teins accumulate in the liver microsomes of the guinea pigs in 35. Chakrabarty, Free Radical Biol. Med., in press spite of the presence of adequate levels of other antioxidants, 36. Zannoni, V. G., Holsztynska, E. J.,and Lau,S. S. (1982)AscorbicAcid: Chemistry, Metabolism, and Uses (Seib, P. A,, and Tolbert, B. M.,eds) pp. 349-368, namely, SOD, catalase, GSH, and a-tocopherol (35). American Chemical Society, Washington, D. C. Activeoxygen species-mediated oxidative damage of pro- 37. Clemetson, C.A. B. (1989) Vitamin C , Vol 11, pp. 213-219, CRC Press Inc. Boca Raton, FL teins, as reflected by protein modification, loss of catalytic acP. E, and Oliver, C. N. (1989) Arch. Biochem.Biophys. 275, tivity, and protein fragmentation are supposed to be physiologi- 38. Starke-Reed, 559-567 cal events not only associated with aging but also with various 39. Garland, D. Zigler, J. S., Jr., and Kinoshitas,J. (1986)Arch.Biochem. Biophys. 251, 771-776 other inflammatory diseases (1, 17, 38-42). If the results ob40. Oliver, C. N., Fulks, R., Levine, R. L., Fucci, L., Rivett, A. J., Roseman, J. E., tained with guinea pigs are extrapolated to humans and if the and Stadtman, E. R. (1984) in Molecular Basis of A g i n g (Roy, A. K., and Chatterjee, P., eds) PP. 235-262, Academic Press. New York process of free metal ion-independent cyt P450-mediated oxi41. Chapman,". L., Rubin,B. R., and Gracy, R.W. (1989) J. Rheumatol. 1 6 , 1 5 1 8 dative damage of proteins occurs in vivo, then AH,,should be 42. Oliver, C. N., Ahn, B. W., Moerman, E. J., Goldstein, S., and Stadtman, E. R. one of the most effectivepreventive measures to protkct us from (1987) J. Biol. Chem. 262,5488-5491
AH,
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