Immunochemical Studies on Electron Transport Chains Involving ...

THE JOURNALOF BIOLOGICALCHEMISTRY Vol. 246, No. 13, Issue of July 10,~~. 4143-4150,1971 Printed

in

U.S.A.

Immunochemical Studies on Electron Involving Cytochrome P-450

Transport

Chains

I. EFFECTS OF ANTIBODIES TO PIG LIVER MICROSOMAL REDUCED NUCLEOTIDE-CYTOCHROME c REDUCTASE AND THE NON-HEME IRON ADRENOCORTICAL MITOCHONDRIA* (Received BETTIE JOSEPH

SUE SILER LOSPALLUTO

MASTERS,~

JEFFREY

From The Department of Biochemistry, Texas 76.255

BARON,

WAYNE

E. TAYLOR,

TRIPHOSPHOPYRIDINE PROTEIN FROM BOVINE

for publication, ELIZABETH

November L.

16, 1970)

ISAACSON,

AND

The University of Texas (Southwestern) Medical School at Dallas, Dallas,

An antibody preparation to purified, homogeneous TPNHcytochrome c reductase from pig liver microsomes was shown to inhibit concomitantly TPNH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) and ethyhnorphine N-demethylase activities in pig liver microsomes. The antibody also inhibited TPNH-cytochrome c reductase and TPNH-cytochrome P-450 reductase activities in rat and pig liver microsomes and bovine adrenocortical microsomes, but it did not inhibit either of these activities in bovine adrenocortical mitochondria. In addition, an antibody to adrenodoxin, the non-heme iron protein isolated from bovine adrenocortical mitochondria, inhibited both TPNH-cytochrome c reductase and TPNH-cytochrome P-450 reductase activities in bovine adrenocortical mitochondria but not in any of the microsomal preparations, including those isolated from adrenal cortex. The results presented establish these antibodies as specific inhibitors which can be utilized in studying hydroxylation reactions mediated by cytochrome P-450. With the use of these specific antibodies in each system studied, either the same or immunochemically similar enzymes were shown to mediate the TPNH-dependent reduction of both cytochrome c and cytochrome P-450, and the flavoprotein functional in the mixed-function oxidations of the mitochondria of adrenal cortex was shown to differ from that found in the microsomes of liver and adrenal cortex. Furthermore, the lack of inhibition of TPNH-dependent electron transport activities in liver and adrenal cortex microsomes, by antibody to adrenodoxin, which strongly inhibits bovine adrenal cortical mitochondrial TPNH-cytochrome c and cytochrome P-450 reductase activities, is further evi*This work was supported by United States Public Health Service Grant 13619 from the Heart and Lung Institute and by Grant 687.56 from the American Heart Association (B. S. S. M.), and by United States Public Health Service Grant iPllGM16488 from the Institute of General Medical Sciences. $ An Established Investigator of the American Heart Association.

dence that a non-heme iron protein similar to adrenodoxin is not involved in cytochrome P-450-mediated reactions in microsomes.

Cytochrome P-450 is involved in the oxidative metabolism of a multitude of drugs, steroids, carcinogens, and fatty acids in hepatic microsomes (l-4), cholesterol side chain cleavage, and ll&hydroxylation of steroids in adrenocortical mitochondria (5, 6), and C-21 hydroxylation of steroids in adrenocortical microsomes (2, 7). In these three systems, TPNH supplies the necessary reducing equivalents for the reduction of cytochrome P-450, the terminal oxidase, via an electron transport chain which includes a flavoprotein as the acceptor of electrons from TPNH (1, 8). This flavoprotein has been generally assumed to be TPNH-cytochrome c reductase (NADPH-cytochrome c oxidoreductase, EC 1.6.2.3) although an additional component, a non-heme iron protein, adrenodoxin, is required for the TPNHdependent reduction of both cytochrome c and cytochrome P-450 in adrenocortical mitochondria (8). The isolation, solubilization, and purification of TPNHcytochrome c reductase from porcine liver microsomes has been reported by Williams and Kamin (9) and Phillips and Langdon (lo), and the mechanism of electron transfer in the purified flavoprotein has been studied in detail by Masters et al. (11-13). After reports from a number of laboratories that TPNH was required for the “mixed-function oxidation” of many drugs (14-16), Omura (17) reported the inhibition of aniline hydroxylation by antibodies to a trypsin-solubilized and purified preparation of TPNH-cytochrome c reductase from rat liver. Wada et al. (18) also demonstrated the inhibition of hepatic w-hydroxylation activity by the antibody supplied by Omura. During their studies on the isolation of the components of the electron transport chain functional in the Ilo-hydroxylation of steroids in adrenocortical mitochondria, Omura et al. (8) observed that the activity of TPNH-cytochrome c reductase in-

4143

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SUMMARY

4144

Immunochemical

Studies on Cytochrome P-QSO-mediated Reactions

MATERIALS

AND

METHODS

TPNH and TPN+ were products of P-L Biochemicals. Cytochrome c (type VI), trisodium isocitrate, and isocitrate dehydrogenase were obtained from Sigma. Ethylmorphine hydrochloride was purchased from Merck Sharp and Dohme. Freund’s complete adjuvant was a product of Difco, and Ouchterlony double diffusion agar plates were obtained from Hyland Laboratories, Los Angeles. DEAE-cellulose (medium mesh, 1 meq per g) from Sigma was washed extensively with acid and base and resuspended in the appropriate buffer. Preparations of iMicrosomes and Mitochondria-Liver microsomes were prepared from untreated and phenobarbital-treated female pigs essentially according to the method of Nelson, Masters, and Peterson (22). Washed microsomes were resuspended to a protein concentration between 35 and 45 mg per ml in 0.05 M potassium phosphate buffer, pH 7.7, containing lop4 M EDTA. Phenobarbital was administered orally in gelatin capsules for 6 to 10 days using a “balling gun.” Each capsule contained sufficient phenobarbital so that a dose of 15 mg of phenobarbital per kg, body weight, was administered. Pigs were fasted for 24 hours before killing. Hepatic microsomes were prepared from untreated and phenobarbital-treated male Holtzman rats (150 to 200 g) which had also been fasted for 24 hours before killing. Rats which were treated with phenobarbital received, intraperitoneally, 40 mg of phenobarbital per kg for 4 days. Rats were killed by decapitation and livers were perfused in situ with ice cold 0.15 M NaCl. The perfused livers were excised and homogenized in 5 volumes of 0.25 M sucrose. The resulting homogenate was centrifuged at

246, No.

13

755 X g for 10 min at 4’ in a refrigerated RC-2B Sorvall centrifuge to sediment unbroken cells, nuclei, and cell debris. The supernatant fraction was then centrifuged for 10 min at 8,700 X g to sediment mitochondria. The resulting supernatant fraction was centrifuged for an additional 10 min at 18,800 X g to ensure complete removal of mitochondrial particles, and the postmitochondrial supernatant fraction obtained was centrifuged for 45 min at 105,000 X g in a Spinco model L2-65B ultracentrifuge. The microsomal pellet was resuspended in 0.15 M KC1 and recentrifuged for an additional 30 min at 105,000 x g to remove hemoglobin contamination. The resulting pellet was finally resuspended in 0.25 M sucrose, containing 50 mM Tris-chloride buffer, pH 7.4, to a protein concentration of 30 mg per ml. Bovine adrenocortical mitochondria were prepared according to the method of Cammer and Estabrook (23), and the microsomal fraction was prepared from the postmitochondrial supernatant fractions in the same manner as rat hepatic microsomes. Both the mitochondrial and microsomal fractions were resuspended in 0.25 M sucrose to a protein concentration between 20 and 30 mg per ml. The mitochondrial preparations were subjected to freezing and thawing several times before use to facilitate penetration of antibodies into the mitochondria. Fraction Sp, a solubilized preparation of flavoprotein and adrenodoxin, was prepared from sonicated mitochondria according to the method of Omura et al. (8). Protein was determined by the biuret reaction (24). Assay for TPNH-Cytochrome c Reductase Actiuity-TPNHcytochrome c reductase activity was assayed essentially according to the method of Masters et al. (19). Each reaction mixture contained the following: 0.9 ml of 0.05 M potassium phosphate buffer, pH 7.7,0.8 ml of 10m4 M cytochrome c in the same buffer, 0.1 ml of 10e2 M KCN, and 0.2 ml of enzyme protein (suitably diluted). Where indicated, preimmune or immune y-globulin was substituted for the appropriate volume of buffer. The reaction was initiated by the addition of 0.2 ml of 10m3 M TPNH, and the rate of reduction of cytochrome c was measured at 550 rnp using a millimolar extinction coefficient of 21 rnM+ cm-r (25) for the difference in absorbance between reduced and oxidized cytochrome c. Assay for TPNH-Cytochrome P-450 Reductase Activity-The activity of TPNH-cytochrome P-450 reductase in the microsomal and mitochondrial preparations was determined by measuring the rate of formation of the carbon monoxide complex of ferrous cytochrome P-450 (cytochrome P-450-CO complex). Each reaction mixture contained 3 mg of microsomal or mitochondrial protein and sufficient 0.1 M potassium phosphate buffer, pH 7.4, to make a 3-ml reaction volume. Where indicated, preimmune or immune y-globulin was substituted for the appropriate volume of buffer. After a 5-min incubation period at room temperature, the reaction mixtures were placed in anaerobic cuvettes and were gassed with argon for 3 min to produce anaerobiosis and then with oxygen-free carbon monoxide for 2 min. The reactions were initiated by the addition of TPNH (final concentration = 200 PM), and the reduction of cytochrome P-450 was followed by measuring the initial rate of formation of the reduced cytochrome P-450-CO complex using the wave length pair of 450 and 490 rnp on an Aminco-Chance dual wave length/split beam recording spectrophotometer. Total cytochrome P-450 content in the 3-ml reaction mixture was determined after the addition of sodium dithionite. A millimolar extinction coefficient of 91 rn~1 cm+ (26) was employed for the difference in absorbance


creased parallel to that of TPNH-cytochrome P-450 reductase during the purification procedure. It was suggested that the same enzyme system was responsible for the TPNH-dependent Omura et al. (8) had also demreduction of both cytochromes. onstrated that TPNH-cytochrome c reductase, purified by Masters, Williams, and Kamin (19) from pig liver microsomes, was unable to substitute for the isolated mitochondrial flavoprotein in the reduction of cytochrome P-450 in the reconstituted electron transport system of bovine adrenocortical mitochondria. These results suggest differences between the adrenocortical mitochondrial and hepatic microsomal flavoproteins. More recently, Sweat et al. (20) suggested that the flavoproteins involved in cytochrome P-450 reduction and steroid hydroxylation in both adrenocortical microsomes and mitochondria are identical. In light of these results, the present studies, employing an antibody to pig liver microsomal TPNH-cytochrome c reductase, were undertaken to determine if immunochemical identity exists among the flavoproteins of the three systems described. Although the system which mediates the TPNH-dependent reduction of cytochrome c and cytochrome P-450 in sdrenocortical mitochondria involves both a flavoprotein and a non-heme iron protein, adrenodoxin (8), the reduction of cytochrome c in microsomes requires only the flavoprotein (21). Furthermore, no evidence has been presented which demonstrates the presence of an adrenodoxin-like non-heme iron protein in either hepatic or adrenocortical microsomes. In view of these observations, the role of adrenodoxin in the electron transport chains involving cytochrome P-450 in these three systems was studied employing an antibody preparation to the non-heme iron protein which had been isolated and purified to homogeneity from bovine adrenocortical mitochondria.

Vol.

Issue of July 10, 1971

Masters,

Baron, Taylor, Isaacson, and LoSpalluto

1 K. Suzuki, T. Kimura, personal communication.

D. Y. Cooper,

and R. W. Estabrook,

0.24

--A

I2

MINUTES

MINUTES

FIG. 1. Inhibition of TPNH-cytochrome c reductase and oxidative demethylation by antibody to reductase in pig liver microsomes. Microsomes from phenobarbital-treated pigs were previously incubated with varying amounts of r-globulin prepared from Dooled antisera to nurified. lioase-solubilized TPNH-cvtochrome c reductase (19). -Aliquo& &ere removed from previokly incubated mixtures containing 61.6 mg of microsomal protein and varying amounts of nonimmune and immune r-globulin and assayed for ethylmorphine demethylase (A) and TPNH-cytochrome c reductase (B) activities. Formaldehyde formation from ethylmorphine was measured at 38” and TPNH-cytochrome c reductase activity was determined at 25’, as described under “Materials and Methods.” The previously incubated mixtures contained no r-globulin (O-O), 25 mg (0-O) or 51 mg (O-0) of preimmune r-globulin, or 33 mg (+m), 66 mg (A-A) or 132 mg (A-A) of immune r-globulin. TPNH-cytochrome c reductase activity was 147 mpmoles min-1 mg-1 and ethylmorphine demethylation activity was 15.1 mkmoles min-1 mg-1 in uninhibited microsomes from phenobarbital-treated pigs.

resulting globulin fraction was further purified by chromatography on DEAE-cellulose using approximately 1 g of DEAEcellulose per 50 mg of protein. The column was eluded with 0.01 M potassium phosphate buffer, pH 7.7, and the fractions containing protein, as determined by absorbance at 280 rnp (A%0 of 1 mg of y-globulin per ml = 1.35) were combined, conThe y-globucentrated, and tested for immunological activity. lin preparations used in the experiments reported in this paper were obtained from antisera collected over a period of time. This resulted in a variable titer of antibody content. RESULTS

Inhibition of TPNH-Cytochrome c Reductase Activity and Ethylmorphine N-Demethylation in P@ Liver Microsomes by Antibody to PuriJied Reductase-Fig. 1 shows the results of an experiment in which pig liver microsomes were previously incubated with varying amounts of the y-globulin fraction prepared by DEAE-cellulose column chromatography of antiserum from rabbits injected with homogeneous pig liver TPNH-cytochrome Aliquots were removed from the previously inc reductase. cubated mixtures and diluted suitably for TPNH-cytochrome c reductase and ethylmorphine N-demethylase assays. The differences between controls with and without similarly purified Fig. 2 shows the results preimmune y-globulin were negligible. plotted as percentage of control activity. The data clearly show the concomitant inhibition of both TPNH-cytochrome c reductase and N-demethylase activities in pig liver microsomes by the antibody prepared to the purified, lipase-solubilized TPNH-cytochrome c reductase prepared from pig liver. Fig. 3 shows the concomitant titration of purified, homogeneous TPNH-cytochrome c reductase from pig liver and TPNHcytochrome c reductase activity in pig hepatic microsomes with


between reduced cytochrome P-450-CO complex and reduced cytochrome P-450, using the wave length pair 450 and 490 mp. Determin,ation of Ethylmorphine N-Demethylase Actiwity-The oxidative metabolism of ethylmorphine by pig liver microsomes was determined by measuring formaldehyde production by the method of Nash (27) as modified by Cochin and Axelrod (28). Assays were performed in a 15.ml total volume in a shaking water bath at 38” using 50-ml flasks containing 5.6 pmoles of TPN+, 128 pmoles of trisodium isocitrate, 150 pmoles of MgClz, 120 pmoles of ethylmorphine, and 1.1 mmole of potassium phosphate buffer, pH 7.5, containing 2.2 pmoles of EDTA. After a 3-min incubation with 0.5 mg of purified isocitrate dehydrogenase, ethylmcrphine N-demethylation was initiated by the addition of microsomes. Purification of TPNH-Cyochrome c Reductase and Adrenodo&n-Microsomal TPNH-cytochrome c reductase was purified according to the method of Masters et al. (19) from lipasesolubilized pig liver microsomes, except, that chromatography on DEAE-cellulose was substituted for hydroxylapatite and subsequent chromatography on Sephadex G-100 was included. This procedure resulted in a homogeneous preparation as determined by disc gel electrophoresis at pH 8.5. Adrenodoxin was isolated and purified to homogeneity from bovine adrenocortical mitochondria by a slight modification of the method of Suzuki et al.’ The resulting purified adrenodoxin preparation gave a single band on disc gel electrophoresis at pH 8.3. The ratio of absorbance of the homogeneous adrenodoxin preparation at 415 rnp to that at 280 rnp was 0.87, a value slightly greater than that reported by Kimura and Suzuki (29). Inoculation of Rabbits and Preparation of Antibodies-To immunize each rabbit, 0.6 mg of purified TPNH-cytochrome c reductase in 0.3 ml of 0.05 M potassium phosphate buffer, pH 7.7, containing 10e4 M EDTA, was mixed with an equal volume of Freund’s complete adjuvant and distributed by injection into the footpad areas and around the lymph nodes in the nape of the neck. This treatment was repeated in each of three rabbits once a week for 3 weeks. After several weeks, booster injections of 0.6 mg of enzyme were administered. Estimates of antibody activity were obtained by the Ouchterlony double diffusion procedure (30) or by inhibition of enzymic activity or both. Then 3.0 to 3.5 mg of adrenodoxin in 1.5 ml of 0.01 M Trischloride buffer, pH 7.4, with 10e3 M EDTA! were mixed with 0.6 ml of Freund’s complete adjuvant and injected into rabbits throughout the areas previously indicated. It was also possible to ascertain antibody titer by the Ouchterlony diffusion techniques since a precipitin reaction was obtained with adrenodoxin and its corresponding antibody. Rabbits were bled from an ear vein weekly to collect 25 to 50 ml of blood per bleeding. Bleedings were obtained prior to immunization with either of the antigens. Immune or preimmune sera from a number of bleedings were pooled and fractionated with (NH&S04 in the following manner. The serum was made 1.75 M in (NH&S04 and stirred for 30 min after which it was centrifuged at 27,000 X g for 10 min. If still contaminated with hemoglobin, the precipitate was resuspended in 0.01 M potassium phosphate buffer, pH 7.5, and sufficient (NH&S04 was added to give a 1.75 M solution which, after stirring for 30 min, was centrifuged. The resulting precipitate was resuspended in and dialyzed overnight against 0.01 M potassium phosphate buffer, pH 7.5. The

4145

4146

Immunochemical

Studies on Cytochrome P-&O-mediated

Reactions

Vol. 246, No. 13

TABLE I to pig liver microsomal TPNH-cytochrome c reductase on activity of TPNH-cytochronze c reductase in rat and pig liver microsomes and bovine adrenocortical mitochondria and microsomes

Effect

of antibody

-

-

l’PNH-cytochrome c reductases

Fraction

Control activity

mg y-globulin/mg p&in

b

I

5 w 0

25 ! I

L

33

mg.

OF

66

IMMUNE

99

132

Y - GLOBULIN

165

IN PREINCUBATION

Fxo. 2. Concomitant inhibition of pig liver microsomal activities. The conditions are as described for Fig. 1. but the data are presented as per cent of control activity to-demonstrate the concomitant inhibition of both activities by antibody to TPNHcytochrome c reductase. TPNH-cytochrome c reductase activity was calculated on the basis of controls without r-globulin (O-O) and with preimmune r-globulin (A--A). Ethylmorphine demethylase activity was calculated on the basis of controls without r-globulin (O----U) and with preimmune r-globulin (A-A).

ml.

OF

ANTISERUM

TO

TPNH-CYTOCHROME

E REDUCTASE

FIQ. 3. Titration of TPNH-cytochrome c reductase activity in nurified enzyme and pig liver microsomes by antiserum to the reductase. TPNH-cyiochrome c reductase activity was assayed as described under “Materials and Methods” with 0.17 mg of microsomal protein and 0.1 rg of purified protein in a 3.0-ml ;olume. Control activities were 112 mpmoles min-1 mgi and 24,000 nqumoles min-1 mgi with microsomes and purified enzyme, respectively. Antiserum was collected from three rabbits and tested; the antiserum yielding greatest inhibition was used. Figure shows purified, TPNH-cytochrome c reductase (O-O), fresh, unsonicated microsomes (A-A), and sonicated microsomes inhibited by antiserum (O---O). Microsomes were used the day after preparation. Sonciated microsomes were prepared using Branson model 975 sonic oscillator for 1 min on output setting 5 and 5 amps. Preliminary experiments on Ouchterlony diffusion plates indicated lack of diffusion of fresh, unsonicated microsomes; sonicated microsomes diffused through agar and produced clear precipitin lines with antiserum to TPNH-cytochrome c reductase. Reductase activity in sonicated and fresh microsomes was within 2% of each other.

%

147 102 34 174

100 70 23 119

164 138 80 138

100 84 49 84t

100 75 57 100

12.2 8.5 6.4 12.2

100 107 110 125

138 148 152 172

-

-

0 Each mixture contained the following: 0.9 ml of 0.05 M potasc, sium phosphate buffer, pH 7.7, 0.8 ml of 10-’ M cytochrome 0.1 ml of 10-z M KCN, 0.2 ml of enzyme protein (suitably diluted), and preimmune or immune -y-globulin substituted for buffer as indicated. The reaction was initiated by the addition of 0.2 ml c was of 10-8 M TPNH, and the rate of reduction of cytochrome measured at 550 mp. All specific activities were calculated on the basis of enzyme protein. The r-globulin preparations used in these experiments were obtained from antisera collected over a period of time resulting in a variable titer of antibody content. The same immune r-globulin preparation was used in the experiments with adrenocortical microsomes and Fraction &. b The apparent inhibition exhibited in the rat liver microsomes experiment by preimmune -y-globulin was not observed with any other preimmune preparation (six or more) in hepatic microsomes so that it is not considered to be a specific or significant phenomenon. Furthermore, the same degree of inhibition was demonstrated with one-seventh the protein concentration in the immune preparation. It has been observed, however, that preimmune r-globulin almost invariably increases the rate of cytochrome c reduction, probably due to nonspecific protein-protein interactions. Since the same amount of antiserum antiserum to the reductase. is required to inhibit TPNH-cytochrome c reductase activity in both systems, the data clearly demonstrate that the antibody affects, either directly or indirectly, the active sites of both the purified and microsomal enzymes in the same manner and to the same extent. These experiments conclusively demonstrate that TPNH-cytochrome c reductase is a component of the elec-


I

Phenobarbital-treated pig liver microsomes 0.00 ...................... 0.53 (immune). ........... 2.13 (immune). ........... 0.82 (preimmune) ........ Phenobarbital-treated rat liver microsomes 0.0 ....................... 0.6 (immune). ............ 3.6 (immune). ............ 4.2 (preimmune) ......... Bovine adrenocortical microsomes 0.0 ...................... 14.2 (immune). ........... 56.8 (immune). ........... 46.0 (preimmune) ........ Fraction Sz of adrenocortical mitochondria 0 ....................... 93 (immune). ............ 186 (immune). ............ 47 (preimmune) .........

Issue

of July

a

10, 1971

Masters,

Baron,

Taylor,

Isaacson,

and LoSpalluto

+ TPNH-cytochrome c reductase in the center we12 is reacted with both nurified liuase-solubilized TPNH-cvtochrome c reductase (L) and purified trypsin-solubilized reductase (T) in the outer wells. When, as in b, antibody is adsorbed with trypsin-solubilized enzyme 1:l (v/v), the major lines of identity begin to disappear. In c, in which antibody is adsorbed with trypsin-solubilized reductase 1:2 (v/v), the major lines of identity have completely disappeared, but minor lines remain characteristic of the lipase-solubilized preparation. The lipase-solubilized reductase preparation catalyzed the reduction of 2200 mpmoles of cytochrome c per min per ml, and the trypsin-solubilized reductase preparation had an activity of 2800mpmoles per min per ml. The trypsin-solubilized enzyme was prepared according to Omura and Takesue (33).

tron transport chain leading to the oxidative metabolism of various drugs in the liver, and that inhibition of this activity in hepatic microsomes leads to inhibition of drug metabolism. Collaborative experiments with Dr. D. M. Ziegler, using antibody to TPNH-cytochrome c reductase, failed to demonstrate either inhibition of TPNH-specific mixed-function amine oxidase (31) activity in pig liver microsomes or of the purified, homogeneous enzyme catalyzing these activities. Furthermore, there was no interaction of the purified mixed-function amine oxidase with antibody in Ouchterlony double diffusion experiments. Effect of Antibody to Porcine Liver Reductase on TPNHCytochmme c Reductase Activities in Rat Liver Microsomes and Bovine Adrenocortical Microsomes and Mitoc?wndria-Figs. 1 to 3 have demonstrated the inhibition of pig liver microsomal TPNHcytochrome c reductase and TPNH-dependent oxidative N-demethylation of ethylmorphine by antibody preparations to the reductase. As seen in Table I, the antibody also inhibits the activity of TPNH-cytochrome c reductase activity in hepatic microsomes of phenobarbital-treated pigs and rats. This is in agreement with the results of Kuriyama et al. (32), who have shown that the trypsin-solubilized enzyme purified from phenobarbital-treated rats is chromatographically and immunochemitally identical with the enzyme normally synthesized by the liver. The data presented in Table I also demonstrate inhibition of TPNH-cytochrome c reductase activity in adrenocortical microsomes, by the antibody to liver microsomal reductase, suggesting that the two enzymic activities are immunochemically similar. Although the antibody inhibited TPNH-cytochrome c reductase activities in rat and pig liver and bovine adrenocortical microsomes, the same antibody preparations failed to inhibit the activity of this enzyme in Fraction St, a solubilized preparation of flavoprotein and adrenodoxin prepared from sonicated mitochondria (8). These data suggest that the flavoprotein which is functional in the mixed-function oxidations occurring in adrenal cortex mitochondria is different from the enzyme or enzymes functioning in liver and adrenocortical microsomes.

This is consistent with the observation that TPNH-cytochrome c reductase purified from pig liver was unable to reconstitute the electron transport chain of bovine adrenocortical mitochondria in the absence of the mitochondrial flavoprotein (8). Fig. 4a demonstrates interactions of antibody to pig liver microsomal TPNH-cytochrome c reductase with hepatic and adrenocortical microsomal preparations on Ouchterlony double diffusion agar plates. The precipitin lines indicate partial immunochemical identity between TPNH-cytochrome c reductase of pig liver microsomes and bovine adrenocortical microsomes. Fig. 48 shows the lack of interaction between the reductase antibody and Fraction St of bovine adrenocortical mitochondria. These data support the results obtained in experiments where inhibition of enzymic activity was used as a criterion of pig liver TPNH-cytochrome c reductase antibody interaction with microsomal fractions from other tissues and species. Fig. 5 shows an Ouchterlony plate in which antibody to lipasesolubilized pig liver TPNH-cytochrome c reductase in the center well was reacted with purified lipase-solubilized and trypsinsoluhilized (33) TPNH-cytochrome c reductase preparations in outer wells. As seen in Fig. 5a, strong continuous precipitin lines of interaction resulted. When, as in Fig. 5b, the antibody is mixed 1:l with the trypsin-solubilized enzyme, added to the center well, and interacted with the lipase-solubilized enzyme in the outer wells, weaker lines of interaction are shown. In Fig. 5c, antibody was mixed 1:2 with trypsin-solubilized enzyme and interacted with the lipase-solubilized enzyme, resulting in the complete adsorbing out of the identical precipitin lines and leaving only those antigen-antibody precipitin lines characteristic of the lipase-solubilized preparation. These data show unequivocally that the purified lipase-solubilized and trypsinsolubilized TPNH-cytochrome c reductases isolated from pig liver are immunochemically similar. Thus, these results suggest that the antibody prepared by Omura (17) to the purified trypsin-solubilized TPNH-cytochrome c reductase and the antibody prepared to the purified lipase-solubilized reductase


FIG. 4 (Zeft). Ouchterlony double diffusion of antibody to pig liver TPNH-cytochrome c reductase against pig liver microsomes and bovine adrenocortical microsomes and bovine adrenocortical mitochondria. Fig. 4ashows the interaction of antibody prepared to purified, lipaze-solubilized TPNH-cytochrome c reductase in the center well to pig liver microsomes (P) and adrenocortical microsomes in the outer wells (&.I). In b, no interaction of the antibody can be seen with adrenocortical mitochondria (AM). Microsomes and mitochondria were prepared as described under “Materials and Methods.” FIG. 5 (right). Ouchterlony double diffusion of antibody to pig liver TPNH-cytochrome c reductase against purified lipasesolubilized and trypsin-solubilized TPNH-cytochrome c reductase preparations from pig liver. In a, antibody to lipase-solubilised

Immunochemical

4148

are immunochemically similar and would the same reactions in various microsomal

Studies on Cytochrome P-&O-mediated

be expected preparations.

to inhibit

Inhibition of TPNH-Cytochrome P-450 ReductaseActivities by ReductaseAntibody-It has been generally assumed that TPNHcytochrome c reductase is responsible, either directly or indirectly, for the reduction of cytochrome P-450. Indeed, based on observations made during the separation and purification of the components of the TPNH-cytochrome c reductase and the TPNH-cytochrome P-450 reductase systems from bovine adrenocortical mitochondria, Omura et al. (8) suggested that the same enzyme system was responsible for the TPNH-dependent reduction of both hemoproteins. It was therefore of interest to determine if the antibody to pig liver TPNH-cytochrome c reductase was capable of inhibiting TPNH-cytochrome P-450 reductase activity. From the data presented in Table II, it can be seen that the activity of TPNH-cytochrome P-450 reductase in various preparations of microsomes was inhibited by antibody TABLE

II

-

I ’TPNH-cytochrome

Fraction

mg y-globulin/3 reaction

P-450

reductas&’

ml

Control pig liver microsomes 0.0.. 24.0 (immune)c. 48.0 (immune). 24.0 (preimmune) Control rat liver microsomes 0.0 2.6 (immune). 7.8 (immune). 18.0 (preimmune) Phenobarbital-treated rat liver microsomes 0.0.. 2.6 (immune). 7.8 (immune). 18.0 (preimmune) Bovine adrenocortical mitochondria 0.0.. 7.8 (immune). 15.6 (immune). . 18.0 (preimmune) Bovine adrenocortical microsomes O.O.................... 2.6 (immune). 7.8 (immune). . 18.0 (preimmune)

I

Total cytochrome

% conlrol

% control

0.92 1.05 1.03 1.05

100 114 112 114

6.5 5.6 3.8 6.3

100 86 59 97

0.60 0.62 0.63 0.60

100 103 105 100

29.0 25.0 19.0 29.0

100 86 65 100

2.00 1.90 2.00 1.80

100 95 100 91

2.9 2.7 3.1 2.7

100 93 108 93

1.22 1.10 1.08 1.10

100 90 88 90

-

100 78 51 91

0.41 0.45 0.43 0.49

246, No.

13

to TPNH-cytochrome c reductase of pig hepatic microsomes. This antibody preparation, however, had no effect on bovine adrenocortical mitochondria, although it exhibited inhibitory properties in bovine adrenocortical microsomes. It should be noted that the antibody did not alter the amount of dithionitereducible cytochrome P-450 present in the reaction mixtures. Thus, the reductase antibody inhibited the TPNH-dependent reduction of cytochrome P-450 only in those systems in which TPNH-cytochrome c reductase activity was inhibited. These results indicate that either the same enzyme is involved in the TPNH-dependent reduction of both cytochrome c and cytochrome P-450 in hepatic and adrenocortical microsomes, or, that if different enzymes mediate the reduction of the two cytochromes they are immunochemically similar. The inability of the reductase antibody to inhibit the activity of TPNH-cytochrome P-450 reductase in adrenocortical mitochondria is consistent with the lack of effect of the antibody on TPNH-cytochrome c reductase activity in this system and further indicates that the electron transport chain functional in mixed-function oxidations in adrenocortical mitochondria differs from that found in hepatic and adrenocortical microsomes. This inability of antibody to inhibit the TPNH-cytochrome P-450 reductase activity in the adrenocortical mitochondrial system cannot be attributed to a lack of penetration of the antibody since there

P-45Ob

100 77 59 100

0.88 0.68 0.45 0.80

Vol.

100 110 105 119

R Each reaction contained the following in a total volume of 3 ml: 3 mg of microsomal or mitochondrial protein, 0.1 M potassium phosphate buffer, pH 7.4, and preimmune or immune r-globulin where indicated. TPNH-cytochrome P-450 reductase activity was determined as described under “Materials and Methods.” b Total cytochrome P-450 in the 3-ml reaction mixture was determined after the addition of Na&O,. c The immune r-globulin fraction used in these experiments had a lower antibody titer than that used in other experiments.

TABLE III Effect of antibody to bovine adrenocortical mitochondrial adrenodoxin on activity of TPNH-cytochrome P-460 reductase in rat liver microsomes and bovine adrenocortical mitochondria and microsomes

1,

Fraction

-_

TPNH-cytochrome P-450 reductase”

mg yglobulin/3-ml reaction

Total cytochrome

P-4SOb

% control

% control

Control rat liver microsomes 0.0.. .. 9.0 (immune) . 18.0 (immune). 18.0 (preimmune) Phenobarbital-treated rat liver microsomes 0.0.. 9.0 (immune). . 18.0 (immune). 18.0 (preimmune)

Bovine adrenocortical somes O.O........................

-

I

6.5 6.2 6.2 6.3

100 95 95 97

0.60 0.59 0.62 0.60

100 98 103 100

29.0 27.4 26.5 29.0

100 95 92 100

2.00 1.90 1.90 1.80

100 95 95 91

0.88 0.82 0.82 0.80

100 93 93 91

0.41 0.42 0.42 0.49

100 102 102 119

2.9 0.43 0.13 2.7

100 15 4 93

1.22 1.12 1.30 1.10

100 92 108 90

micro-

9 .O (immune). . 18.0 (immune). 18.0 (preimmune) Bovine adrenocortical mitochondria 0.0.. _, _, _, 9.0 (immune). . 18.0 (immune). . 18.0 (preimmune)

-

a Each reaction contained the following in a total volume of 3 ml: 3 mg of mitochondrial or microsomal protein, 0.1 M potassium phosphate buffer, pH 7.4, and preimmune or immune r-globulin where indicated. b Total cytochrome P-450 in the 3-ml reaction mixture was determined after the addition of Na&04.


Effect of an antibody to pig liver microsomal TPNH-cytochrome c reductase on activity of TPNH-cytochrome P-460 reductase in rat and pig liver microsomes and bovine adrenocortical mitochondria and microsomes

Reactions

Masters, Baron, Taylor, Isaacson, and LoSpalluto

Issue of July 10, 1971

DISCUSSION

Evidence has been presented in the present report which conclusively demonstrates that TPNH-cytochrome c reductase is indeed the flavoprotein component involved in the mixed-funcThe immunotion oxidations occurring in hepatic microsomes. chemical similarity of TPNH-cytochrome c reductase, solubilized from pig liver microsomes by pancreatic lipase and purified to homogeneity, to the TPNH-dependent flavoprotein involved in hepatic microsomal mixed-function oxidations is clearly demonstrated by the concomitant inhibition of both TPNH-cytochrome c reductase and ethylmorphine N-demethylase activities by the antibody to the reductase (Figs. 1 and 2). Although purified to a form which no longer functions in the w-hydroxylation of fatty acids (4) the reductase retained antigenic determinants in common with the “native” flavoprotein (Fig. 3). These results are in agreement with those of Omura (17), who demonstrated that antibodies to the trypsin-solubilized reductase of rat liver microsomes inhibited aniline hydroxylation and of Wada et al. (18), who reported the inhibition of w-hydroxylation by these antibodies in rat liver microsomes. The antibody to homogeneous pig liver TPNH-cytochrome c reductase was also found to inhibit the TPNH-dependent reduction of cytochrome c in hepatic microsomes prepared from phenobarbital-treated pigs and rats, an observation consistent with the results of Kuriyama et al. (32), who have shown that the reductase which is induced in rat liver by phenobarbital is chromatographically and immunochemically identical with that These results also demonstrate which is normally synthesized. the immunochemical similarity of TPNH-cytochrome c reductase of pig and rat liver microsomes. Furthermore, this same antibody was also observed to inhibit TPNH-cytochrome c reductase activity in microsomes isolated from autopsy samples of human liver.2 Ouchterlony double diffusion experiments demonstrate precipitin reactions between antibody to pig liver 2 E. B. Nelson,

published

P. Raj,

observations,

K. J. Belfi,

and manuscript

and B. S. S. Masters,

in preparation.

un-

TPNH-cytochrome c reductase and microsomal preparations from pig liver and bovine adrenal cortex. It should also be noted that the reductase antibody did not inhibit the TPNHdependent mixed-function amine oxidase activity of pig liver microsomes or of the purified flavoprotein responsible for this activity (34). These observations thus indicate that the observed inhibition was not due to a nonspecific inhibition of microsomal enzymes and that the inhibition of TPNH-cytochrome c reductase activity by the antibody resulted from a specific immunochemical interaction between antigen and antibody. Thus, by employing an antibody prepared to homogeneous TPNH-cytochrome c reductase from pig liver, the immunochemical similarity of the flavoproteins involved in cytochrome P-450mediated reactions in hepatic endoplasmic reticulum of various species has been demonstrated for the first time. In addition, the activity of TPNH-cytochrome c reductase in bovine adrenocortical microsomes was also inhibited by this antibody. This observation and the Ouchterlony double diffusion data indicate that the flavoproteins of both hepatic and adrenocortical endoplasmic reticulum are immunochemically similar. If one assumes that the suggestion of Sweat et al. (20) is correct, that is, that the flavoproteins involved in cytochrome P-450mediated mixed-function oxidations in adrenocortical microsomes and mitochondria are identical, then the antibody to pig liver TPNH-cytochrome c reductase would also be expected to inhibit the activity of this enzyme in adrenocortical mitochondria. However, when TPNH-cytochrome c reductase activity was examined in either adrenocortical mitochondria or in Fraction SZ, the solubilized preparation of flavoprotein and non-heme iron protein obtained from sonicated mitochondria, there was no apparent inhibitory effect exerted by the reductase antibody. These results indicate that the TPNH-dependent flavoprotein involved in those reactions mediated by cytochrome P-450 in mitochondria of the adrenal cortex differs from the enzyme or enzymes functioning in hepatic and adrenocortical microsomes and are consistent with the observations of Omura et a.1. (8) who showed that TPNH-cytochrome c reductase which had been purified from pig liver was unable to substitute for the mitochondrial flavoprotein in the reconstituted electron transport system of bovine adrenocortical mitochondria. Furthermore, the observation that a non-heme iron protein, adrenodoxin, is required in addition to the flavoprotein for the TPNH-dependent reduction of cytochrome c in adrenocortical mitochondria (8) strengthens the suggestion that the flavoprotein of adrenocortical mitochondria differs from those occurring in the endoplasmic reticulum of the liver and adrenal cortex. Furthermore, experiments performed on the purilied flavoproteins isolated and purified from pig liver microsomes and bovine adrenocortical mitochondria indicate entirely different reaction mechanisms for reduction by TPNH and reoxidation (13). Indeed, when an antibody to adrenodoxin was employed, inhibition of TPNHcytochrome c reductase activity was observed only in adrenocortical mitochondria, but not in hepatic or adrenocortical microsomes. Although it is generally assumed that TPNH-cytochrome c reductase is the flavoprotein involved in the reduction of cytochrome P-450, until now there has been no experimental evidence presented which conclusively demonstrates that this assumption is correct. Employing an antibody prepared to homogeneous pig liver TPNH-cytochrome c reductase, it was observed that inhibition of both TPNH-cytochrome c reductase and TPNH-cytochrome P-450 reductase activities occurred in


was no inhibition of TPNH-cytochrome c reductase activity in the solubilized preparation (Fraction SJ. Egect of Antibody to Adrenodoxin on TPNH-Cytochrome c Reductase and TPNH-Cytochrome P-450 Reductase ActivitiesFrom the data in Table III, it can be seen that TPNH-cytochrome P-450 reductase activities measured in hepatic microsomes from control and phenobarbital-treated rats and in bovine adrenocortical microsomes were unaffected by the antibody to adrenodoxin. The TPNH-cytochrome c reductase activity in these microsomal preparations was similarly unaffected by the antibody to adrenodoxin. When the anti-adrenodoxin y-globulin fraction was tested in adrenocortical mitochondrial TPNHcytochrome P-450 reductase assays, 96% inhibition was obtained with the higher level of antibody showing that the antibody could penetrate and inhibit in the mitochondrial system. Therefore, inaccessibility of antibody to mitochondria could not explain the lack of inhibition by the anti-TPNH-cytochrome c reductase y-globulin in the mitochondrial assays (Tables I and II). Furthermore, TPNH-cytochrome c reductase activity in adrenocortical mitochondria was also similarly inhibited by the antibody to adrenodoxin. It will also be noted that, although TPNH-cytochrome P-450 reductase activity was markedly inhibited in mitochondria, the amount of dithionite-reducible cytochrome P-450 remained relatively constant.

4149

4150

Immunochemical

Studies on Cytochrome P-4.60~mediated Reactions

the presenceof the antibody in hepatic and adrenocortical microsomes, but that no inhibition of either ensymic activity was found in preparations of adrenal cortex mitochondria. Furthermore, when an antibody to homogeneous adrenodoxin prepared from adrenal cortex mitochondria was employed, inhibition of both activities was observed in mitochondrial preparations, whereas no inhibition was found in microsomal preparations of liver or adrenal cortex. These results therefore indicate (1) that within a given subcellular fraction, the same enzyme or enzyme system is involved in the TPNH-dependent reduction of both cytochrome c and cytochrome P-450, (2) that, while the electron transport chains functional in mixed-function oxidations in liver and adrenal cortex endoplasmic reticulum appear to be the same, the electron transport chain operative in cytochrome P-450-mediated reactions in adrenocortical mitochondria is different, and (3) that the non-heme iron protein, adrenodoxin, is involved in cytochrome P-450-mediated reactions only in adrenocortical mitochondria, and not in microsomes of liver or adrenal cortex.

REFERENCES 1. OMURA, T., SATO, R., COOPER, D. Y., ROSENTHAL, O., AND ESTABROOK. R. W.. Fed. Proc.. 24. 1181 (1965). 2. COOPER, D. 5!., LEVI&, S., NARA&&JLU, g., ROSENTHAL, O., AND ESTABROOK, R. W., Science, 147,400 (1965). 3. CONNEY, A. H., IKEDA, M., LEVIN, S., COOPER, D. Y., ROSENTHAL, O., AND ESTABROOK, R. W., Fed. PTOC., 26, 462 (1967). 4. Lu, A. Y. H., JUNK, K. W., AND COON, M. J., J. Biol. Chem., 244, 3714 (1969). 5. SIMPSON, E. R.. AND BOYD. G. S.. Eur. J. Biochem., 2, 275 (1967): 6. COOPER, D. Y., NOVACK, B., FOROFF, O., SLADE, A., SANDERS, E., NARASIMHULU, S., AND ROSENTHAL, O., Fed. Proc., 26, 341 (1967). 7. ESTABROOK, R. W., COOPER, D. Y., AND ROSENTHAL, O., Biochem. 2.. 338. 741 (1963). 8. OMURA, T., SANDERS, E.; E&ABROOK, R. W., COOPER, D. Y., AND ROSENTHAL, O., Arch. Biochem. Biophys., 11’7, 660 (1966).

9. WILLIAMS, C. H., JR., AND KAMIN, H., J. Biol. Chem., 237,587 (1962). 10. PHILLIPS, A. H., AND LANODON, R. G., J. Biol. Chem., 237, 2652 (1962). 11. MASTERS, B. S. S., KAMIN, H., GIBSON, Q. H., AND WILLIAMS, C. H., JR., J. Biol. Chem., 249,921 (1965). 12. MASTERS, B. S. S., BILIMORIA, M. H., KAMIN, H., AND GIBSON, Q. H., J. Biol. Chem., 249, 4081 (1965). 13. KAMIN, H., MASTERS, B. S. S., AND GIBSON, Q. H., in E. C. SLAT~R (Editor), Flavins and jlavoproteins, ~Elsevier Publishing Comuanv. Amsterdam. 1966. DD. 306 to 324. 14. GILLETTE, J. lk, &ODIE, B. B., ‘AND I;&, B. N., J. Pharmacol. Exp. Ther., 119,532 (1957). 15. AXELROD, J., J. Pharmacol. Exp. Ther., 110, 315 (1954). 16. AXELROD, J., J. Biol. Chem., 214, 753 (1955). 17. OMURA, T., iu J. R. GILLETTE, A. H. CONNEY, G. J. COSMIDES, R. W. ESTARROOK. J. R. FOIJTS. AND G. J. MANNERIN~ (Editors), Microsomes and drug ozidations, Academic Press, New York, 1969, pp. 160 to 162. 18. WADA, F., SHIBATA, H., GOTO, M., AND SAKAMOTO, Y., Biochim. Biophys. Acta, 162. 518 (1968). 19. MASTERS, I% #. S., WILLIAMS, Cl H., JR., AND KAMIN, H., in R. W. ESTABROOK AND M. E. PULLMAN (Editors). Methods in enzumelosu. Vol. X. Academic Press,‘New York, 1967, pp. 565 to 5%: ’ 20. SWEAT, M. L., DUTCHER, J. S., YOUNO, R. B., AND BRYSON, M. J.. Biochemistry, 6. 4956 (1969). 21. KAMIN,‘H., MASTER~,~B; S. S.,‘GIB~ON, Q. H., AND WILLIAMS, C. H., JR., Fed. Proc., 24, 1164 (1965). 22. NELSON, E. B., MASTERS, B. S. S., AND PETERSON, J. A., Anal. Biochem., 39, 128 (1971). 23. CAMMER, W., AND ESTABROOK, R. W., Arch. Biochem. Biophys. 122, 721 (1967). 24. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem., 177, 751 (1949). 25. MASSEY, V., Biochim. Biophys. Acta, 34, 255 (1959). 26. OMURA, T., AND SATO, R., J. Biol. Chem., 239, 2379 (1964). 27. NASH, T., Biochem. J., 66, 416 (1953). 28. COCHIN, J., AND AXELROD, J., J. Pharmacol. Exp. Ther., 126, 105 (1959). 29. KIMURA, T., AND SUZUKI, K., J. Biol. Chem., 242,485 (1967). 30. OUCHTERLONY, O., Progr. Allergy, 6, 1 (1958). 31. ZIEGLER, D. M., AND PETTIT, F. H., Biochem. Biophys. Res. Commun., 16, 188 (1964). 32. KURIYAMA, Y., OMURA, T., SIEKEVITZ, P., AND PALADE, G. E., J. Biol. Chem., 244, 2017 (1969). 33. OMURA, T., AND TAKESUE, S., J. Biochem. (Tokyo), 67, 249 (1970). 34. ZIEQLER, D. M., MITCHELL, C. H., AND JOLLOW, D., in J. R. GILLETTE, A. H. CONNEY, G. J. COSMIDES, R. W. ESTABROOK, J.‘R. FOUTS, AND G. J. MANNERINQ (Editors), Microsomes and druo oxidations, Academic Press, New York, 1969,

pp. 173 to 183:


Aclcnowledgmcnts-We thank Mr. Earl F. Calahan for his skilled handling of the rabbits used in the production of antibodies. The able technical assistance of Mr. George Nunn, Miss Carol Phelps, and Mrs. Karen Kohl is gratefully acknowledged. We also wish to thank Dr. Ronald W. Estabrook for his critical evaluation and encouragement throughout these studies. We are indebted to Mrs. Margaret Franklin for her skill in typing this manuscript.

Vol. 246, No. 13