ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
170,
160-168
The Role of NADPH-Cytochrome Hydroxylation R. A. PROUGH Department
ofBiochemistry,
(1975)
c Reductase Reaction@ AND
in Microsomal
M. D. BURKE
The University of Texas Health Science Center, Southwestern 5323 Harry Hines Boulevard, Dallas, Texas 75235 Received
May
Medical
School,
1, 1975
A specific antibody elicited against NADPH-cytochrome c reductase inhibited cytochrome P-450- and NADPH-dependent hydroxylation of biphenyl by rodent liver and lung microsomal preparations. The inhibition profiles suggested that both the 2- and 4hydroxylation of biphenyl were mediated by a common NADPH-cytochrome c reductase (NADPH-cytochrome P-450 reductase) and that the same flavoprotein species operated in liver and lung microsomes of corn oil- or 3-methylcholanthrene-pretreated rats and hamsters. An immunochemically identical NADPH-cytochrome c reductase also apparently functioned in the NADPH-supported metabolism of benzo(a)pyrene and ethylmorphine. NADH supported the microsomal metabolism of benzo(a)pyrene and ethylmorphine in liver and biphenyl in liver and lung, but the maximal rates of reaction were slower than when supported with NADPH. The K, of NADH for biphenyl 2- and 4hydroxylations in control hamster liver microsomes were approximately 5 mM. AntiNADPH-cytochrome c reductase globulin inhibited NADH-supported biphenyl2and 4hydroxylase activities in corn oil- or 3-methylcholanthrene-pretreated rats and hamsters, even at NADH concentrations as low as 0.25 mM. These results indicate that the same flavoprotein reductase species which mediated NADPH-dependent biphenyl hydroxylase donated at least one electron for the NADH-supported hydroxylation.
A number of aromatic hydroxylation reactions have been reported to be catalyzed by rodent liver microsomes, but several activities, such as benzo(a)pyrene hydroxylase, yield multiple products which are difficult to characterize or quantitate. The metabolite pattern of biphenyl hydroxylation has been well characterized using a sensitive fluorimetric assay and is known to yield predominantly the 2- and 4-hydroxylated products (1, 2). Although 2,2’- and 4,4’-dihydroxybiphenyl are minor products, the 2- and 4-hydroxybiphenyl products represent >85% of total metabolites as determined by radiochromatographic analysis (3). This report will show confirmatory data which indicates that 2- and 4hydroxybiphenyl together account for ap1 Supported by National Cancer Institute tract NO1 CP33362 (RWEIRAP), a USPHS Investigator Pulmonary Research Grant HL17134 (RAP), and USPHS Grant GM16488. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
proximately 95% of total biphenyl metabolites in a 5-min incubation mixture with rodent liver microsomes. Pretreatment of animals with drugs, such as phenobarbital, preferentially induces biphenyl 4-hydroxylation, while pretreatmeht with polycyclic hydrocarbon carcinogens induces either both 2- and 4-hydroxylation in hamsters or preferentially 2-hydroxylation in rats (3). The simple metabolite pattern and the extremely sensitive fluorimetric determination of both major metabolites of biphenyl hydroxylation allow this reaction to be favorably compared with other reactions catalyzed by the cytochrome P-450dependent monoxygenase systems in liver and lung microsomes. Two distinct electron transport chains exist in the microsomal fraction of liver: the fatty acid desaturase system (4) consisting of NADH-cytochrome bs reductase, cytochrome bs and the desaturase and the cytochrome P-450 system (5) consisting of
ConYoung I R23 160
NADH-
AND
NADPH-SUPPORTED
NADPH-cytochrome c (P-450) reductase and cytochrome P-450. Using chemical and immunochemical (6, 7) inhibitors, a number of NADPH-mediated monoxygenase reactions have been shown to require NADPH-cytochrome c reductase as the flavoprotein reductase. Several reports indicate that NADH-cytochrome b, reductase and cytochrome b5 may be involved in some NADH-sustained cytochrome P-450reactions (8, 9). The synergistic enhancement of NADPH-mediated reactions by the presence of NADH has been shown to involve cytochrome b, by directly measuring changes in the steady-state reduction of the cytochrome bs during metabolism (lo), by inhibition of the synergism in the presence of stearyl CoA (ll), and by inhibition of the synergism by a specific inhibitory antibody to cytochrome b5 (12, 13). This result suggests an interaction of the two electron transport systems in the presence of both reduced pyridine nucleotides and implicates cytochrome b5 as a second electron donor to cytochrome P-450 under these conditions. The participation of cytochrome b5 in transfer of electrons to cytochrome P-450 in NADPH-dependent hydroxylation reactions remains unclear. Studies with purified NADPH-cytochrome c reductase (EC 1.6.2.4) have suggested that NADH can serve as an electron donor to the reductase but with a larger K, compared to NADPH (14, 15). Although no data have been published, T. Omura (16) was quoted as suggesting that the NADPH-cytochrome c reductase must be involved in NADH-dependent hydroxylation reactions. Recently, we reported the apparent similarity in rate of cytochrome P-450 reduction by NADH or NADPH in rodent lung microsomes (17). In this study, we have used a specific immunochemical inhibitor, an antibody to rat liver NADPH-cytochrome c reductase, to elucidate the role of the flavoprotein NADPH-cytochrome c reductase in the NADPH- and NADH-supported microsomal hydroxylation of several drug or carcinogen substrates by liver and lung microsomes. ’ 2 Throughout this study, NADHor NADPH-supported lations as being microsomal
the authors or sustained hydroxylation
refer to hydroxyreactions
MICROSOMAL
161
HYDROXYLATIONS MATERIALS
AND
METHODS
Materials. NADH, NADPH, and NADP+ were purchased from the Sigma Chemical Company and “Chromatopure” NADH was obtained from P.L. Biochemicals. Biphenyl, P-hydroxybiphenyl, and 4-hydroxybiphenyl were obtained from K & K Laboratories, Inc. and recrystallized from petroleum ether; the biphenyl compounds were judged to be pure from analysis using gas chromatography and mass spectrometry (See following section). All other compounds were obtained in analytical grade from commercial sources. Preparation of microsomes. Male Golden Syrian hamsters W-130 g) and male Sprague-Dawley rats (150-225 g) were used in this study. The methods of pretreatment of animals and preparation of liver and lung microsomes were previously described (17). Assay of microsomal enzyme activities. NADPHcytochrome c reductase activity was measured as described by Masters et al. (18). Benzo(a)pyrene hydroxylase and ethylmorphine demethylase assays were performed using the methods of Nebert and Gelboin (19) and Masters et al. (71, respectively. All reactions which included anti-reductase globulin were incubated with antibody at 0°C for 10 min prior to initiation. To establish the metabolic products of biphenyl hydroxylation activity in hamster or rat liver microsomes, we analyzed 20 ml of the heptane extract of a 15min (5 mg/ml) incubation mixture by derivatizing the hydroxybiphenyls with 5 ml ofN,O-bis-(trimethylsilyl)-acetamide at 50°C for 2 h, evaporating to dryness, and redissolving the residue in heptane (0.2 ml). The derivatized samples were injected onto a 6 ft 3% OV-I gas chromatograph column and the samples were eluted using a temperature program which included a 2-min postinjection period at lOOC, a temperature increase to 170°C over a 12-min period, and a final temperature of 170°C for 6 min. The following elution times were determined for the underivatized substrate and derivatised hydroxy standards: Biphenyl (3.8 min), 2-hydroxybiphenyl (8.0 mini, 2,2’-dihydroxybiphenyl (9.5 min), 3-hydroxybiphenyl (10.7 min), 4-hydroxybiphenyl (11.6 min), and 4,4’-dihydroxybiphenyl (16.4 min). These standards were ~98% pure as judged by their individual chromatograms. Analysis of the derivatized heptane extracts of a 15 min incubation mixture yielded only two major and two minor metabolite peaks as determined by flame ionization. Their identities, based on retention time and mass spectrometry analysis, suggest that the major metabolites were 2- and 4-hydroxybiphenyl and the minor carried out in the presence of only NADH or only NADPH, respectively. This designation does not refer to the case of NADH stimulation (synergism) of NADPH-dependent reactions described by other workers (10-13).
162
PROUGH
metabolites were 2,2’- and 4,4’-dihydroxybiphenyl. Analysis of the area under the peak on flame ionization detection indicated that 2- and ri-hydroxybiphenyl together accounted for approximately 95% of the total biphenyl metabolites after a 5-min incubation with rodent liver microsomes. In agreement with Bridges, Creaven, and Williams (20), 3-hydroxybiphenyl has 10-X% of the fluorescence of 2-hydroxyand 4-hydroxybiphenyl at their respective fluorescent settings. However, the gas chromatograpy-mass spectrometry analysis of the incubation mixtures indicate that 3-hydroxybiphenyl formation by liver microsomes must be less than 5% of the formation of the 2-hydroxyand 4-hydroxybiphenyl. The biphenyl hydroxylase assay was performed by a slight modification of the method of Creaven, Parke, and Williams (1). The reaction mixtures contained, in a total volume of 2 ml, 1 mM biphenyl, 0.37% (w/v) Tween 80, 3 mM n&socitric acid, 0.8 U isocitrate dehydrogenase, 5 mM MgSO,, 1 mg microsomal protein/ml, and 0.1 M potassium phosphate buffer, pH 7.6. The reaction was initiated with NADPH or NADH after 1.5 min preincubation at 37°C and was linear for 5 min at 37°C under all conditions. Initiation of the reaction with either NADPH, biphenyl, or microsomal protein yielded identical hydroxylation rates. The rates of NADH-supported biphenyl hydroxylation were not dependent on the presence or absence of a regenerating system for NADH or NADPH. Authentic 2- and 4-hydroxybiphenyl were routinely added to quenched reaction mixtures to serve as standards for the assay. Preparation of rabbit-antiserum against rat liver NADPH-cytochrome c reductase. NADPH-cytochrome c reductase was purified from liver microsomes of Sprague-Dawley rats using pancreatic lipase as reported by Prough and Masters (21). This flavoprotein reductase yielded a single protein band upon polyacrylamide gel electrophoresis and was stable on storage at -5°C. The protein band of the reductase was excised from the gel and used as the challenging antigen. Three weekly injections of 1 mg of purified reductase in Freund’s Adjuvant (Difco Co.) were administered to young white rabbits at multiple injection sites in the nape of the neck. The blood of the rabbits was obtained biweekly from ear veins and tested for specific antibody against rat liver NADPH-cytochrome c reductase by measuring the percentage of inhibition (titer) of rat liver microsomal NADPHcytochrome c reductase activity. Sera, with high antibody titer, from several bleedings were pooled and the immunoglobulins were precipitated from the serum by addition of ammonium sulfate (1.75 M), redissolved in 0.05 M potassium phosphate buffer, pH 7.7, and dialyzed against the same buffer. These globulin solutions were stored frozen. Certain globulin preparations were further purified on DEAEcellulose using a KC1 gradient; this resulted in glob
AND
BURKE ulin fractions with higher titer. Control globulins were prepared both from the rabbits prior to immunochemical challenge (preimmune) and from untreated rabbits (nonimmune); no differences were seen between the two sources of control globulins. The specificities of the anti-NADPH-cytochrome c reductase globulins were tested by employing them as inhibitors against NADHand NADPHdependent microsomal cytochrome c reduction. The immunoglobulin inhibited only the NADPH-dependent cytochrome c reductase. The anti-NADPH-cytochrome c reductase globulin did not affect microsoma1 NADH-dependent cytochrome b, reduction but did inhibit microsomal NADPH-dependent cytochrome b, reductase. When examined by Ouchterlony double diffusion tests in agar gel (221, the immunoglobulin elicited a single precipitation line against purified rat liver NADPH-cytochrome c reductase but not against trypsin-solubilized rat liver cyto chrome b, or lysosomally-solubilized rat liver cytochrome 6, reductase. The anti-reductase globulins were equally effective at inhibiting either liver or lung microsomal NADPH-cytochrome c reductase activity of either hamsters or rats. This result suggests a strong immunochemical similarity of the NADPH-cytochrome c reductase of hamster and rat, liver and lung. RESULTS
Normally, the NADPH-cytochrome c reductase assay is run at much lower concentrations of microsomal protein (l/20) than are the biphenyl, benzo(a)pyrene, and ethylmorphine oxygenase reactions. We have established the validity of a comparison of anti-NADPH-cytochrome c reductase globulin effects on the reductase and oxygenases by determining that the concentration of the antibody which gave 50% inhibition of the reductase did not vary over the range of microsomal protein concentrations used. The effectiveness of the immunoglobulin was expressed as the ratio of milligrams of antibody to milligrams of microsomal protein which gave 50% inhibition of the NADPH-cytochrome c reduction reaction (I,,,). Figure 1 shows that the ISo of the immunoglobulin purified by DEAE-cellulose chromatography was independent of microsomal protein concentrations from 0.05 to 0.8 mg/ml. Animal pretreatment did not affect the independence of the I,, value from microsomal protein concentration, but did alter this ratio of milligrams of antibody to milligrams of microsomal protein giving 50% inhibition
NADH,
00
AND
NADPH-SUPPORTED
1
I
1
MICROSOMAL
I
1
o-0
0.2 PROTEIN
o-
I
I
0.4 CONCENTRATION
I
0.6
8
I
0.6
i
(mg/ml)
FIG. 1. The inhibition by the anti-NADPH-cytochrome c reductase globulin at varying microsomal protein concentrations. The ratio of milligrams antibody to milligrams microsomal protein to give 50% inhibition (I& of microsomal NADPH-cytochrome c reductase, sp act = 110 nmol min-’ mg-’ (Methods section), was determined at several concentrations of rat liver microscmal protein.
of microsomal NADPH-cytochrome c reductase activity. The change in Iso observed using liver microsomes from animals pretreated with drugs i such as phenobarbital, was consistent with the increase of the specific activity of the microsomal reductase under these same conditions of pretreatment. The Is0 obtained with immunoglobulin from different animals or bleedings varied but was independent of microsomal protein concentration. The effect of the anti-NADPH-cytochrome c reductase globulin on NADPHdependent biphenyl 2- and 4-hydroxylase activities of liver microsomes from hamsters injected with corn oil or 3-methylcholanthrene can be seen in Fig. 2a and b. With microsomes of corn oil-treated hamsters, the inhibition profiles of the biphenyl 2- and 4-hydroxylation reactions were similar with each other but were not parallel with the inhibition profile of NADPH-cytochrome c reduction; in contrast, with liver microsomes from 3-methylcholanthrene-injected hamsters, the hydroxylation and NADPH-cytochrome c reduction reactions were inhibited in parallel fashion by the immunoglobulin. In both cases NADPH-dependent cytochrome P-
163
HYDROXYLATIONS
450 reductase activity was inhibited parallel with NADPH-cytochrome c reduction (R. A. Prough, unpublished results). Since the antibody had the same relative effectiveness regardless of protein concentration, the lack of parallel inhibition between hydroxylation and cytochrome reduction activities may reflect 3-methylcholanthrene-induced differences in the molar ratio of the flavoprotein and the hydroxylase. The inhibition of benzo(a)pyrene hydroxylase activity by the anti-NADPH-cytochrome c reductase globulin was seen to parallel the inhibition of biphenyl2- and 4hydroxylase activity in liver microsomes from either corn oil-injected or 3-methylcholanthrene-injected hamsters (maximum inhibition at 8 mg globulin/mg protein = 75%). The activity of hamster liver microsomal benzo(a)pyrene hydroxylase (0.65 nmoles/min/mg microsomal protein) was not appreciably induced by animal pretreatment with 3-methylcholanthrene; benzo(a)pyrene hydroxylase activity was induced two- to threefold in rat liver micro-
1
2
4
6
B
m g GLOBULIN/mgPROTEIN
FIG. 2. The effect of anti-NADPH-cytochrome c reductase globulin on NADPH-dependent biphenyl hydroxylase and cytochrome c reductase activity of hamster liver microsomes. The enzyme activities were determined as described in the Methods section. Figure 2a shows the inhibition of NADPHdependent microsomal biphenyl 2-hydroxylase activity (0, 100% = 1.0 nmol min-’ mg-‘), biphenyl 4hydroxylase activity (0, 100% = 23 nmol min-’ mg-‘1, and NADPH-cytochrome c reductase (A, 100% = 210 nmol mine1 mg-‘) from corn oil-injected hamsters. Figure 2b shows the inhibition of NADPH-sustained microsomal biphenyl 2-hydroxylase activity (0, 100% = 2.7 nmol min-’ mg?, biphenyl I-hydroxylase activity (0, 100% = 7.9 nmol min-’ mg-‘), and cytochrome c reductase activity (A, 100% = 218 nmol min-’ mg-‘) from 3-methylcholanthrene-pretreated hamsters. No effect of nonimmune globulin was seen on any of the activities (A).
164
PROUGH
somes by pretreatment with 3-methylcholanthrene. Reduced pyridine nucleotide specificity of liver microsomal biphenyl2and 4-hydroxylase activity. The dependence of biphenyl 2- and 4-hydroxylase activities on NADPH and NADH concentration can be seen in Fig. 3. Although the reactions were saturated in the micromolar range with NADPH (KmaPP 5 40 FM), approximately 10 mM NADH was required to saturate the hydroxylase reactions of hamster liver microsomes from corn oil-pretreated hamsters (KmaPP for NADH = 5.2 mM). Similar results were obtained when highly purified NADH, P.L. Biochemicals “Chromatopure” NADH, was used. The apparently high ‘K,,, of the hydroxylases for NADH suggests that NADH-cytochrome b, reductase was probably not involved, since 100 PM NADH should have been sufficient to cause maximal steady-state reduction of the NADH-cytochrome b, reductase (23). Effect of anti-NADPH cytochrome c reductase globulin on NADH-sustained liver and lung biphenyl hydroxylase activ-
mMOLAR
3. The
NADtPJH
reduced pyridine nucleotide specificity of liver microsomal biphenyl hydroxylase activity. The reactions were run as described in the Methods section but initiated with various concentrations of NADH or NADPH as indicated. The figure shows biphenyl I-hydroxylase activity supported by NADPH (0) or NADH (0) and biphenyl a-hydroxylase activity supported by NADPH (A) or NADH (A). The liver microsomes were obtained from corn oilinjected hamsters; identical results were obtained with liver microsomes from 3-methylcholanthreneinjected hamsters. FIG.
AND
BURKE
1
25
1 mgGLOBULIN/mg
PROTEIN
FIG. 4. The effect of anti-cytochrome c reductase globulin on NADH-dependent liver biphenyl 4-hydroxylase activity. The figure shows the inhibition of microsomal biphenyl I-hydroxylase activity (Methods section) mediated by 0.25 mM NADH (A, 100% = 1.0 nmol min-’ mg-9, 10 mM NADH (0, 100% = 3.0 nmol min-’ mg-I), and 0.25 mM NADPH (0, 100% = 8.0 nmol min-’ mg-*). The effect of nonimmune globulin on 0.25 mM NADH(A) and 0.25 mM NADPH(M) sustained microsomal biphenyl Chydroxylase activity is shown. The liver microsomes were obtained from 3-methylcholanthrene-injected hamsters.
ity. Using the specific immunoglobulin for rat liver NADPH-cytochrome c reductase, the NADH-dependent biphenyl 2- and 4hydroxylase activities of hamster liver microsomes were tested for involvement of NADPH-cytochrome c reductase. As shown in Fig. 4, the anti-NADPH-cytochrome c reductase globulin inhibited NADH-sustained biphenyl 4-hydroxylase activity of liver microsomes from 3-methylcholanthrene-pretreated hamsters; the inhibition profiles for 2- and 4-hydroxylation were almost identical. At 0.25 mM NADH, the hydroxylase activities were inhibited approximately 45%. Parallel profiles were observed for inhibition, by the anti-reductase globulins, of rat or hamster liver microsomal biphenyl hydroxylation and cytochrome c reduction supported by saturating concentrations of either NADPH or NADH (Table I). The effect of antiNADPH-cytochrome c reductase globulin on hamster liver microsomal benzo(a)pyrene hydroxylase and ethylmorphine demethyl-
NADH-
AND
NADPH-SUPPORTED
MICROSOMAL
BIPHENYL
MICROSOMAL TABLE
INHIBITION
Species
Hamster
Rat
OF LIVER
Activity
measured
Cytochrome c reduction Biphenyl4-hydroxylation Cytochrome c reduction Biphenyl 4-hydroxylation
I
HYDROXYLASE AND NADPH OR NADH
CYTOCHROME
Reduced” pyridine nucleotide
c REDUCTION
SUPPORTED
Control
Immune globulin 49 470 2.4 0.9 16 712 0.8 0.4
215 490 8.0 3.0 110 727 3.9 1.4
“The concentrations of NADH or NADPH were 10 and treated with 3-methylcholanthrene. * Both control and immune globulin reaction mixtures anti-rat liver NADPH-cytochrome c reductase globulin per The activity is expressed as nanomoles product per minute
0.25 mM,
BY
% Inhibition
Activityb
NADPH NADH NADPH NADH NADPH NADH NADPH NADH
ase activities sustained by NADH was to inhibit these reactions in relatively parallel fashion to the inhibition of biphenyl hydroxylase reactions (Table II). Hamster or rat lung microsomal NADPH-dependent .biphenyl 4-hydroxylase activity was of the same order of magnitude as liver microsomal biphenyl 4-hydroxylase activity of corn oil-pretreated animals (1.0 and 2.0 nmol/min/mg microsoma1 protein in lung or liver, respectively). Furthermore, the relative rates of NADHand NADPH-dependent biphenyl li-hydroxylase activities in lung microsomes were also similar to those shown for liver microsomes in Fig. 3. Typical rates for liver and lung NADHand NADPH-sustained biphenyl4-hydroxylase activities are shown in Table III. The inhibiting effect of antiNADPH-cytochrome c reductase on NADHor NADPH-supported lung biphenyl 4-hydroxylase activity was nearly identical to that shown for the liver hydroxylase (Fig. 5). The 2-hydroxylase activity of lung microsomes from 3-methylcholanthrene-pretreated hamsters was also inhibited in a manner similar to the 4-hydroxylase activity (78% maximal inhibition). The effect of NADP+ on NADH-sustained biphenyl hydroxylase activity. As was shown by Williams and Kamin (61, NADP+ is an inhibitor of NADPH-cyto-
165
HYDROXYLATIONS
respectively.
The
78 4 70 71 86 2 80 72
animals
were
pre-
contained a 8:l ratio of nonimmune globulin milligram of microsomal protein, respectively. per milligram of microsomal protein. TABLE
INHIBITION
OF
NADH-
BENZO(a)PYRENE PHINE DEMETHYLASE CORN OIL INJECTED
Activity measured
II OR
NADPH-SUPPORTED
HYDROXYLASE OF LIVER HAMSTERS
NADPH-CYTOCHROME
AND ETHYLMORMICROSOMES FROM BY ANTI-RAT LIVER
c REDUCTASE
Reduced” pyridine Nucleotide
or
GLOBULIN
Activityb Control
Immune
% Inhibition
fizBenzo(a)pyrene Hydroxylase Ethylmorphine Demethylase
NADPH NADH NADPH NADH
2.78 0.95 4.3 1.8
0.47 0.29 0.7 0.7
83 70 84 60
@The concentrations of NADH and NADPH were 10 and 0.25 mM, respectively. b Both control and immune globulin reaction mixtures contained a 8:l ratio of nonimmune globulin or anti-rat liver NADPH-cytochrome c reductase globulin per milligram of microsomal protein, respectively. The activity is expressed as nanomoles product formed per minute per milligram ‘of microsomal protein.
chrome c reductase-catalyzed reactions. Using NADP+ as a specific chemical inhibitor, one can see in Table IV that NADP+ effectively inhibited NADH- and NADPHdependent liver microsomal biphenyl hydroxylase activity. This effect of NADP+
166
PROUGH
THE BIPHENYL Organ source of microsomes
HYDROXYLASE
Reduced” pyridine nucleotide
Activities Corn
oil
Lung
D The concentrations * Nanomoles product
2.0 1.2 1.0 0.5
1.0 0.3 co.1 co.1
of NADH and NADPH formed/minute/milligram
LIVER
after
AND LUNG
animal
MICRO~~MES
pretreatment”
Phenobarbital
2Hydroxylase
ylase NADPH NADH NADPH NADH
BURKE
TABLE III ACTIVITY OF HAMSTER
:;;;I
Liver
AND
4-Hydroxylase
3-Methyl cholanthrene 2-
4-Hy-
“{Z;-
7.8 2.5 0.9 -
dKi:
1.3 0.3 co.1 -
2H;f;;;-
8.1 2.9 1.2 0.6
2.9 0.9 0.13 co.1
were 10 and 0.25 mM, respectively. of microsomal protein.
TABLE IV THE EFFECT OF NADP+ ON LIVER MICROSOMAL NADHAND NADPH-DEPENDENT BIPHENYL HYDROXYLASE ACTIVITY OF CORN OIL INJECTED HAMSTERS” Pyridine nucleotide (mM) NADPH NADH
NADP+ bnM)
0.25 0.25 1 1 10 10
4 4 40
4-Hydroxylase” activity
lasd activity
2.10 0.80 0.54 0.38 0.94 0.11
0.90 0.18 0.08 0.02 0.30 0.05
’ No pyridine nucleotide regenerating was included in these experiments. b Nanomoles product formed/minute/milligram microsomal protein,
2-HydrOXy-
2
4 mgGLOSULlN/mg
system of
on NADH-sustained hydroxylation reactions is consistent with the anti-reductase effects of the preceding section in implicating an important role for NADPH-@ochrome c reductase in NADH-sustained microsomal biphenyl hydroxylation. DISCUSSION
We have chosen the well-characterized reaction of biphenyl to monitor microsoma1 aryl hydroxylation. As reported in the Methods section, the 2- and 4-hydroxybiphenyl metabolites account for approximately 95% of the total metabolites of a 5min in vitro incubation mixture. Biphenyl
6
8
PROTEIN
FIG. 5. The effect of anti-reductase globulin on lung biphenyl 4-hydroxylase activity. The figure shows the inhibition of microsomal biphenyl 4-hydroxylase activity (Methods) media&d by 0.25 mM NADPH (A, 100% = 0.90 nmol min-’ mg-‘) or 10 mu NADH (0, 100% = 0.45 nmol min-’ mg?. The corresponding effect of nonimmune globulin was noted: 0.25 mM NADPH (A) and 10 mM NADH (0). The lung microsomes were prepared from corn oilinjected hamsters.
hydroxylase activities of liver and lung microsomes are, respectively, approximately equal and relatively large compared to other activities, such as benzo(a)pyrene hydroxylase (24) and ethylmorphine demethylase @‘rough and Burke, unpublished results). Specific antibodies have been elicited against several liver microsomal proteins in order to test their involvement in the electron transport chains of microsomes from numerous organs. Japanese research-
NADH-
AND
NADPH-SUPPORTED
ers have elucidated the roles of NADHcytochrome b, reductase, NADPH-cytochrome c reductase, and cytochrome b, in NADHand NADPH-dependent microsoma1 stearyl CoA desaturase activity (25 27). Masters et al. (7) established the role of NADPH-cytochrome c reductase in cytochrome P-450 linked dealkylation reactions in pig liver microsomes and in steroid hydroxylation reactions of bovine adrenocortical microsomes. The possible role of cytochrome b, in lauric acid oxidation by kidney cortex or liver microsomes and in ethanolamine plasmalogen synthesis by pork spleen microsomes has been elucidated recently using specific antibodies (28, 29). The role of cytochrome b, in NADH-synergism of NADPH-sustained reactions have also been shown (12, 13). A lack of specific chemical inhibitors for the components of the microsomal enzyme system led us to an immunochemical approach to evaluate the role of NADPHcytochrome c reductase in NADH-dependent microsomal hydroxylases. This study is dependent on the specificity of the antireductase globulin to the NADPH-cytochrome c reductase and on the ability to measure the total metabolites of an aromatic hydroxylation reaction. Since the specificity of the antibody was established in the Methods section and the metabolite pattern for liver microsomal biphenyl hydroxylase activity was confirmed, we have shown that both requirements have been met. We also noted no effect of antiNADPH-cytochrome c reductase globulin on the apparent binding of benzphetamine or biphenyl to hamster liver microsomal cytochrome P-450 as measured by the apparent K, and AA maxof their Type I interaction spectra. Type I interaction spectra may reflect the formation of a substratecytochrome P-450 complex (30). The similarity in the apparent K, for NADH between purified NADPH-cytochrome c reductase and microsomal biphenyl hydroxylase activities of liver or lung indicates that the reductase may be involved in NADH-dependent hydroxylation reactions. The stoichiometric reduction of the flavoprotein by NADH further supports the ability of NADH to act as an electron donor to NADPH-cytochrome c re-
MICROSOMAL
HYDROXYLATIONS
167
ductase (15). The biphenyl 4-hydroxylase activity supported by 0.25 mM NADH was maximally inhibited 45% while that supported by either 0.25 mM NADPH or 10 mM NADH was maximally inhibited 70% by anti-NADPH-cytochrome c reductase globulin. The inhibition of NADH-dependent biphenyl or benzo(a)pyrene hydroxylase and ethylmorphine demethylase activities by anti-reductase globulin and biphenyl hydroxylase activity by NADP+ strongly suggests that at least one electron of NADH-dependent hydroxylation reactions must be donated via NADPH-cytochrome c reductase, in agreement with the suggestion of Omura (16). On the basis of the experiments presented in this paper, no evidence can be given to indicate the number of electrons donated to cytochrome P-450. However, the roles of NADPH-cytochrome c reductase in NADH- or NADPHdependent microsomal hydroxylation reactions of liver or lung appear to be identical, and the apparant enhanced rate of NADHreduction of lung microsomal cytochrome P-450 (17) may be the result of a difference in the molar ratio of microsomal electron transport components of lung compared to those of liver. Our observations that titration with the anti-reductase globulin yielded identical inhibition profiles for liver microsomal ethylmorphine demethylation, benzo(a)pyrene hydroxylation, and biphenyl2and 4-hydroxylation suggest that all four microsomal mixed function oxygenases utilize an immunochemically similar species of the NADPH-cytochrome c reductase. Furthermore, this NADPH-cytochrome c reductase appears to operate in both liver and lung microsomes from either the hamster or the rat. However, in the case of hamster liver, microsomal biphenyl2- and 4-hydroxylase activities, the molar ratio of the flavoprotein reductase to other components of the hydroxylase appears to be diminished by pretreatment of the animal with 3-methylcholanthrene. ACKNOWLEDGMENTS The authors express their appreciation to Ms. Nell Mock and Ms. Betty Paul for their expert technical help, and to Dr. Ronald Estabrook and Dr. Bettie Sue Masters for their encouragement.
168
PROUGH
REFERENCES 1. CREAVEN, P. J., PARKE, D. V., AND WILLIAMS, R. T. (1965) Biochem. J. 96, 8’79-885. 2. RAE, VON P., AND AMMON, R. (1970) Drug. Res. 9, 1266-1269. 3. BURKE, M. D., AND BRIDGES, J. W. (1975)&n&otica, 5, 357-376. 4. STRITTMATTER, P., SPATZ, L., CORCORAN, D., ROGERS, M. J., SETLOW, B., AND REDLINE, R. (1974) Proc. Nat. Acad. Sci. USA 71, 456% 4569. 5. Lu, A. Y. H., JUNK, K. W., AND COON, M. J. (1969) J. Biol. Chem. 244, 3714. 6. WILLIAMS, C. H., JR., AND KAMIN, H. (1962) J. Biol. Chem. 237,587-595. 7. MASTERS, B. S. S., BARON, J., TAYLOR, W. E., ISAACSON, E. L., AND LOSPALLUTO, J. (1971) J. Biol. Chem. 246, 4143-4150. 8. WEST, S. B., LEVIN, W., RYAN, D., VORE, M., AND Lu, A. Y. H. (1974) Biochem. Biophys. Res. Commun. 58, 516-522. 9. HRYCAY, E. G., AND PROUGH, R. A. (1974)Arch. Biochem. Biophys., 165, 331-339. 10. HILDEBRANDT, A., AND ESTABROOK, R. W. (1971) Arch. Biochem. Biophys. 143,66-79. 11. CORREIA, M. A., AND MANNEIUNG, G. J. (1973) Mol. Pharmacol. 9, 455-469. 12. SASAME, H. A., MITCHELL, J. R., THORGEIRSSON, S., AND GILLETTE, J. R. (1973) Drug. Met. Disp. 1, 150-155. 13. MANNERING, G. J., KUWAHARA, S., AND OMURA, T. (1974) Biochem. Biophys. Res. Commun. 57,476-481. 14. ICHIKAWA, Y., AND YAMANO, T. (1969) J. Biochem. 66, 351-360. 15. PROUGH, R. A., AND MASTERS, B. S. S. (1975) in Fifth International Symposium on Flavins
AND
BURKE
16. 17.
18.
19. 20. 21. 22. 23. 24.
25. 26. 27. 28.
29.
30.
and Flavoproteins (T. P. Singer, ed.). In press. GILLETTE, J. R., DAVIS, D. C., AND SASAME, H. A. (1972) Ann. Rev. Pharm. 12, 57-84. MATSUBARA, T., PROUGH, R. A., BURKE, M. D., AND ESTABROOK, R. W. (1974) Cancer Res. 34, 2196-2203. MASTERS, B. S. S., WILLIAMS, C. H., JR., AND KAMIN, H. (1967) Methods Enzymol. 10, 565573. NEBERT, D. W., AND GELBOIN, H. V. (1968) J. Biol. Chem. 243,6242-6249. BRIDGES, J. W., CREAVEN, P. J., AND WILLIAMS, R. T. 11965) Biochem. J. 96,872-878. PROUGH, R. A., AND MASTERS, B. S. (1973)Ann. N.Y. Acad. Sci. 212, 89-93. OUCHTERLONY, 0. (1949) Ark. Kemi 1,43-54. STRITTMATTER, P. AND VELICH, S. F. (1957) J. Biol. Chem. 228, 785-799. WIEBEL, F. J., LENTZ, J. C., DIAMOND, L., AND GELBOIN, H. V. (1971) Arch. Biochem. Biophys. 144, 78-86. OSHINO, N., IMAI, Y., AND SATO, R. (1966) Biochim. Biophys. Acta 128, 13-29. OSHINO, N., IMAI, Y., AND SATO, R. (1971) J. Biochem. 69, 155-167. OSHINO, N., AND OMURA, T. (1973) Arch. Biochem. Biophys. 157.395-404. SASAME, H. A., THORGEIRSSON, S. S., MITCHELL, J. R., AND GILLETTE, J. R. (1974) Life Sci. 14, 35-46. PALTAUF, F., PROUGH, R. A., MASTERS, B. S. S., AND JOHNSTON, J. M. (1974) J. Biol. Chem. 249,2661-2662. SCHENKKAN, J. B., REMMER, H., AND ESTABROOK, R. W. (1967) Mol. Pharmacol. 3, 113123.
OF BIOCHEMISTRY
AND
BIOPHYSICS
170,
160-168
The Role of NADPH-Cytochrome Hydroxylation R. A. PROUGH Department
ofBiochemistry,
(1975)
c Reductase Reaction@ AND
in Microsomal
M. D. BURKE
The University of Texas Health Science Center, Southwestern 5323 Harry Hines Boulevard, Dallas, Texas 75235 Received
May
Medical
School,
1, 1975
A specific antibody elicited against NADPH-cytochrome c reductase inhibited cytochrome P-450- and NADPH-dependent hydroxylation of biphenyl by rodent liver and lung microsomal preparations. The inhibition profiles suggested that both the 2- and 4hydroxylation of biphenyl were mediated by a common NADPH-cytochrome c reductase (NADPH-cytochrome P-450 reductase) and that the same flavoprotein species operated in liver and lung microsomes of corn oil- or 3-methylcholanthrene-pretreated rats and hamsters. An immunochemically identical NADPH-cytochrome c reductase also apparently functioned in the NADPH-supported metabolism of benzo(a)pyrene and ethylmorphine. NADH supported the microsomal metabolism of benzo(a)pyrene and ethylmorphine in liver and biphenyl in liver and lung, but the maximal rates of reaction were slower than when supported with NADPH. The K, of NADH for biphenyl 2- and 4hydroxylations in control hamster liver microsomes were approximately 5 mM. AntiNADPH-cytochrome c reductase globulin inhibited NADH-supported biphenyl2and 4hydroxylase activities in corn oil- or 3-methylcholanthrene-pretreated rats and hamsters, even at NADH concentrations as low as 0.25 mM. These results indicate that the same flavoprotein reductase species which mediated NADPH-dependent biphenyl hydroxylase donated at least one electron for the NADH-supported hydroxylation.
A number of aromatic hydroxylation reactions have been reported to be catalyzed by rodent liver microsomes, but several activities, such as benzo(a)pyrene hydroxylase, yield multiple products which are difficult to characterize or quantitate. The metabolite pattern of biphenyl hydroxylation has been well characterized using a sensitive fluorimetric assay and is known to yield predominantly the 2- and 4-hydroxylated products (1, 2). Although 2,2’- and 4,4’-dihydroxybiphenyl are minor products, the 2- and 4-hydroxybiphenyl products represent >85% of total metabolites as determined by radiochromatographic analysis (3). This report will show confirmatory data which indicates that 2- and 4hydroxybiphenyl together account for ap1 Supported by National Cancer Institute tract NO1 CP33362 (RWEIRAP), a USPHS Investigator Pulmonary Research Grant HL17134 (RAP), and USPHS Grant GM16488. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
proximately 95% of total biphenyl metabolites in a 5-min incubation mixture with rodent liver microsomes. Pretreatment of animals with drugs, such as phenobarbital, preferentially induces biphenyl 4-hydroxylation, while pretreatmeht with polycyclic hydrocarbon carcinogens induces either both 2- and 4-hydroxylation in hamsters or preferentially 2-hydroxylation in rats (3). The simple metabolite pattern and the extremely sensitive fluorimetric determination of both major metabolites of biphenyl hydroxylation allow this reaction to be favorably compared with other reactions catalyzed by the cytochrome P-450dependent monoxygenase systems in liver and lung microsomes. Two distinct electron transport chains exist in the microsomal fraction of liver: the fatty acid desaturase system (4) consisting of NADH-cytochrome bs reductase, cytochrome bs and the desaturase and the cytochrome P-450 system (5) consisting of
ConYoung I R23 160
NADH-
AND
NADPH-SUPPORTED
NADPH-cytochrome c (P-450) reductase and cytochrome P-450. Using chemical and immunochemical (6, 7) inhibitors, a number of NADPH-mediated monoxygenase reactions have been shown to require NADPH-cytochrome c reductase as the flavoprotein reductase. Several reports indicate that NADH-cytochrome b, reductase and cytochrome b5 may be involved in some NADH-sustained cytochrome P-450reactions (8, 9). The synergistic enhancement of NADPH-mediated reactions by the presence of NADH has been shown to involve cytochrome b, by directly measuring changes in the steady-state reduction of the cytochrome bs during metabolism (lo), by inhibition of the synergism in the presence of stearyl CoA (ll), and by inhibition of the synergism by a specific inhibitory antibody to cytochrome b5 (12, 13). This result suggests an interaction of the two electron transport systems in the presence of both reduced pyridine nucleotides and implicates cytochrome b5 as a second electron donor to cytochrome P-450 under these conditions. The participation of cytochrome b5 in transfer of electrons to cytochrome P-450 in NADPH-dependent hydroxylation reactions remains unclear. Studies with purified NADPH-cytochrome c reductase (EC 1.6.2.4) have suggested that NADH can serve as an electron donor to the reductase but with a larger K, compared to NADPH (14, 15). Although no data have been published, T. Omura (16) was quoted as suggesting that the NADPH-cytochrome c reductase must be involved in NADH-dependent hydroxylation reactions. Recently, we reported the apparent similarity in rate of cytochrome P-450 reduction by NADH or NADPH in rodent lung microsomes (17). In this study, we have used a specific immunochemical inhibitor, an antibody to rat liver NADPH-cytochrome c reductase, to elucidate the role of the flavoprotein NADPH-cytochrome c reductase in the NADPH- and NADH-supported microsomal hydroxylation of several drug or carcinogen substrates by liver and lung microsomes. ’ 2 Throughout this study, NADHor NADPH-supported lations as being microsomal
the authors or sustained hydroxylation
refer to hydroxyreactions
MICROSOMAL
161
HYDROXYLATIONS MATERIALS
AND
METHODS
Materials. NADH, NADPH, and NADP+ were purchased from the Sigma Chemical Company and “Chromatopure” NADH was obtained from P.L. Biochemicals. Biphenyl, P-hydroxybiphenyl, and 4-hydroxybiphenyl were obtained from K & K Laboratories, Inc. and recrystallized from petroleum ether; the biphenyl compounds were judged to be pure from analysis using gas chromatography and mass spectrometry (See following section). All other compounds were obtained in analytical grade from commercial sources. Preparation of microsomes. Male Golden Syrian hamsters W-130 g) and male Sprague-Dawley rats (150-225 g) were used in this study. The methods of pretreatment of animals and preparation of liver and lung microsomes were previously described (17). Assay of microsomal enzyme activities. NADPHcytochrome c reductase activity was measured as described by Masters et al. (18). Benzo(a)pyrene hydroxylase and ethylmorphine demethylase assays were performed using the methods of Nebert and Gelboin (19) and Masters et al. (71, respectively. All reactions which included anti-reductase globulin were incubated with antibody at 0°C for 10 min prior to initiation. To establish the metabolic products of biphenyl hydroxylation activity in hamster or rat liver microsomes, we analyzed 20 ml of the heptane extract of a 15min (5 mg/ml) incubation mixture by derivatizing the hydroxybiphenyls with 5 ml ofN,O-bis-(trimethylsilyl)-acetamide at 50°C for 2 h, evaporating to dryness, and redissolving the residue in heptane (0.2 ml). The derivatized samples were injected onto a 6 ft 3% OV-I gas chromatograph column and the samples were eluted using a temperature program which included a 2-min postinjection period at lOOC, a temperature increase to 170°C over a 12-min period, and a final temperature of 170°C for 6 min. The following elution times were determined for the underivatized substrate and derivatised hydroxy standards: Biphenyl (3.8 min), 2-hydroxybiphenyl (8.0 mini, 2,2’-dihydroxybiphenyl (9.5 min), 3-hydroxybiphenyl (10.7 min), 4-hydroxybiphenyl (11.6 min), and 4,4’-dihydroxybiphenyl (16.4 min). These standards were ~98% pure as judged by their individual chromatograms. Analysis of the derivatized heptane extracts of a 15 min incubation mixture yielded only two major and two minor metabolite peaks as determined by flame ionization. Their identities, based on retention time and mass spectrometry analysis, suggest that the major metabolites were 2- and 4-hydroxybiphenyl and the minor carried out in the presence of only NADH or only NADPH, respectively. This designation does not refer to the case of NADH stimulation (synergism) of NADPH-dependent reactions described by other workers (10-13).
162
PROUGH
metabolites were 2,2’- and 4,4’-dihydroxybiphenyl. Analysis of the area under the peak on flame ionization detection indicated that 2- and ri-hydroxybiphenyl together accounted for approximately 95% of the total biphenyl metabolites after a 5-min incubation with rodent liver microsomes. In agreement with Bridges, Creaven, and Williams (20), 3-hydroxybiphenyl has 10-X% of the fluorescence of 2-hydroxyand 4-hydroxybiphenyl at their respective fluorescent settings. However, the gas chromatograpy-mass spectrometry analysis of the incubation mixtures indicate that 3-hydroxybiphenyl formation by liver microsomes must be less than 5% of the formation of the 2-hydroxyand 4-hydroxybiphenyl. The biphenyl hydroxylase assay was performed by a slight modification of the method of Creaven, Parke, and Williams (1). The reaction mixtures contained, in a total volume of 2 ml, 1 mM biphenyl, 0.37% (w/v) Tween 80, 3 mM n&socitric acid, 0.8 U isocitrate dehydrogenase, 5 mM MgSO,, 1 mg microsomal protein/ml, and 0.1 M potassium phosphate buffer, pH 7.6. The reaction was initiated with NADPH or NADH after 1.5 min preincubation at 37°C and was linear for 5 min at 37°C under all conditions. Initiation of the reaction with either NADPH, biphenyl, or microsomal protein yielded identical hydroxylation rates. The rates of NADH-supported biphenyl hydroxylation were not dependent on the presence or absence of a regenerating system for NADH or NADPH. Authentic 2- and 4-hydroxybiphenyl were routinely added to quenched reaction mixtures to serve as standards for the assay. Preparation of rabbit-antiserum against rat liver NADPH-cytochrome c reductase. NADPH-cytochrome c reductase was purified from liver microsomes of Sprague-Dawley rats using pancreatic lipase as reported by Prough and Masters (21). This flavoprotein reductase yielded a single protein band upon polyacrylamide gel electrophoresis and was stable on storage at -5°C. The protein band of the reductase was excised from the gel and used as the challenging antigen. Three weekly injections of 1 mg of purified reductase in Freund’s Adjuvant (Difco Co.) were administered to young white rabbits at multiple injection sites in the nape of the neck. The blood of the rabbits was obtained biweekly from ear veins and tested for specific antibody against rat liver NADPH-cytochrome c reductase by measuring the percentage of inhibition (titer) of rat liver microsomal NADPHcytochrome c reductase activity. Sera, with high antibody titer, from several bleedings were pooled and the immunoglobulins were precipitated from the serum by addition of ammonium sulfate (1.75 M), redissolved in 0.05 M potassium phosphate buffer, pH 7.7, and dialyzed against the same buffer. These globulin solutions were stored frozen. Certain globulin preparations were further purified on DEAEcellulose using a KC1 gradient; this resulted in glob
AND
BURKE ulin fractions with higher titer. Control globulins were prepared both from the rabbits prior to immunochemical challenge (preimmune) and from untreated rabbits (nonimmune); no differences were seen between the two sources of control globulins. The specificities of the anti-NADPH-cytochrome c reductase globulins were tested by employing them as inhibitors against NADHand NADPHdependent microsomal cytochrome c reduction. The immunoglobulin inhibited only the NADPH-dependent cytochrome c reductase. The anti-NADPH-cytochrome c reductase globulin did not affect microsoma1 NADH-dependent cytochrome b, reduction but did inhibit microsomal NADPH-dependent cytochrome b, reductase. When examined by Ouchterlony double diffusion tests in agar gel (221, the immunoglobulin elicited a single precipitation line against purified rat liver NADPH-cytochrome c reductase but not against trypsin-solubilized rat liver cyto chrome b, or lysosomally-solubilized rat liver cytochrome 6, reductase. The anti-reductase globulins were equally effective at inhibiting either liver or lung microsomal NADPH-cytochrome c reductase activity of either hamsters or rats. This result suggests a strong immunochemical similarity of the NADPH-cytochrome c reductase of hamster and rat, liver and lung. RESULTS
Normally, the NADPH-cytochrome c reductase assay is run at much lower concentrations of microsomal protein (l/20) than are the biphenyl, benzo(a)pyrene, and ethylmorphine oxygenase reactions. We have established the validity of a comparison of anti-NADPH-cytochrome c reductase globulin effects on the reductase and oxygenases by determining that the concentration of the antibody which gave 50% inhibition of the reductase did not vary over the range of microsomal protein concentrations used. The effectiveness of the immunoglobulin was expressed as the ratio of milligrams of antibody to milligrams of microsomal protein which gave 50% inhibition of the NADPH-cytochrome c reduction reaction (I,,,). Figure 1 shows that the ISo of the immunoglobulin purified by DEAE-cellulose chromatography was independent of microsomal protein concentrations from 0.05 to 0.8 mg/ml. Animal pretreatment did not affect the independence of the I,, value from microsomal protein concentration, but did alter this ratio of milligrams of antibody to milligrams of microsomal protein giving 50% inhibition
NADH,
00
AND
NADPH-SUPPORTED
1
I
1
MICROSOMAL
I
1
o-0
0.2 PROTEIN
o-
I
I
0.4 CONCENTRATION
I
0.6
8
I
0.6
i
(mg/ml)
FIG. 1. The inhibition by the anti-NADPH-cytochrome c reductase globulin at varying microsomal protein concentrations. The ratio of milligrams antibody to milligrams microsomal protein to give 50% inhibition (I& of microsomal NADPH-cytochrome c reductase, sp act = 110 nmol min-’ mg-’ (Methods section), was determined at several concentrations of rat liver microscmal protein.
of microsomal NADPH-cytochrome c reductase activity. The change in Iso observed using liver microsomes from animals pretreated with drugs i such as phenobarbital, was consistent with the increase of the specific activity of the microsomal reductase under these same conditions of pretreatment. The Is0 obtained with immunoglobulin from different animals or bleedings varied but was independent of microsomal protein concentration. The effect of the anti-NADPH-cytochrome c reductase globulin on NADPHdependent biphenyl 2- and 4-hydroxylase activities of liver microsomes from hamsters injected with corn oil or 3-methylcholanthrene can be seen in Fig. 2a and b. With microsomes of corn oil-treated hamsters, the inhibition profiles of the biphenyl 2- and 4-hydroxylation reactions were similar with each other but were not parallel with the inhibition profile of NADPH-cytochrome c reduction; in contrast, with liver microsomes from 3-methylcholanthrene-injected hamsters, the hydroxylation and NADPH-cytochrome c reduction reactions were inhibited in parallel fashion by the immunoglobulin. In both cases NADPH-dependent cytochrome P-
163
HYDROXYLATIONS
450 reductase activity was inhibited parallel with NADPH-cytochrome c reduction (R. A. Prough, unpublished results). Since the antibody had the same relative effectiveness regardless of protein concentration, the lack of parallel inhibition between hydroxylation and cytochrome reduction activities may reflect 3-methylcholanthrene-induced differences in the molar ratio of the flavoprotein and the hydroxylase. The inhibition of benzo(a)pyrene hydroxylase activity by the anti-NADPH-cytochrome c reductase globulin was seen to parallel the inhibition of biphenyl2- and 4hydroxylase activity in liver microsomes from either corn oil-injected or 3-methylcholanthrene-injected hamsters (maximum inhibition at 8 mg globulin/mg protein = 75%). The activity of hamster liver microsomal benzo(a)pyrene hydroxylase (0.65 nmoles/min/mg microsomal protein) was not appreciably induced by animal pretreatment with 3-methylcholanthrene; benzo(a)pyrene hydroxylase activity was induced two- to threefold in rat liver micro-
1
2
4
6
B
m g GLOBULIN/mgPROTEIN
FIG. 2. The effect of anti-NADPH-cytochrome c reductase globulin on NADPH-dependent biphenyl hydroxylase and cytochrome c reductase activity of hamster liver microsomes. The enzyme activities were determined as described in the Methods section. Figure 2a shows the inhibition of NADPHdependent microsomal biphenyl 2-hydroxylase activity (0, 100% = 1.0 nmol min-’ mg-‘), biphenyl 4hydroxylase activity (0, 100% = 23 nmol min-’ mg-‘1, and NADPH-cytochrome c reductase (A, 100% = 210 nmol mine1 mg-‘) from corn oil-injected hamsters. Figure 2b shows the inhibition of NADPH-sustained microsomal biphenyl 2-hydroxylase activity (0, 100% = 2.7 nmol min-’ mg?, biphenyl I-hydroxylase activity (0, 100% = 7.9 nmol min-’ mg-‘), and cytochrome c reductase activity (A, 100% = 218 nmol min-’ mg-‘) from 3-methylcholanthrene-pretreated hamsters. No effect of nonimmune globulin was seen on any of the activities (A).
164
PROUGH
somes by pretreatment with 3-methylcholanthrene. Reduced pyridine nucleotide specificity of liver microsomal biphenyl2and 4-hydroxylase activity. The dependence of biphenyl 2- and 4-hydroxylase activities on NADPH and NADH concentration can be seen in Fig. 3. Although the reactions were saturated in the micromolar range with NADPH (KmaPP 5 40 FM), approximately 10 mM NADH was required to saturate the hydroxylase reactions of hamster liver microsomes from corn oil-pretreated hamsters (KmaPP for NADH = 5.2 mM). Similar results were obtained when highly purified NADH, P.L. Biochemicals “Chromatopure” NADH, was used. The apparently high ‘K,,, of the hydroxylases for NADH suggests that NADH-cytochrome b, reductase was probably not involved, since 100 PM NADH should have been sufficient to cause maximal steady-state reduction of the NADH-cytochrome b, reductase (23). Effect of anti-NADPH cytochrome c reductase globulin on NADH-sustained liver and lung biphenyl hydroxylase activ-
mMOLAR
3. The
NADtPJH
reduced pyridine nucleotide specificity of liver microsomal biphenyl hydroxylase activity. The reactions were run as described in the Methods section but initiated with various concentrations of NADH or NADPH as indicated. The figure shows biphenyl I-hydroxylase activity supported by NADPH (0) or NADH (0) and biphenyl a-hydroxylase activity supported by NADPH (A) or NADH (A). The liver microsomes were obtained from corn oilinjected hamsters; identical results were obtained with liver microsomes from 3-methylcholanthreneinjected hamsters. FIG.
AND
BURKE
1
25
1 mgGLOBULIN/mg
PROTEIN
FIG. 4. The effect of anti-cytochrome c reductase globulin on NADH-dependent liver biphenyl 4-hydroxylase activity. The figure shows the inhibition of microsomal biphenyl I-hydroxylase activity (Methods section) mediated by 0.25 mM NADH (A, 100% = 1.0 nmol min-’ mg-9, 10 mM NADH (0, 100% = 3.0 nmol min-’ mg-I), and 0.25 mM NADPH (0, 100% = 8.0 nmol min-’ mg-*). The effect of nonimmune globulin on 0.25 mM NADH(A) and 0.25 mM NADPH(M) sustained microsomal biphenyl Chydroxylase activity is shown. The liver microsomes were obtained from 3-methylcholanthrene-injected hamsters.
ity. Using the specific immunoglobulin for rat liver NADPH-cytochrome c reductase, the NADH-dependent biphenyl 2- and 4hydroxylase activities of hamster liver microsomes were tested for involvement of NADPH-cytochrome c reductase. As shown in Fig. 4, the anti-NADPH-cytochrome c reductase globulin inhibited NADH-sustained biphenyl 4-hydroxylase activity of liver microsomes from 3-methylcholanthrene-pretreated hamsters; the inhibition profiles for 2- and 4-hydroxylation were almost identical. At 0.25 mM NADH, the hydroxylase activities were inhibited approximately 45%. Parallel profiles were observed for inhibition, by the anti-reductase globulins, of rat or hamster liver microsomal biphenyl hydroxylation and cytochrome c reduction supported by saturating concentrations of either NADPH or NADH (Table I). The effect of antiNADPH-cytochrome c reductase globulin on hamster liver microsomal benzo(a)pyrene hydroxylase and ethylmorphine demethyl-
NADH-
AND
NADPH-SUPPORTED
MICROSOMAL
BIPHENYL
MICROSOMAL TABLE
INHIBITION
Species
Hamster
Rat
OF LIVER
Activity
measured
Cytochrome c reduction Biphenyl4-hydroxylation Cytochrome c reduction Biphenyl 4-hydroxylation
I
HYDROXYLASE AND NADPH OR NADH
CYTOCHROME
Reduced” pyridine nucleotide
c REDUCTION
SUPPORTED
Control
Immune globulin 49 470 2.4 0.9 16 712 0.8 0.4
215 490 8.0 3.0 110 727 3.9 1.4
“The concentrations of NADH or NADPH were 10 and treated with 3-methylcholanthrene. * Both control and immune globulin reaction mixtures anti-rat liver NADPH-cytochrome c reductase globulin per The activity is expressed as nanomoles product per minute
0.25 mM,
BY
% Inhibition
Activityb
NADPH NADH NADPH NADH NADPH NADH NADPH NADH
ase activities sustained by NADH was to inhibit these reactions in relatively parallel fashion to the inhibition of biphenyl hydroxylase reactions (Table II). Hamster or rat lung microsomal NADPH-dependent .biphenyl 4-hydroxylase activity was of the same order of magnitude as liver microsomal biphenyl 4-hydroxylase activity of corn oil-pretreated animals (1.0 and 2.0 nmol/min/mg microsoma1 protein in lung or liver, respectively). Furthermore, the relative rates of NADHand NADPH-dependent biphenyl li-hydroxylase activities in lung microsomes were also similar to those shown for liver microsomes in Fig. 3. Typical rates for liver and lung NADHand NADPH-sustained biphenyl4-hydroxylase activities are shown in Table III. The inhibiting effect of antiNADPH-cytochrome c reductase on NADHor NADPH-supported lung biphenyl 4-hydroxylase activity was nearly identical to that shown for the liver hydroxylase (Fig. 5). The 2-hydroxylase activity of lung microsomes from 3-methylcholanthrene-pretreated hamsters was also inhibited in a manner similar to the 4-hydroxylase activity (78% maximal inhibition). The effect of NADP+ on NADH-sustained biphenyl hydroxylase activity. As was shown by Williams and Kamin (61, NADP+ is an inhibitor of NADPH-cyto-
165
HYDROXYLATIONS
respectively.
The
78 4 70 71 86 2 80 72
animals
were
pre-
contained a 8:l ratio of nonimmune globulin milligram of microsomal protein, respectively. per milligram of microsomal protein. TABLE
INHIBITION
OF
NADH-
BENZO(a)PYRENE PHINE DEMETHYLASE CORN OIL INJECTED
Activity measured
II OR
NADPH-SUPPORTED
HYDROXYLASE OF LIVER HAMSTERS
NADPH-CYTOCHROME
AND ETHYLMORMICROSOMES FROM BY ANTI-RAT LIVER
c REDUCTASE
Reduced” pyridine Nucleotide
or
GLOBULIN
Activityb Control
Immune
% Inhibition
fizBenzo(a)pyrene Hydroxylase Ethylmorphine Demethylase
NADPH NADH NADPH NADH
2.78 0.95 4.3 1.8
0.47 0.29 0.7 0.7
83 70 84 60
@The concentrations of NADH and NADPH were 10 and 0.25 mM, respectively. b Both control and immune globulin reaction mixtures contained a 8:l ratio of nonimmune globulin or anti-rat liver NADPH-cytochrome c reductase globulin per milligram of microsomal protein, respectively. The activity is expressed as nanomoles product formed per minute per milligram ‘of microsomal protein.
chrome c reductase-catalyzed reactions. Using NADP+ as a specific chemical inhibitor, one can see in Table IV that NADP+ effectively inhibited NADH- and NADPHdependent liver microsomal biphenyl hydroxylase activity. This effect of NADP+
166
PROUGH
THE BIPHENYL Organ source of microsomes
HYDROXYLASE
Reduced” pyridine nucleotide
Activities Corn
oil
Lung
D The concentrations * Nanomoles product
2.0 1.2 1.0 0.5
1.0 0.3 co.1 co.1
of NADH and NADPH formed/minute/milligram
LIVER
after
AND LUNG
animal
MICRO~~MES
pretreatment”
Phenobarbital
2Hydroxylase
ylase NADPH NADH NADPH NADH
BURKE
TABLE III ACTIVITY OF HAMSTER
:;;;I
Liver
AND
4-Hydroxylase
3-Methyl cholanthrene 2-
4-Hy-
“{Z;-
7.8 2.5 0.9 -
dKi:
1.3 0.3 co.1 -
2H;f;;;-
8.1 2.9 1.2 0.6
2.9 0.9 0.13 co.1
were 10 and 0.25 mM, respectively. of microsomal protein.
TABLE IV THE EFFECT OF NADP+ ON LIVER MICROSOMAL NADHAND NADPH-DEPENDENT BIPHENYL HYDROXYLASE ACTIVITY OF CORN OIL INJECTED HAMSTERS” Pyridine nucleotide (mM) NADPH NADH
NADP+ bnM)
0.25 0.25 1 1 10 10
4 4 40
4-Hydroxylase” activity
lasd activity
2.10 0.80 0.54 0.38 0.94 0.11
0.90 0.18 0.08 0.02 0.30 0.05
’ No pyridine nucleotide regenerating was included in these experiments. b Nanomoles product formed/minute/milligram microsomal protein,
2-HydrOXy-
2
4 mgGLOSULlN/mg
system of
on NADH-sustained hydroxylation reactions is consistent with the anti-reductase effects of the preceding section in implicating an important role for NADPH-@ochrome c reductase in NADH-sustained microsomal biphenyl hydroxylation. DISCUSSION
We have chosen the well-characterized reaction of biphenyl to monitor microsoma1 aryl hydroxylation. As reported in the Methods section, the 2- and 4-hydroxybiphenyl metabolites account for approximately 95% of the total metabolites of a 5min in vitro incubation mixture. Biphenyl
6
8
PROTEIN
FIG. 5. The effect of anti-reductase globulin on lung biphenyl 4-hydroxylase activity. The figure shows the inhibition of microsomal biphenyl 4-hydroxylase activity (Methods) media&d by 0.25 mM NADPH (A, 100% = 0.90 nmol min-’ mg-‘) or 10 mu NADH (0, 100% = 0.45 nmol min-’ mg?. The corresponding effect of nonimmune globulin was noted: 0.25 mM NADPH (A) and 10 mM NADH (0). The lung microsomes were prepared from corn oilinjected hamsters.
hydroxylase activities of liver and lung microsomes are, respectively, approximately equal and relatively large compared to other activities, such as benzo(a)pyrene hydroxylase (24) and ethylmorphine demethylase @‘rough and Burke, unpublished results). Specific antibodies have been elicited against several liver microsomal proteins in order to test their involvement in the electron transport chains of microsomes from numerous organs. Japanese research-
NADH-
AND
NADPH-SUPPORTED
ers have elucidated the roles of NADHcytochrome b, reductase, NADPH-cytochrome c reductase, and cytochrome b, in NADHand NADPH-dependent microsoma1 stearyl CoA desaturase activity (25 27). Masters et al. (7) established the role of NADPH-cytochrome c reductase in cytochrome P-450 linked dealkylation reactions in pig liver microsomes and in steroid hydroxylation reactions of bovine adrenocortical microsomes. The possible role of cytochrome b, in lauric acid oxidation by kidney cortex or liver microsomes and in ethanolamine plasmalogen synthesis by pork spleen microsomes has been elucidated recently using specific antibodies (28, 29). The role of cytochrome b, in NADH-synergism of NADPH-sustained reactions have also been shown (12, 13). A lack of specific chemical inhibitors for the components of the microsomal enzyme system led us to an immunochemical approach to evaluate the role of NADPHcytochrome c reductase in NADH-dependent microsomal hydroxylases. This study is dependent on the specificity of the antireductase globulin to the NADPH-cytochrome c reductase and on the ability to measure the total metabolites of an aromatic hydroxylation reaction. Since the specificity of the antibody was established in the Methods section and the metabolite pattern for liver microsomal biphenyl hydroxylase activity was confirmed, we have shown that both requirements have been met. We also noted no effect of antiNADPH-cytochrome c reductase globulin on the apparent binding of benzphetamine or biphenyl to hamster liver microsomal cytochrome P-450 as measured by the apparent K, and AA maxof their Type I interaction spectra. Type I interaction spectra may reflect the formation of a substratecytochrome P-450 complex (30). The similarity in the apparent K, for NADH between purified NADPH-cytochrome c reductase and microsomal biphenyl hydroxylase activities of liver or lung indicates that the reductase may be involved in NADH-dependent hydroxylation reactions. The stoichiometric reduction of the flavoprotein by NADH further supports the ability of NADH to act as an electron donor to NADPH-cytochrome c re-
MICROSOMAL
HYDROXYLATIONS
167
ductase (15). The biphenyl 4-hydroxylase activity supported by 0.25 mM NADH was maximally inhibited 45% while that supported by either 0.25 mM NADPH or 10 mM NADH was maximally inhibited 70% by anti-NADPH-cytochrome c reductase globulin. The inhibition of NADH-dependent biphenyl or benzo(a)pyrene hydroxylase and ethylmorphine demethylase activities by anti-reductase globulin and biphenyl hydroxylase activity by NADP+ strongly suggests that at least one electron of NADH-dependent hydroxylation reactions must be donated via NADPH-cytochrome c reductase, in agreement with the suggestion of Omura (16). On the basis of the experiments presented in this paper, no evidence can be given to indicate the number of electrons donated to cytochrome P-450. However, the roles of NADPH-cytochrome c reductase in NADH- or NADPHdependent microsomal hydroxylation reactions of liver or lung appear to be identical, and the apparant enhanced rate of NADHreduction of lung microsomal cytochrome P-450 (17) may be the result of a difference in the molar ratio of microsomal electron transport components of lung compared to those of liver. Our observations that titration with the anti-reductase globulin yielded identical inhibition profiles for liver microsomal ethylmorphine demethylation, benzo(a)pyrene hydroxylation, and biphenyl2and 4-hydroxylation suggest that all four microsomal mixed function oxygenases utilize an immunochemically similar species of the NADPH-cytochrome c reductase. Furthermore, this NADPH-cytochrome c reductase appears to operate in both liver and lung microsomes from either the hamster or the rat. However, in the case of hamster liver, microsomal biphenyl2- and 4-hydroxylase activities, the molar ratio of the flavoprotein reductase to other components of the hydroxylase appears to be diminished by pretreatment of the animal with 3-methylcholanthrene. ACKNOWLEDGMENTS The authors express their appreciation to Ms. Nell Mock and Ms. Betty Paul for their expert technical help, and to Dr. Ronald Estabrook and Dr. Bettie Sue Masters for their encouragement.
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PROUGH
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