Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 558-563
April 30, 1991
COMPARISON OF RAINBOW TROUT AND MAMMALIAN CYTOCHROME P450 ENZYMES: EVIDENCE FOR STRUCTURAL SIMILARITY BETWEEN TROUT P450 LMC5 AND HUMAN P450111A4 C.L. Miranda 1,2, J.-L. Wang 1.2, M.C. Henderson ~ X. Zhao ~, F.P. Guengerich 3 and D.R. Buhler 1,2 1Department of Agricultural Chemistry and 2Marine Freshwater Biomedical Center Oregon State University Corvallis, OR 97331 3Department of Biochemistry and Center in Molecular Toxicology Vanderbilt University School of Medicine Nashville, TN 37232 Received March 9, 1991
SUMMARY: Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450111Aenzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450111A1 in dexamethasone-induced rat liver microsomes and with P450111A4in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450111A1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450111A4IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6/?-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by antiP450111A4and by gestodene, a mechanism-based inactivator of P450111A4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450111A4. © 1991 A c a d e m i c
Press,
Inc.
Rainbow trout liver contains at least five constitutive cytochrome P450 isozymes (P450s LMC1 to LMC5) and one aromatic hydrocarbon-inducible form, P450 LM4b or P4501A1 (1,2). Trout P4501A1 appears to be orthologous to both rat P4501A1 and P4501A2 on the basis of immunological properties and amino acid sequences (3,4). Recently, we have shown that trout P450 LMC1 may be structurally related to phenobarbital (PB)-inducible rat P450s such as P45011B1 (5). Anti-P450 LMC1 IgG was found to cross-react strongly with the PB-induced P450s in rat liver microsomes and with purified rat P45011B1 (5). It is not known, however, whether or not other forms of trout P450 are related to mammalian P450s. The existence in trout of other P450 forms related to mammalian P450s can be deduced in part from their catalytic activity.
Trout P450 LMC5 may be similar to certain
members of the mammalian P450111Asubfamily on the basis of its ability to catalyze the 6flhydroxylation of steroid hormones such as progesterone and testosterone (1). 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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P450111Aenzymes, particularly P450111A1 and P450111A4,are now recognized as the major catalysts for steroid ~-hydroxylation (6). To determine whether trout P450 LMC5 is structurally related to mammalian P450111Aenzymes, studies were carried out to: 1) determine if rabbit anti-LMC5 IgG cross-reacts strongly with rat and human P450111Aenzymes in immunoblots and conversely, if antibodies to mammalian P450111A enzymes recognize trout P450 LMC5 in immunoblots; 2) establish whether or not anti-P450111A4 IgG is capable of inhibiting the 6fl-hydroxylase activity of trout liver microsomes; and 3) reveal whether or not a selective inhibitor of P450111A4, gestodene, is capable of inhibiting 6/Y-hydroxylase activity of trout liver microsomes. In addition, the crossreactivity of anti-P450 LMC1 IgG with members of the mammalian P450111A subfamily was investigated. The results of these immunochemical and enzyme inhibition studies establish for the first time that the trout P450 enzyme, LMC5, is structurally and functionally related to human P450111A4. MATERIALS AND METHODS Antibodies and liver microsomes. Anti-rat P450111A1 (P4500) IgG, prepared by injecting rabbits with purified P450111A-triacetyloleandomycin complex (7), was generously provided by Dr. Jan Hulla, Battelle Pacific Northwest Labs, Richland, WA. Anti-human P450111A4 IgG was raised in rabbits and has been characterized previously (8). Rabbit anti-P450 LMC1 IgG and anti-P450 LMC5 IgG were prepared according to a previously published method (1). Liver microsomes from untreated rainbow trout, trout pretreated with dexamethasone (50 mg/kg, i.p., for 3 days), untreated adult male Sprague-Dawley rats, phenobarbital-treated (80 mg/kg, i.p., for 3 days) rats and dexamethasone-treated (50 mg/kg, i.p., for 3 days) rats were prepared as described previously (1). Human liver micros0mes were isolated as described by Wang et al. (9) from organ donors obtained through Tennessee Donor Services (Nashville, TN).
Immunoblotting.
Blotting was performed according to the method of Burnette (10) with some modifications. Microsomal proteins and individual P450s were electrophoresed on polyacrylamide gel containing sodium dodecyl sulfate (11) and transferred onto 0.45pro nitrocellulose sheets for 30 min at 4°C using a Genie Electrophoretic blotter (Idea Scientific, Corvallis, OR). After blocking with 2% bovine serum albumin in TBS/Tween (Tris buffered saline containing 0.05% Tween 20), the nitrocellulose sheets were incubated with the primary antibody for 1 hr at room temperature. The nitrocellulose sheets were subsequently incubated with [1251]-Protein A (ICN Biomedicals, Irvine, CA) for 1 hr. The protein bands were visualized by autoradiography on Kodak XAR film. Micresomal progesterone 6p-hydroxylaseactivity. Steroid 6#-hydroxylase activity of trout liver microsomes was measured following a 30-min incubation at 30°C in the following medium: 0.1 M sodium phosphate buffer, pH 7.4, 0.5 mg microsomal protein, 50pM [14C]-progesterone (New England Nuclear, Boston, MA) and 1 mM NADPH in a total volume of 0.5 ml. The reaction was terminated by the addition of 3 ml methylene chloride. After a second extraction with methylene chloride, the combined extracts were evaporated with N2. The residue was dissolved in methanol for HPLC analysis. Inhibition of 6~-hydroxylase activity by anti-human P450111A4IgG and anti-LMC5 IgG was performed as described above except that the microsomes were first preincubated with the antibody for 20 rain at 30°C prior to the addition of other components of the incubation mixture. Inhibition of 6/~-hydroxylase activity by gestodene (gift from Dr. H. Kuhl, J.W. Goethe University, Frankfurt, Federal Republic of Germany) was carried out by preincubating the mixture with 100pM gestodene in the absence of progesterone. After preincubation at 30°C for 30 min, progesterone was added to the reaction mixture and the latter was further incubated for 30 min. HPLC analysis of progesterone and its metabolite, 6/~-hydroxyprogesterone, was performed on an Altex Ultrasphere C18 column (4.6 x 250 mm). The 6~6-hydroxyprogesterone 559
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metabolite was identified by co-elution with a known standard. The column was eluted isocratically with 55% A (water) and 45% B (44% tetrahydrofuran, 29% acetonitrile and 27% methanol) (12). RESULTS AND DISCUSSION Polyclonal antibodies to trout P450 LMC5 were found to recognize P450 LMC5 (Mr59,000) in trout liver microsomes and to crossreact with a protein (Mr 51,000) induced by both dexamethasone and phenobarbital in rat liver microsomes (Fig. 1). No protein was recognized by anti-LMC5 IgG in control rat liver microsomes.
A protein (Mr 53,000) in human liver
microsomal sample HL115 was also recognized by anti-LMC5 IgG but only a trace was detected in sample HL101. HL101 and HL115 microsomes vary appreciably in their P450111A4 content as indicated by their activity towards nifedipine. Nifedipine oxidase activity, a reaction catalyzed by P450111A4 (8), was 0.39 nmol/min/mg protein for HL101 and 3.25 nmol/min/mg protein for HL115. Treatment of trout with dexamethasone did not produce any appreciable increase in immunoreactive LMC5 in liver microsomes (Fig. 1) suggesting that this enzyme is probably not induced by dexamethasone in trout. In contrast to anti-LMC5 IgG, anti-LMC1 IgG which binds P450 LMC1 (Mr 50,000), did not crossreact with rat microsomal proteins induced by dexamethasone (Fig. 2). In phenobarbitalinduced rat liver microsomes, two bands were strongly recognized by anti-LMC1 IgG confirming our previous findings (5). From these results, we can conclude that the proteins induced by phenobarbital and recognized by anti-LMC1 IgG are not the same as those induced in the rat by dexamethasone. Anti-LMC1 IgG has little or no affinity toward protein in human liver microsomes (HL101 and HLl15), since only faint bands were seen after staining with this IgG (Fig. 2). The proteins induced by dexamethasone in rat liver microsomes recognized by anti-LMC5 IgG could be one of the gene products belonging to P450111A. P450111Aincorporates at least two gene products, namely, P450p (P450PCN1, P450PCNa or P45OrAo) and P450PCN-E (P450PB2a
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Figure 1.
Immunoblot of liver microsomes from rainbow trout, rat, and human probed with anti-LMC5 IgG with detection by [1251]-ProteinA. The microsomes (20 p.g/well)and P450 LMC5 (1 pmol/well) were applied to the wells as follows: A. Control rat; B. Dexamethasone-treated rat; C. Phenobarbital-treated rat; D. P450 LMC5; E. Controltrout; F. Dexamethasone-treatedtrout;G. Human HL101 ; and H. Human HL115.
Figure 2.
Immunoblot of liver microsomes from rainbow trout, rat, and human probed with anti-LMC1 IgG with detection by [12Sl]-ProteinA. The microsomes (20 i~g/well) and P450 LMC1 (1 pmol/well) were applied to the wells as follows: A. Control rat; B. Dexamethasone-treated rat; C. Phenobarbital-treated rat; D. P450 LMC1; E. Control Trout; F. Dexamethasone-treated trout; G. Human HL101; and H. Human HL115.
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Figure 3.
Immunoblot of liver microsomes from rainbow trout, rat, and human probed with anti-rat P450111A1 IgG with detection by [~2~l]-Protein A. The microsomes (20 i~g/well) and purified trout cytochrome P450s (2 pmol/well) were applied as follows: A. Control rat; B. Dexamethasone-treated rat; C. P450 LMC1; D. P450 LMC5; E. Control trout; F. Dexamethasone-treatedtrout; G. Human HL101 ; and H. Human HL115.
Figure 4.
Immunoblot of liver microsomes from trout and humans probed with anti-human P450111A4IgG with detection [~2Sl]-ProteinA. The microsomes (20 pg/well for wells A,E,F, and G; 5 i~g/wellfor wells B,C, and H) and purified P450 LMC1 or P450 LMC5 (2 pmol/well) were applied as follows: A. Control rat; B. Dexamethasonetreated rat; C. Phenobarbital-treated rat; D. P450 LMC5; E. Control trout; F. Dexamethasone-treated trout; G. Human HL101; H. Human HLl15; and I. P450 LMCI.
or P450PCNb)
(7).
I
Fig. 3 shows that anti-P450111A1 IgG recognized a single band in
dexamethasone-induced but not in uninduced rat liver microsomes. This protein which represents P450111A1, has the same mobility as the protein stained with anti-LMC5 IgG shown in Fig. 1. However, anti-rat P450111A1 IgG failed to recognize LMC5 or a corresponding protein in trout liver microsomes (Fig. 3). The results of these reciprocal blots suggest that there is at least one common epitope in trout P450 LMC5 and rat P450111A1 recognized by anti-trout P450 LMC5 but the epitope recognized by anti-P450111A1 IgG is absent in trout P450 LMC5. On the basis of cross-reaction between anti-LMC5 IgG and rat P450111A1, we hypothesize that the protein in human liver microsomes (HLl15) recognized by anti-LMC5 IgG may also belong to the P450111A subfamily. Immunoblots (Fig. 4) using anti-P450111A4 IgG show that a reactive protein is found in much higher concentration in HLl15 than in HL101. This protein is most likely P450111A4 and may correspond to the protein stained by anti-LMC5 IgG shown in Fig. 1. Anti-P450111A4 IgG also recognized P450 LMC5 and a protein in trout liver microsomes with the same mobility as purified P450 LMC5. Thus, trout P450 LMC5 and P450ilIA4 may share a common epitope recognized by both anti-P450 LMC5 IgG and anti-P450111A4 IgG. In addition, P450 LMC1 and a protein in trout liver microsomes with the same Mr as P450 LMC1 were recognized by anti-P450111A4 IgG (Fig. 4). tmmunoinhibition studies were performed to determine whether the epitope(s) in P450 LMC5 recognized by anti-P450111A4 are involved in the biological activities of the enzyme. AntiP450111A4 IgG, at a concentration of 10 mg/nmol P450, almost completely inhibited (93% reduction) the 6/Y-hydroxylation of progesterone by trout liver microsomes, an effect similarly produced by anti-P450 LMC5 IgG (Table 1). These findings suggest that the epitopes in P450 LMC5 recognized by anti-P450111A4 are involved in the catalytic process.
In addition to the
structural similarity between P450 LMC5 and P450111A4 as indicated by the immunoblot studies, 561
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Table 1. In Vitro Inhibition of Trout Liver Microsomal £~8-Hydroxylationof Progesterone by Gestodene and by Rabbit Anti-Cytochrome P450111A4IgG or Rabbit Anti-Cytochrome P450 LMC5 IgG a
Progesterone cqS-Hydroxylase (% Inhibition)
Additions Gestodene (100 I~M)
83
Pre-immune IgG (10 mg/nmol P-450)
12
Anti-P450Nr (P450111A4)IgG 2 mg/nmol of P-450
28
10 mg/nmol of P-450
93
Anti-P450 LMC5 IgG 10 mg/nmol of P-450
92
Activity of uninhibited liver microsomes (14-month male trout) was 0.58 nmol/min/mg protein. Values represent the mean of duplicate determinations.
there could be similarities in the chemical regulation ofthese enzymes. Gestodene, a mechanismbased inactivator of P450111A4(13), was found to strongly inhibit the progesterone 6fl-hydroxylase activity of trout liver microsomes (Table 1).
This activity, chiefly mediated by P450 LMC5,
however, was not induced by dexamethasone in trout (Table 2) which is consistent with the observed lack of effect of dexamethasone on P450 LMC5 levels (Fig. 1). Interestingly, we have found that pretreatment of rainbow trout with 3,4,5,3'4',5'-hexachlorobiphenyl (1 mg/kg) significantly increases the progesterone 6#-hydroxylase activity of trout liver microsomes with no effect on P450 LMC5 levels (14). In conclusion, the immunoblot and enzyme inhibition studies provide evidence that there is strong similarity in structure and function between the constitutive trout P450 LMC5 which is noninducible by dexamethasone pretreatment and dexamethasone-inducible human P450111A4 and less so in the structure between P450 LMC5 and rat P450111A1. Further studies on the amino acid sequence of P450 LMC5 would be needed to establish that this trout enzyme belongs to the P450111A subfamily.
Table 2. Effect of dexamethasona Pretreatment of Trout on 6]Y-Hydroxylationof Progesterone by Trout Liver Microsomes a Progesterone 6#-Hydroxylase Treatment
nmol/min/mg/protein
nmol/min/nmol P450
Control
0.20 + 0.01
0.48 __+0.02
Dexamethasone (50 mg/kg, ip for 3 days)
0.14 -I- 0.01b
0.45 __+0.02
a
b
Values are the mean and S.E. of five animals. P < 0.05 vs. control.
562
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This study was supported by NIH grants ES-00210 and ES-03850 (DRB), and CA44353 (FPG). This manuscript was issued as Technical Paper 9455 from the Oregon Agricultural Experiment Station, Oregon State University, Corvallis, OR. REFERENCES 1. 2,
3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
Miranda, C.L., Wang, J.-L., Henderson, M.C., and Buhler, D.R. (1989). Arch. Biochem. Biophys. 268, 227-238. Williams, D.E., and Buhler, D.R. (1984). Biochem. Pharmacol. 33, 3743-3753. Heilmann, L.J., Sheen, Y.-Y., Bigelow, S.W., and Nebert, D.W. (1988). DNA, 7, 379-387. Stegeman, J.J. (1989). Xenobiotica 19, 1093-1110. Miranda, C.L., Wang, J.-L., Henderson, M.C. and Buhler, D.R. (1990). Biochem. Biophys. Acta, 1037, 155-160. Waxman, D.J., Attisano, C., Guengerich, F.P., and Lapenson, D.P. (1988). Arch. Biochem. Biophys. 263, 424-436. Hulla, J., and Juchua, M.R. (1989). Biochemistry 28, 4871-4879. Guengerich, F.P., Martin, M.V., Beaune, P.H., Kremers, P., Wolff, T., and Waxman, D.J. (1986). J. Biol. Chem. 261, 5051-5060. Wang, P.P., Beaune, P., Kaminsky, L.S., Dannan, G.A., Kadlubar, F.F., Larrey, D., and Guengerich, F.P. (1983). Biochemistry 22, 5375-5383. Burnette, W.N. (1981). Anal. Biochem. 112, 195-203. Laemmli, U.K. (1970). Nature (London) 227, 680-685. Waxman, D.J. (1984). J. Biol. Chem. 259, 15481-15490. Guengerich, F.P. (1990). Chem. Res. Toxicol. 3, 363-371. Miranda, C.L., Wang, J.-L., Chang, H.-S., and Buhler, D.R. (1990). Biochem. Pharmacol. 40, 387-390.
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COMPARISON OF RAINBOW TROUT AND MAMMALIAN CYTOCHROME P450 ENZYMES: EVIDENCE FOR STRUCTURAL SIMILARITY BETWEEN TROUT P450 LMC5 AND HUMAN P450111A4 C.L. Miranda 1,2, J.-L. Wang 1.2, M.C. Henderson ~ X. Zhao ~, F.P. Guengerich 3 and D.R. Buhler 1,2 1Department of Agricultural Chemistry and 2Marine Freshwater Biomedical Center Oregon State University Corvallis, OR 97331 3Department of Biochemistry and Center in Molecular Toxicology Vanderbilt University School of Medicine Nashville, TN 37232 Received March 9, 1991
SUMMARY: Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450111Aenzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450111A1 in dexamethasone-induced rat liver microsomes and with P450111A4in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450111A1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450111A4IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6/?-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by antiP450111A4and by gestodene, a mechanism-based inactivator of P450111A4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450111A4. © 1991 A c a d e m i c
Press,
Inc.
Rainbow trout liver contains at least five constitutive cytochrome P450 isozymes (P450s LMC1 to LMC5) and one aromatic hydrocarbon-inducible form, P450 LM4b or P4501A1 (1,2). Trout P4501A1 appears to be orthologous to both rat P4501A1 and P4501A2 on the basis of immunological properties and amino acid sequences (3,4). Recently, we have shown that trout P450 LMC1 may be structurally related to phenobarbital (PB)-inducible rat P450s such as P45011B1 (5). Anti-P450 LMC1 IgG was found to cross-react strongly with the PB-induced P450s in rat liver microsomes and with purified rat P45011B1 (5). It is not known, however, whether or not other forms of trout P450 are related to mammalian P450s. The existence in trout of other P450 forms related to mammalian P450s can be deduced in part from their catalytic activity.
Trout P450 LMC5 may be similar to certain
members of the mammalian P450111Asubfamily on the basis of its ability to catalyze the 6flhydroxylation of steroid hormones such as progesterone and testosterone (1). 0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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P450111Aenzymes, particularly P450111A1 and P450111A4,are now recognized as the major catalysts for steroid ~-hydroxylation (6). To determine whether trout P450 LMC5 is structurally related to mammalian P450111Aenzymes, studies were carried out to: 1) determine if rabbit anti-LMC5 IgG cross-reacts strongly with rat and human P450111Aenzymes in immunoblots and conversely, if antibodies to mammalian P450111A enzymes recognize trout P450 LMC5 in immunoblots; 2) establish whether or not anti-P450111A4 IgG is capable of inhibiting the 6fl-hydroxylase activity of trout liver microsomes; and 3) reveal whether or not a selective inhibitor of P450111A4, gestodene, is capable of inhibiting 6/Y-hydroxylase activity of trout liver microsomes. In addition, the crossreactivity of anti-P450 LMC1 IgG with members of the mammalian P450111A subfamily was investigated. The results of these immunochemical and enzyme inhibition studies establish for the first time that the trout P450 enzyme, LMC5, is structurally and functionally related to human P450111A4. MATERIALS AND METHODS Antibodies and liver microsomes. Anti-rat P450111A1 (P4500) IgG, prepared by injecting rabbits with purified P450111A-triacetyloleandomycin complex (7), was generously provided by Dr. Jan Hulla, Battelle Pacific Northwest Labs, Richland, WA. Anti-human P450111A4 IgG was raised in rabbits and has been characterized previously (8). Rabbit anti-P450 LMC1 IgG and anti-P450 LMC5 IgG were prepared according to a previously published method (1). Liver microsomes from untreated rainbow trout, trout pretreated with dexamethasone (50 mg/kg, i.p., for 3 days), untreated adult male Sprague-Dawley rats, phenobarbital-treated (80 mg/kg, i.p., for 3 days) rats and dexamethasone-treated (50 mg/kg, i.p., for 3 days) rats were prepared as described previously (1). Human liver micros0mes were isolated as described by Wang et al. (9) from organ donors obtained through Tennessee Donor Services (Nashville, TN).
Immunoblotting.
Blotting was performed according to the method of Burnette (10) with some modifications. Microsomal proteins and individual P450s were electrophoresed on polyacrylamide gel containing sodium dodecyl sulfate (11) and transferred onto 0.45pro nitrocellulose sheets for 30 min at 4°C using a Genie Electrophoretic blotter (Idea Scientific, Corvallis, OR). After blocking with 2% bovine serum albumin in TBS/Tween (Tris buffered saline containing 0.05% Tween 20), the nitrocellulose sheets were incubated with the primary antibody for 1 hr at room temperature. The nitrocellulose sheets were subsequently incubated with [1251]-Protein A (ICN Biomedicals, Irvine, CA) for 1 hr. The protein bands were visualized by autoradiography on Kodak XAR film. Micresomal progesterone 6p-hydroxylaseactivity. Steroid 6#-hydroxylase activity of trout liver microsomes was measured following a 30-min incubation at 30°C in the following medium: 0.1 M sodium phosphate buffer, pH 7.4, 0.5 mg microsomal protein, 50pM [14C]-progesterone (New England Nuclear, Boston, MA) and 1 mM NADPH in a total volume of 0.5 ml. The reaction was terminated by the addition of 3 ml methylene chloride. After a second extraction with methylene chloride, the combined extracts were evaporated with N2. The residue was dissolved in methanol for HPLC analysis. Inhibition of 6~-hydroxylase activity by anti-human P450111A4IgG and anti-LMC5 IgG was performed as described above except that the microsomes were first preincubated with the antibody for 20 rain at 30°C prior to the addition of other components of the incubation mixture. Inhibition of 6/~-hydroxylase activity by gestodene (gift from Dr. H. Kuhl, J.W. Goethe University, Frankfurt, Federal Republic of Germany) was carried out by preincubating the mixture with 100pM gestodene in the absence of progesterone. After preincubation at 30°C for 30 min, progesterone was added to the reaction mixture and the latter was further incubated for 30 min. HPLC analysis of progesterone and its metabolite, 6/~-hydroxyprogesterone, was performed on an Altex Ultrasphere C18 column (4.6 x 250 mm). The 6~6-hydroxyprogesterone 559
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metabolite was identified by co-elution with a known standard. The column was eluted isocratically with 55% A (water) and 45% B (44% tetrahydrofuran, 29% acetonitrile and 27% methanol) (12). RESULTS AND DISCUSSION Polyclonal antibodies to trout P450 LMC5 were found to recognize P450 LMC5 (Mr59,000) in trout liver microsomes and to crossreact with a protein (Mr 51,000) induced by both dexamethasone and phenobarbital in rat liver microsomes (Fig. 1). No protein was recognized by anti-LMC5 IgG in control rat liver microsomes.
A protein (Mr 53,000) in human liver
microsomal sample HL115 was also recognized by anti-LMC5 IgG but only a trace was detected in sample HL101. HL101 and HL115 microsomes vary appreciably in their P450111A4 content as indicated by their activity towards nifedipine. Nifedipine oxidase activity, a reaction catalyzed by P450111A4 (8), was 0.39 nmol/min/mg protein for HL101 and 3.25 nmol/min/mg protein for HL115. Treatment of trout with dexamethasone did not produce any appreciable increase in immunoreactive LMC5 in liver microsomes (Fig. 1) suggesting that this enzyme is probably not induced by dexamethasone in trout. In contrast to anti-LMC5 IgG, anti-LMC1 IgG which binds P450 LMC1 (Mr 50,000), did not crossreact with rat microsomal proteins induced by dexamethasone (Fig. 2). In phenobarbitalinduced rat liver microsomes, two bands were strongly recognized by anti-LMC1 IgG confirming our previous findings (5). From these results, we can conclude that the proteins induced by phenobarbital and recognized by anti-LMC1 IgG are not the same as those induced in the rat by dexamethasone. Anti-LMC1 IgG has little or no affinity toward protein in human liver microsomes (HL101 and HLl15), since only faint bands were seen after staining with this IgG (Fig. 2). The proteins induced by dexamethasone in rat liver microsomes recognized by anti-LMC5 IgG could be one of the gene products belonging to P450111A. P450111Aincorporates at least two gene products, namely, P450p (P450PCN1, P450PCNa or P45OrAo) and P450PCN-E (P450PB2a
Q
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D
E
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G
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~
A
B
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D
E
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Figure 1.
Immunoblot of liver microsomes from rainbow trout, rat, and human probed with anti-LMC5 IgG with detection by [1251]-ProteinA. The microsomes (20 p.g/well)and P450 LMC5 (1 pmol/well) were applied to the wells as follows: A. Control rat; B. Dexamethasone-treated rat; C. Phenobarbital-treated rat; D. P450 LMC5; E. Controltrout; F. Dexamethasone-treatedtrout;G. Human HL101 ; and H. Human HL115.
Figure 2.
Immunoblot of liver microsomes from rainbow trout, rat, and human probed with anti-LMC1 IgG with detection by [12Sl]-ProteinA. The microsomes (20 i~g/well) and P450 LMC1 (1 pmol/well) were applied to the wells as follows: A. Control rat; B. Dexamethasone-treated rat; C. Phenobarbital-treated rat; D. P450 LMC1; E. Control Trout; F. Dexamethasone-treated trout; G. Human HL101; and H. Human HL115.
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Figure 3.
Immunoblot of liver microsomes from rainbow trout, rat, and human probed with anti-rat P450111A1 IgG with detection by [~2~l]-Protein A. The microsomes (20 i~g/well) and purified trout cytochrome P450s (2 pmol/well) were applied as follows: A. Control rat; B. Dexamethasone-treated rat; C. P450 LMC1; D. P450 LMC5; E. Control trout; F. Dexamethasone-treatedtrout; G. Human HL101 ; and H. Human HL115.
Figure 4.
Immunoblot of liver microsomes from trout and humans probed with anti-human P450111A4IgG with detection [~2Sl]-ProteinA. The microsomes (20 pg/well for wells A,E,F, and G; 5 i~g/wellfor wells B,C, and H) and purified P450 LMC1 or P450 LMC5 (2 pmol/well) were applied as follows: A. Control rat; B. Dexamethasonetreated rat; C. Phenobarbital-treated rat; D. P450 LMC5; E. Control trout; F. Dexamethasone-treated trout; G. Human HL101; H. Human HLl15; and I. P450 LMCI.
or P450PCNb)
(7).
I
Fig. 3 shows that anti-P450111A1 IgG recognized a single band in
dexamethasone-induced but not in uninduced rat liver microsomes. This protein which represents P450111A1, has the same mobility as the protein stained with anti-LMC5 IgG shown in Fig. 1. However, anti-rat P450111A1 IgG failed to recognize LMC5 or a corresponding protein in trout liver microsomes (Fig. 3). The results of these reciprocal blots suggest that there is at least one common epitope in trout P450 LMC5 and rat P450111A1 recognized by anti-trout P450 LMC5 but the epitope recognized by anti-P450111A1 IgG is absent in trout P450 LMC5. On the basis of cross-reaction between anti-LMC5 IgG and rat P450111A1, we hypothesize that the protein in human liver microsomes (HLl15) recognized by anti-LMC5 IgG may also belong to the P450111A subfamily. Immunoblots (Fig. 4) using anti-P450111A4 IgG show that a reactive protein is found in much higher concentration in HLl15 than in HL101. This protein is most likely P450111A4 and may correspond to the protein stained by anti-LMC5 IgG shown in Fig. 1. Anti-P450111A4 IgG also recognized P450 LMC5 and a protein in trout liver microsomes with the same mobility as purified P450 LMC5. Thus, trout P450 LMC5 and P450ilIA4 may share a common epitope recognized by both anti-P450 LMC5 IgG and anti-P450111A4 IgG. In addition, P450 LMC1 and a protein in trout liver microsomes with the same Mr as P450 LMC1 were recognized by anti-P450111A4 IgG (Fig. 4). tmmunoinhibition studies were performed to determine whether the epitope(s) in P450 LMC5 recognized by anti-P450111A4 are involved in the biological activities of the enzyme. AntiP450111A4 IgG, at a concentration of 10 mg/nmol P450, almost completely inhibited (93% reduction) the 6/Y-hydroxylation of progesterone by trout liver microsomes, an effect similarly produced by anti-P450 LMC5 IgG (Table 1). These findings suggest that the epitopes in P450 LMC5 recognized by anti-P450111A4 are involved in the catalytic process.
In addition to the
structural similarity between P450 LMC5 and P450111A4 as indicated by the immunoblot studies, 561
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1. In Vitro Inhibition of Trout Liver Microsomal £~8-Hydroxylationof Progesterone by Gestodene and by Rabbit Anti-Cytochrome P450111A4IgG or Rabbit Anti-Cytochrome P450 LMC5 IgG a
Progesterone cqS-Hydroxylase (% Inhibition)
Additions Gestodene (100 I~M)
83
Pre-immune IgG (10 mg/nmol P-450)
12
Anti-P450Nr (P450111A4)IgG 2 mg/nmol of P-450
28
10 mg/nmol of P-450
93
Anti-P450 LMC5 IgG 10 mg/nmol of P-450
92
Activity of uninhibited liver microsomes (14-month male trout) was 0.58 nmol/min/mg protein. Values represent the mean of duplicate determinations.
there could be similarities in the chemical regulation ofthese enzymes. Gestodene, a mechanismbased inactivator of P450111A4(13), was found to strongly inhibit the progesterone 6fl-hydroxylase activity of trout liver microsomes (Table 1).
This activity, chiefly mediated by P450 LMC5,
however, was not induced by dexamethasone in trout (Table 2) which is consistent with the observed lack of effect of dexamethasone on P450 LMC5 levels (Fig. 1). Interestingly, we have found that pretreatment of rainbow trout with 3,4,5,3'4',5'-hexachlorobiphenyl (1 mg/kg) significantly increases the progesterone 6#-hydroxylase activity of trout liver microsomes with no effect on P450 LMC5 levels (14). In conclusion, the immunoblot and enzyme inhibition studies provide evidence that there is strong similarity in structure and function between the constitutive trout P450 LMC5 which is noninducible by dexamethasone pretreatment and dexamethasone-inducible human P450111A4 and less so in the structure between P450 LMC5 and rat P450111A1. Further studies on the amino acid sequence of P450 LMC5 would be needed to establish that this trout enzyme belongs to the P450111A subfamily.
Table 2. Effect of dexamethasona Pretreatment of Trout on 6]Y-Hydroxylationof Progesterone by Trout Liver Microsomes a Progesterone 6#-Hydroxylase Treatment
nmol/min/mg/protein
nmol/min/nmol P450
Control
0.20 + 0.01
0.48 __+0.02
Dexamethasone (50 mg/kg, ip for 3 days)
0.14 -I- 0.01b
0.45 __+0.02
a
b
Values are the mean and S.E. of five animals. P < 0.05 vs. control.
562
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BIOCHEMICAL AND BIOPHYSICALRESEARCH COMMUNICATIONS ACKNOWLEDGMENTS
This study was supported by NIH grants ES-00210 and ES-03850 (DRB), and CA44353 (FPG). This manuscript was issued as Technical Paper 9455 from the Oregon Agricultural Experiment Station, Oregon State University, Corvallis, OR. REFERENCES 1. 2,
3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
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