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Substrate Influence on Interaction between Cytochrome P450 and Cytochrome b5in Microsomes
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 325, No. 2, January 15, pp. 265–269, 1996 Article No. 0033
Substrate Influence on Interaction between Cytochrome P450 and Cytochrome b5 in Microsomes Ingela Jansson and John B. Schenkman1 Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030-1505
Received May 8, 1995, and in revised form August 24, 1995
We have been able to demonstrate that cytochrome b5 interacts closely with cytochrome P450 in the microsomal membrane, and that substrates can serve to order the interaction: Using the water-soluble carbodiimide EDC we could crosslink cytochrome b5 and CYP2B4 in microsomes from phenobarbital-treated rabbits and cytochrome b5 and CYP1A2 in microsomes from b-naphthoflavone-treated animals. The substrate benzphetamine increased the specific interaction between cytochrome b5 and CYP2B4, decreasing the formation of higher molecular weight oligomeric complexes with cytochrome b5 . In contrast, no ordering of the interactions were obtained on addition of 7-ethoxycoumarin, a substrate of CYP1A2, or of benzphetamine to microsomes of b-naphthoflavone-treated animals in the presence of EDC. Of interest, although evidence could be shown for complementary chargepairing between cytochrome b5 and a number of other microsomal proteins in the membranes, and while the extent of CYP1A2 and CYP2B4 interaction with cytochrome b5 each exceeded 30% in the presence of substrate, no significant complexation of the P450s was obtained with any other microsomal proteins. q 1996 Academic Press, Inc.
The role of cytochrome b5 in cytochrome P450-catalyzed reactions has been the topic of many papers since the original suggestion by Hildebrandt and Estabrook (1) that cytochrome b5 may be involved in the monooxygenation reaction. Their suggestion was that the input of the second of the two electrons needed for oxygen activation was via cytochrome b5 . Other suggestions have been that cytochrome b5 exerts a sparing effect preventing uncoupling (2), or causing metabolic switch1 To whom correspondence should be addressed. Fax: (203) 6792473.
ing, thereby increasing product formation while decreasing hydrogen peroxide formation (3, 4). A more recent suggestion has been made (5) that at least with some forms of cytochrome P450 cytochrome b5 binds and serves as an electron buffer; the two-hemoprotein complex becomes a two-electron acceptor, thereby obviating the need for two sequential interactions with the reductase. Data for such a suggestion was obtained using purified, reconstituted systems. Such studies have implied a physiological role for cytochrome b5 in cytochrome P450 monooxygenations. Omata et al. (6) have recently shown that immunopurification of cytochromes P450 1A1, 2B1, and 2E1 from rat liver microsomes resulted in copurification of cytochrome b5 with P450s 2B1 and 2E1 but not with 1A1. This finding may suggest a close but selective interaction in the microsomal membrane between cytochrome b5 and some forms of cytochrome P450. Watersoluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) have been used with purified proteins as zero-distance crosslinking reagents to covalently link complementary charge pairing amino and carboxyl groups located at the interface of interacting redox proteins (7–13). In fact we have been able to show such interaction between certain purified forms of cytochrome P450 and cytochrome b5 by generating a covalent, functionally active heterodimer with EDC (12). Therefore, in this study we chose to use EDC as a means of trapping charge-pairing proteins in order to examine cytochrome b5 interaction with cytochrome P450 forms. In this paper we have attempted to determine whether the two proteins interact similarly in the liver microsomal membrane as with the reconstituted system. We have used the carbodiimide EDC to crosslink complexes formed in the microsomes in the absence and presence of substrates of the different forms of cytochrome P450. We report here that in the presence of a substrate that is metabolized faster in the reconsti265
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tuted system with cytochrome b5 present the specificity of the interaction is increased. MATERIALS AND METHODS Chemicals. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC, was purchased from Sigma Chemical Co. b-Naphthoflavone (b-NF) and 7-ethoxycoumarin were purchased from Aldrich Chemical Co. Benzphetamine HCl was obtained from Upjohn Co. All reagents were of highest purity available and were not purified further. Preparation of microsomes and purification of proteins. Phenobarbital-induced rabbit liver microsomes were prepared from 3 kg male New Zealand rabbits after daily treatments with 80 mg of sodium phenobarbital per kilogram intraperitoneally for 4 days. b-NF-induced liver microsomes were prepared after the rabbits had been treated with 17 mg of b-NF per kilogram daily for 2 days. CYP1A2 was purified according to Voznesensky and Schenkman (14). Cytochrome b5 was purified as described (15). Chemical crosslinking. Crosslinking of proteins of the microsomal membrane with EDC was performed at 37 or 247C. Microsomes were first incubated in the presence or absence of substrate for 30 min at room temperature after which the water soluble carbodiimide EDC [1-ethyl 3-(3-dimethylaminopropyl)carbodiimide] was added slowly over a 45-min time period to 9 mM final concentration. The microsomal proteins were then separated by SDS–PAGE. Purified proteins were incorporated into dilauroylphosphatidylcholine (DLPC) vesicles at a molar lipid:protein ratio of 160:1 before treatment with EDC. Electrophoretic separation and detection of microsomal proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) was performed according to Laemmli (16). A 9% crosslinked polyacrylamide gel was used for protein separation. After transfer to nitrocellulose in 25 mM Tris–glycine, pH 8.3, containing 10% methanol, Western blotting was performed as described (17) with the exception that the antibodies to CYP2B4 (LM2), CYP1A2 (LM4), and cytochrome b5 had been raised in chickens and the IgG fraction was isolated from the egg yolks and purified according to Polson et al. (18). Spectral studies. Spectral studies were done at room temperature using a Shimadzu UV3000 Dual Wavelength Double Beam recording spectrophotometer. Spectral changes of cytochrome P450 upon cytochrome b5 additions in the absence or presence of substrate were measured as described (19). Briefly, after establishing a baseline of equal light absorbance between sample and reference cuvettes each containing 45 mg/ml of DLPC in 50 mM sodium phosphate buffer, pH 7.4, 20% glycerol, the absolute spectrum of P450 1A2 (0.75 mM) in the same medium was recorded. Ligand binding using either cytochrome b5 or 7-ethoxycoumarin was performed by adding equal volumes of stock solution to both sample and reference cuvettes. This procedure enables clear visualization of the ligand-induced spectral change free from spectral interference from either ligand absorbance. Calculations of high spin content were done as described (19, 20), De390 – 417 nm Å 126 mM01 cm01 and e407 nm Å 90 mM01 cm01.
RESULTS AND DISCUSSION
Chemical Crosslinking in Microsomes from Phenobarbital-Treated Rabbits Microsomes from phenobarbital-treated rabbits were preincubated with 1 mM benzphetamine and the watersoluble carbodiimide EDC was added to covalently link together charge-pairing amino and carboxyl groups in
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FIG. 1. Crosslinking of microsomal proteins in microsomes from phenobarbital-treated rabbits. Tracks 1–4 are Western blots using antibody to P450 2B4, Tracks 5–8 are Western blots using antibody to cytochrome b5 . Molecular weight standards (12.5–92 kDa) were visualized using Ponceau S reversible protein stain. Tracks 1 and 5, microsomes; Tracks 2 and 6, microsomes plus EDC; Tracks 3 and 7, microsomes plus benzphetamine and EDC; Track 4, CYP2B4 in DLPC plus EDC; Track 8, cytochrome b5 in DLPC plus EDC. Crosslinking was performed at 377C.
amide bonds. As shown in Fig. 1 a new 66-kDa protein band appeared in Western blots after treatment with antibody to CYP2B4 (lanes 2, 3, arrow). In the absence of EDC this band was not present (lane 1), nor was it formed when purified CYP2B4 in DLPC was incubated with the EDC (lane 4). With the purified cytochrome P450 2B4 a trace of homodimer was seen at 96 kDa (barely detectable in lane 4). The new protein band had the mass of a covalent complex between CYP2B4 and cytochrome b5 (66 kDa); such a complex was formed earlier in this laboratory using purified cytochrome P450 2B4 and cytochrome b5 (11–12). In those studies a ratio of cytochrome P450 2B4 to cytochrome b5 of 1:1 was used resulting in about 30% of the cytochrome P450 in the complex. In the phenobarbital-treated rabbit liver microsomes used in the present study the ratio between cytochrome b5 and total cytochrome 2B4 was 1:5 and the proportion of cytochrome P450 in the complex formed in the presence of benzphetamine was 32% ({0.3, n Å 6) (Fig. 1, lane 3), an amount similar to that obtained when purified CYP2B4 was incubated with cytochrome b5 and benzphetamine in the presence of EDC. No other significant levels of reaction were seen with antibody to CYP2B4. That the complex formed actually was a combination of cytochrome P450 and cytochrome b5 can be seen in lane 7 (Fig. 1, arrow), where the immunodetection was carried out using antibody to cytochrome b5 . Cytochrome b5-containing bands were formed (lanes 6, 7) with the same apparent electrophoretic mobility (66 kDa) as the bands visual-
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ized with antibody to CYP2B4 (lanes 2, 3). The bands just above the arrow (lane 7) detected by antibody to cytochrome b5 but not by the antibody to CYP2B4 appear to be complexes between CYP1A2 and cytochrome b5 , as determined by antibody reaction (data not shown): Although phenobarbital-treatment induces cytochrome P450 2B4 in the microsomes preferentially, an appreciable amount of cytochrome P450 1A2 is present constitutively in these microsomes and apparently charge-pairs with cytochrome b5 . When complex formation was attempted in the microsomes in the absence of benzphetamine (lanes 2 and 6) the same major heterodimer complex was formed, along with a trace of several higher molecular weight oligomeric complexes which were detected by both antibodies to CYP2B4 and cytochrome b5 . In the absence of benzphetamine the band attributed to the b5-P450 heterodimer was consistently lighter staining, showing an 18% complex formation ({0.9, n Å 6) vs 32% in the presence of benzphetamine (lane 2 vs lane 3), and the amount of high mass cytochrome b5 complexes appeared larger (lane 6 vs lane 7), indicating a lower proportion of the CYP2B4 was coupled to cytochrome b5 in the absence of benzphetamine. The high mass protein bands reacting with antibodies to cytochrome b5 were not homooligomers as seen by lack of their formation with purified cytochrome b5 in DLPC in the presence of EDC; complexes were seen between 51 and 67 kDa (lane 8). These findings are explainable in terms of the studies by Tamburini et al. (19) and Chiang (21), where benzphetamine was shown to increase the affinity of purified cytochrome b5 for purified CYP2B4 by an order of magnitude. Chemical Crosslinking in Microsomes from b-Naphthoflavone-Treated Rabbits Similar crosslinking experiments were done with microsomes from b-NF-treated rabbits. These microsomes contained cytochrome b5 and cytochrome P450 at a ratio of 1:1.7. The major form of cytochrome P450 was CYP1A2 (LM4). As shown in Fig. 2, the addition of EDC caused the formation of a P450-b5 complex at 68 kDa detected by antibody to CYP1A2 (lanes 2, 3) as well as by antibody to cytochrome b5 (lanes 5, 6), plus many very faint, higher molecular weight bands. Based upon densitometric calculations it would appear that about 36% of the CYP1A2 was in heterodimeric complex with cytochrome b5 and an insignificant amount was complexed with other proteins. There was no formation of any of these bands in the absence of EDC (lanes 1 and 4). Contrary to the results with microsomes from phenobarbital-treated animals with benzphetamine, there was no difference in the complexes formed in the presence of the CYP1A2 substrate 7-ethoxycoumarin (lane
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FIG. 2. Crosslinking of microsomal proteins in microsomes from bnaphthoflavone-treated rabbits. Tracks 1–3 are Western blots using antibody to P450 1A2, Tracks 4–6 are Western blots using antibody to cytochrome b5 , and Tracks 7–8 are protein stained with amidoblack after transfer. Molecular weight standards (12.5–92 kDa) were visualized using Ponceau S reversible protein stain. Tracks 1, 4, and 7, microsomes; Tracks 2 and 5, microsomes plus EDC; Tracks 3, 6, and 8, microsomes plus 7-ethoxycoumarin and EDC.
2 vs 3 and lane 5 vs 6), nor did the substitution of benzphetamine for 7-ethoxycoumarin cause a change in the proportion of heterodimer formed (not shown). The presence of substrate did not decrease the formation of higher molecular weight bands that react with antibodies to cytochrome b5 or antibodies to CYP1A2, nor was there an increase in the amount of antibody responsive complex formation in the 68-kDa heterodimer region. The complex formation can also be seen by protein staining (lane 8 vs lane 7). Lack of Influence of 7-Ethoxycoumarin on the Spectrophotometrically Measured Interaction between Purified Cytochrome b5 and CYP1A2 The lack of substrate-induced organization of the cytochrome P450 and cytochrome b5 proteins in the b-NFmicrosomes agrees with a recent paper by Mayuzumi et al. (22), where cytochrome b5 was shown to increase the rate of 7-ethoxycoumarin O-deethylation while the Km for the reaction was unchanged. To further investigate the effect of substrate on the affinity of cytochrome b5 for CYP1A2 we studied the spectrophotometrically detectable binding of cytochrome b5 to the purified P450 1A2 in the absence and presence of 7-ethoxycoumarin. As seen in Fig. 3 in the absence of ligand cytochrome P450 is a mostly high spin hemoprotein with a peak at 393 nm and a shoulder at 417 nm. The addition of saturating levels of cytochrome b5 (ratio 5:1) caused a spectral shift toward the high spin state (dashed line). When 7-ethoxycoumarin, a substrate of P450 1A2, was added there was a slightly higher shift toward high
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spin. Table 1 quantifies the influence of substrate and cytochrome b5 on the spin equilibrium of CYP1A2. There was no significant difference in the maximal attainable spectral change in the presence of both ligands regardless of the order of addition of the two ligands. In the presence of either ligand alone, however, the high spin content of CYP1A2 was always highest in the presence of substrate alone, and the addition of cytochrome b5 consistently caused a small shift in the spin equilibrium toward low spin. It was not possible to determine the influence of 7-ethoxycoumarin on the binding constants for cytochrome b5 because of the very small spectral changes obtained and the changes in the spin equilibrium encountered. These results are very different from those obtained with the binding of cytochrome b5 to P450 2B4 (LM2) in the presence and absence of benzphetamine (Fig. 3, inset), in which saturating levels of substrate or cytochrome b5 caused a similar extent of high spin shift. In the presence of both, however, the magnitude of spin shift was greater than that of either ligand alone (19, 21). Our results,
TABLE I
High Spin–Low Spin Ratio of Cytochrome P450 1A2 in the Absence and Presence of liganda Ligand None Cytochrome b5 5:1 b5 / 7-Ethoxycoumarin 7-Ethoxycoumarin 7-Ethoxycoumarin / b5
% High spin 64.7 76.3 84.5 86.6 85.3
{ { { { {
0.3 0.6 0.5 0.4 0.6
(9) (3) (3) (6) (6)
a Conditions were as described in Fig. 3 and under Materials and Methods.
thus, show that the binding of cytochrome b5 to different forms of cytochrome P450 is differentially influenced by interaction of substrates, suggesting that substrates can exert an ordering effect on interactions between the proteins of the monooxygenase system. In conclusion, cytochrome P450 forms interact closely with cytochrome b5 in the microsomal membrane with no indication of significant interactions with other microsomal proteins. The substrate benzphetamine increased the specific interaction between cytochrome b5 and CYP2B4, while no such effects were obtained with 7-ethoxycoumarin, a substrate of CYP1A2, on the interaction of this form of cytochrome P450 and cytochrome b5 . In contrast, cytochrome b5 , which functions in a number of non-P450 microsomal reactions, also forms complexes with a number of other microsomal proteins. ACKNOWLEDGMENTS The authors thank Wendy Clarke for the purification of the antibodies used in this study and Dr. Andrei I. Voznesensky for the generous contribution of purified CYP1A2.
REFERENCES 1. Hildebrandt, A., and Estabrook, R. W. (1971) Arch. Biochem. Biophys. 143, 66–79. 2. Staudt, H., Lichtenberger, F., and Ullrich, V. (1974) Eur. J. Biochem. 46, 99–106. 3. Gorsky, L. D., Koop, D. R., and Coon, M. J. (1984) Drug Metab. Dispos. 9, 461–465. FIG. 3. Spectral interaction between cytochrome P450 1A2 and cytochrome b5 in the absence and presence of 7-ethoxycoumarin. Visible spectra of P450 1A2 (0.75 mM) in the absence (—) and presence of either 3.75 mM cytochrome b5 (---) or 3.75 mM cytochrome b5 plus 0.1 mM 7-ethoxycoumarin (rrr). Line at bottom of graph represents a baseline of equal absorbance between sample and reference cuvette before the addition of cytochrome P450. Insert shows CYP2B4 spectra with cytochrome b5 with and without benzphetamine (modified from Ref. 19). The dashed line in the insert is the same with saturating levels of either cytochrome b5 or benzphetamine.
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4. Jansson, I., and Schenkman, J. B. (1987) Drug Metab. Dispos. 15, 344–348. 5. Schenkman, J. B., Voznesensky, A. I., and Jansson, I. (1994) Arch. Biochem. Biophys. 314, 234–241. 6. Omata, Y., Robinson, R. C., Gelboin, H. V., Pincus, M. R., and Friedman, F. K. (1994) FEBS Lett. 346, 241–245. 7. Hackett, C. S., and Strittmatter, P. (1984) J. Biol. Chem. 259, 3275–3282. 8. Lambeth, J. D., Geren, L. M., and Millett, F. (1984) J. Biol. Chem. 259, 10025–10029.
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BENZPHETAMINE INCREASES CYP2B4 BINDING TO b5 IN MICROSOMES 9. Waldmeyer, B., and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5184–5190. 10. Nisimoto, Y., and Lambeth, J. D. (1985) Arch. Biochem. Biophys. 241, 386–396. 11. Tamburini, P. P., MacFarquar, S., and Schenkman, J. B. (1986) Biochem. Biophys. Res. Commun. 134, 519–526. 12. Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. USA 84, 11–15. 13. Nisimoto, Y., and Otsuka-Murakami, H. (1988) Biochemistry 27, 5869–5876. 14. Voznesensky, A. I., and Schenkman, J. B. (1994) J. Biol. Chem. 269, 15724–15731.
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15. Jansson, I., Mole, J., and Schenkman, J. B. (1995) Arch. Biochem. Biophys. 316, 275–284. 16. Laemmli, U. K. (1970) Nature 227, 680–685. 17. Favreau, L. V., and Schenkman, J. B. (1988) Diabetes 37, 577– 584. 18. Polson, A., von Wechmar, M. B., and van Regenmortel, M. H. V. (1980) Immunol. Commun. 9, 475–493. 19. Tamburini, P. P., White, R. E., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 4007–4015. 20. Jansson, I., Tamburini, P. P., Favreau, L. V., and Schenkman, J. B. (1985) Drug Metab. Dispos. 13, 453–458. 21. Chiang, J. Y. L. (1981) Arch. Biochem. Biophys. 211, 662–673. 22. Mayuzumi, H., Shimizu, T., Sambongi, C., Hiroya, K., and Hatano, M. (1994) Arch. Biochem. Biophys. 310, 367–372.
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Vol. 325, No. 2, January 15, pp. 265–269, 1996 Article No. 0033
Substrate Influence on Interaction between Cytochrome P450 and Cytochrome b5 in Microsomes Ingela Jansson and John B. Schenkman1 Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030-1505
Received May 8, 1995, and in revised form August 24, 1995
We have been able to demonstrate that cytochrome b5 interacts closely with cytochrome P450 in the microsomal membrane, and that substrates can serve to order the interaction: Using the water-soluble carbodiimide EDC we could crosslink cytochrome b5 and CYP2B4 in microsomes from phenobarbital-treated rabbits and cytochrome b5 and CYP1A2 in microsomes from b-naphthoflavone-treated animals. The substrate benzphetamine increased the specific interaction between cytochrome b5 and CYP2B4, decreasing the formation of higher molecular weight oligomeric complexes with cytochrome b5 . In contrast, no ordering of the interactions were obtained on addition of 7-ethoxycoumarin, a substrate of CYP1A2, or of benzphetamine to microsomes of b-naphthoflavone-treated animals in the presence of EDC. Of interest, although evidence could be shown for complementary chargepairing between cytochrome b5 and a number of other microsomal proteins in the membranes, and while the extent of CYP1A2 and CYP2B4 interaction with cytochrome b5 each exceeded 30% in the presence of substrate, no significant complexation of the P450s was obtained with any other microsomal proteins. q 1996 Academic Press, Inc.
The role of cytochrome b5 in cytochrome P450-catalyzed reactions has been the topic of many papers since the original suggestion by Hildebrandt and Estabrook (1) that cytochrome b5 may be involved in the monooxygenation reaction. Their suggestion was that the input of the second of the two electrons needed for oxygen activation was via cytochrome b5 . Other suggestions have been that cytochrome b5 exerts a sparing effect preventing uncoupling (2), or causing metabolic switch1 To whom correspondence should be addressed. Fax: (203) 6792473.
ing, thereby increasing product formation while decreasing hydrogen peroxide formation (3, 4). A more recent suggestion has been made (5) that at least with some forms of cytochrome P450 cytochrome b5 binds and serves as an electron buffer; the two-hemoprotein complex becomes a two-electron acceptor, thereby obviating the need for two sequential interactions with the reductase. Data for such a suggestion was obtained using purified, reconstituted systems. Such studies have implied a physiological role for cytochrome b5 in cytochrome P450 monooxygenations. Omata et al. (6) have recently shown that immunopurification of cytochromes P450 1A1, 2B1, and 2E1 from rat liver microsomes resulted in copurification of cytochrome b5 with P450s 2B1 and 2E1 but not with 1A1. This finding may suggest a close but selective interaction in the microsomal membrane between cytochrome b5 and some forms of cytochrome P450. Watersoluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) have been used with purified proteins as zero-distance crosslinking reagents to covalently link complementary charge pairing amino and carboxyl groups located at the interface of interacting redox proteins (7–13). In fact we have been able to show such interaction between certain purified forms of cytochrome P450 and cytochrome b5 by generating a covalent, functionally active heterodimer with EDC (12). Therefore, in this study we chose to use EDC as a means of trapping charge-pairing proteins in order to examine cytochrome b5 interaction with cytochrome P450 forms. In this paper we have attempted to determine whether the two proteins interact similarly in the liver microsomal membrane as with the reconstituted system. We have used the carbodiimide EDC to crosslink complexes formed in the microsomes in the absence and presence of substrates of the different forms of cytochrome P450. We report here that in the presence of a substrate that is metabolized faster in the reconsti265
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tuted system with cytochrome b5 present the specificity of the interaction is increased. MATERIALS AND METHODS Chemicals. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC, was purchased from Sigma Chemical Co. b-Naphthoflavone (b-NF) and 7-ethoxycoumarin were purchased from Aldrich Chemical Co. Benzphetamine HCl was obtained from Upjohn Co. All reagents were of highest purity available and were not purified further. Preparation of microsomes and purification of proteins. Phenobarbital-induced rabbit liver microsomes were prepared from 3 kg male New Zealand rabbits after daily treatments with 80 mg of sodium phenobarbital per kilogram intraperitoneally for 4 days. b-NF-induced liver microsomes were prepared after the rabbits had been treated with 17 mg of b-NF per kilogram daily for 2 days. CYP1A2 was purified according to Voznesensky and Schenkman (14). Cytochrome b5 was purified as described (15). Chemical crosslinking. Crosslinking of proteins of the microsomal membrane with EDC was performed at 37 or 247C. Microsomes were first incubated in the presence or absence of substrate for 30 min at room temperature after which the water soluble carbodiimide EDC [1-ethyl 3-(3-dimethylaminopropyl)carbodiimide] was added slowly over a 45-min time period to 9 mM final concentration. The microsomal proteins were then separated by SDS–PAGE. Purified proteins were incorporated into dilauroylphosphatidylcholine (DLPC) vesicles at a molar lipid:protein ratio of 160:1 before treatment with EDC. Electrophoretic separation and detection of microsomal proteins. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS– PAGE) was performed according to Laemmli (16). A 9% crosslinked polyacrylamide gel was used for protein separation. After transfer to nitrocellulose in 25 mM Tris–glycine, pH 8.3, containing 10% methanol, Western blotting was performed as described (17) with the exception that the antibodies to CYP2B4 (LM2), CYP1A2 (LM4), and cytochrome b5 had been raised in chickens and the IgG fraction was isolated from the egg yolks and purified according to Polson et al. (18). Spectral studies. Spectral studies were done at room temperature using a Shimadzu UV3000 Dual Wavelength Double Beam recording spectrophotometer. Spectral changes of cytochrome P450 upon cytochrome b5 additions in the absence or presence of substrate were measured as described (19). Briefly, after establishing a baseline of equal light absorbance between sample and reference cuvettes each containing 45 mg/ml of DLPC in 50 mM sodium phosphate buffer, pH 7.4, 20% glycerol, the absolute spectrum of P450 1A2 (0.75 mM) in the same medium was recorded. Ligand binding using either cytochrome b5 or 7-ethoxycoumarin was performed by adding equal volumes of stock solution to both sample and reference cuvettes. This procedure enables clear visualization of the ligand-induced spectral change free from spectral interference from either ligand absorbance. Calculations of high spin content were done as described (19, 20), De390 – 417 nm Å 126 mM01 cm01 and e407 nm Å 90 mM01 cm01.
RESULTS AND DISCUSSION
Chemical Crosslinking in Microsomes from Phenobarbital-Treated Rabbits Microsomes from phenobarbital-treated rabbits were preincubated with 1 mM benzphetamine and the watersoluble carbodiimide EDC was added to covalently link together charge-pairing amino and carboxyl groups in
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FIG. 1. Crosslinking of microsomal proteins in microsomes from phenobarbital-treated rabbits. Tracks 1–4 are Western blots using antibody to P450 2B4, Tracks 5–8 are Western blots using antibody to cytochrome b5 . Molecular weight standards (12.5–92 kDa) were visualized using Ponceau S reversible protein stain. Tracks 1 and 5, microsomes; Tracks 2 and 6, microsomes plus EDC; Tracks 3 and 7, microsomes plus benzphetamine and EDC; Track 4, CYP2B4 in DLPC plus EDC; Track 8, cytochrome b5 in DLPC plus EDC. Crosslinking was performed at 377C.
amide bonds. As shown in Fig. 1 a new 66-kDa protein band appeared in Western blots after treatment with antibody to CYP2B4 (lanes 2, 3, arrow). In the absence of EDC this band was not present (lane 1), nor was it formed when purified CYP2B4 in DLPC was incubated with the EDC (lane 4). With the purified cytochrome P450 2B4 a trace of homodimer was seen at 96 kDa (barely detectable in lane 4). The new protein band had the mass of a covalent complex between CYP2B4 and cytochrome b5 (66 kDa); such a complex was formed earlier in this laboratory using purified cytochrome P450 2B4 and cytochrome b5 (11–12). In those studies a ratio of cytochrome P450 2B4 to cytochrome b5 of 1:1 was used resulting in about 30% of the cytochrome P450 in the complex. In the phenobarbital-treated rabbit liver microsomes used in the present study the ratio between cytochrome b5 and total cytochrome 2B4 was 1:5 and the proportion of cytochrome P450 in the complex formed in the presence of benzphetamine was 32% ({0.3, n Å 6) (Fig. 1, lane 3), an amount similar to that obtained when purified CYP2B4 was incubated with cytochrome b5 and benzphetamine in the presence of EDC. No other significant levels of reaction were seen with antibody to CYP2B4. That the complex formed actually was a combination of cytochrome P450 and cytochrome b5 can be seen in lane 7 (Fig. 1, arrow), where the immunodetection was carried out using antibody to cytochrome b5 . Cytochrome b5-containing bands were formed (lanes 6, 7) with the same apparent electrophoretic mobility (66 kDa) as the bands visual-
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BENZPHETAMINE INCREASES CYP2B4 BINDING TO b5 IN MICROSOMES
ized with antibody to CYP2B4 (lanes 2, 3). The bands just above the arrow (lane 7) detected by antibody to cytochrome b5 but not by the antibody to CYP2B4 appear to be complexes between CYP1A2 and cytochrome b5 , as determined by antibody reaction (data not shown): Although phenobarbital-treatment induces cytochrome P450 2B4 in the microsomes preferentially, an appreciable amount of cytochrome P450 1A2 is present constitutively in these microsomes and apparently charge-pairs with cytochrome b5 . When complex formation was attempted in the microsomes in the absence of benzphetamine (lanes 2 and 6) the same major heterodimer complex was formed, along with a trace of several higher molecular weight oligomeric complexes which were detected by both antibodies to CYP2B4 and cytochrome b5 . In the absence of benzphetamine the band attributed to the b5-P450 heterodimer was consistently lighter staining, showing an 18% complex formation ({0.9, n Å 6) vs 32% in the presence of benzphetamine (lane 2 vs lane 3), and the amount of high mass cytochrome b5 complexes appeared larger (lane 6 vs lane 7), indicating a lower proportion of the CYP2B4 was coupled to cytochrome b5 in the absence of benzphetamine. The high mass protein bands reacting with antibodies to cytochrome b5 were not homooligomers as seen by lack of their formation with purified cytochrome b5 in DLPC in the presence of EDC; complexes were seen between 51 and 67 kDa (lane 8). These findings are explainable in terms of the studies by Tamburini et al. (19) and Chiang (21), where benzphetamine was shown to increase the affinity of purified cytochrome b5 for purified CYP2B4 by an order of magnitude. Chemical Crosslinking in Microsomes from b-Naphthoflavone-Treated Rabbits Similar crosslinking experiments were done with microsomes from b-NF-treated rabbits. These microsomes contained cytochrome b5 and cytochrome P450 at a ratio of 1:1.7. The major form of cytochrome P450 was CYP1A2 (LM4). As shown in Fig. 2, the addition of EDC caused the formation of a P450-b5 complex at 68 kDa detected by antibody to CYP1A2 (lanes 2, 3) as well as by antibody to cytochrome b5 (lanes 5, 6), plus many very faint, higher molecular weight bands. Based upon densitometric calculations it would appear that about 36% of the CYP1A2 was in heterodimeric complex with cytochrome b5 and an insignificant amount was complexed with other proteins. There was no formation of any of these bands in the absence of EDC (lanes 1 and 4). Contrary to the results with microsomes from phenobarbital-treated animals with benzphetamine, there was no difference in the complexes formed in the presence of the CYP1A2 substrate 7-ethoxycoumarin (lane
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FIG. 2. Crosslinking of microsomal proteins in microsomes from bnaphthoflavone-treated rabbits. Tracks 1–3 are Western blots using antibody to P450 1A2, Tracks 4–6 are Western blots using antibody to cytochrome b5 , and Tracks 7–8 are protein stained with amidoblack after transfer. Molecular weight standards (12.5–92 kDa) were visualized using Ponceau S reversible protein stain. Tracks 1, 4, and 7, microsomes; Tracks 2 and 5, microsomes plus EDC; Tracks 3, 6, and 8, microsomes plus 7-ethoxycoumarin and EDC.
2 vs 3 and lane 5 vs 6), nor did the substitution of benzphetamine for 7-ethoxycoumarin cause a change in the proportion of heterodimer formed (not shown). The presence of substrate did not decrease the formation of higher molecular weight bands that react with antibodies to cytochrome b5 or antibodies to CYP1A2, nor was there an increase in the amount of antibody responsive complex formation in the 68-kDa heterodimer region. The complex formation can also be seen by protein staining (lane 8 vs lane 7). Lack of Influence of 7-Ethoxycoumarin on the Spectrophotometrically Measured Interaction between Purified Cytochrome b5 and CYP1A2 The lack of substrate-induced organization of the cytochrome P450 and cytochrome b5 proteins in the b-NFmicrosomes agrees with a recent paper by Mayuzumi et al. (22), where cytochrome b5 was shown to increase the rate of 7-ethoxycoumarin O-deethylation while the Km for the reaction was unchanged. To further investigate the effect of substrate on the affinity of cytochrome b5 for CYP1A2 we studied the spectrophotometrically detectable binding of cytochrome b5 to the purified P450 1A2 in the absence and presence of 7-ethoxycoumarin. As seen in Fig. 3 in the absence of ligand cytochrome P450 is a mostly high spin hemoprotein with a peak at 393 nm and a shoulder at 417 nm. The addition of saturating levels of cytochrome b5 (ratio 5:1) caused a spectral shift toward the high spin state (dashed line). When 7-ethoxycoumarin, a substrate of P450 1A2, was added there was a slightly higher shift toward high
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JANSSON AND SCHENKMAN
spin. Table 1 quantifies the influence of substrate and cytochrome b5 on the spin equilibrium of CYP1A2. There was no significant difference in the maximal attainable spectral change in the presence of both ligands regardless of the order of addition of the two ligands. In the presence of either ligand alone, however, the high spin content of CYP1A2 was always highest in the presence of substrate alone, and the addition of cytochrome b5 consistently caused a small shift in the spin equilibrium toward low spin. It was not possible to determine the influence of 7-ethoxycoumarin on the binding constants for cytochrome b5 because of the very small spectral changes obtained and the changes in the spin equilibrium encountered. These results are very different from those obtained with the binding of cytochrome b5 to P450 2B4 (LM2) in the presence and absence of benzphetamine (Fig. 3, inset), in which saturating levels of substrate or cytochrome b5 caused a similar extent of high spin shift. In the presence of both, however, the magnitude of spin shift was greater than that of either ligand alone (19, 21). Our results,
TABLE I
High Spin–Low Spin Ratio of Cytochrome P450 1A2 in the Absence and Presence of liganda Ligand None Cytochrome b5 5:1 b5 / 7-Ethoxycoumarin 7-Ethoxycoumarin 7-Ethoxycoumarin / b5
% High spin 64.7 76.3 84.5 86.6 85.3
{ { { { {
0.3 0.6 0.5 0.4 0.6
(9) (3) (3) (6) (6)
a Conditions were as described in Fig. 3 and under Materials and Methods.
thus, show that the binding of cytochrome b5 to different forms of cytochrome P450 is differentially influenced by interaction of substrates, suggesting that substrates can exert an ordering effect on interactions between the proteins of the monooxygenase system. In conclusion, cytochrome P450 forms interact closely with cytochrome b5 in the microsomal membrane with no indication of significant interactions with other microsomal proteins. The substrate benzphetamine increased the specific interaction between cytochrome b5 and CYP2B4, while no such effects were obtained with 7-ethoxycoumarin, a substrate of CYP1A2, on the interaction of this form of cytochrome P450 and cytochrome b5 . In contrast, cytochrome b5 , which functions in a number of non-P450 microsomal reactions, also forms complexes with a number of other microsomal proteins. ACKNOWLEDGMENTS The authors thank Wendy Clarke for the purification of the antibodies used in this study and Dr. Andrei I. Voznesensky for the generous contribution of purified CYP1A2.
REFERENCES 1. Hildebrandt, A., and Estabrook, R. W. (1971) Arch. Biochem. Biophys. 143, 66–79. 2. Staudt, H., Lichtenberger, F., and Ullrich, V. (1974) Eur. J. Biochem. 46, 99–106. 3. Gorsky, L. D., Koop, D. R., and Coon, M. J. (1984) Drug Metab. Dispos. 9, 461–465. FIG. 3. Spectral interaction between cytochrome P450 1A2 and cytochrome b5 in the absence and presence of 7-ethoxycoumarin. Visible spectra of P450 1A2 (0.75 mM) in the absence (—) and presence of either 3.75 mM cytochrome b5 (---) or 3.75 mM cytochrome b5 plus 0.1 mM 7-ethoxycoumarin (rrr). Line at bottom of graph represents a baseline of equal absorbance between sample and reference cuvette before the addition of cytochrome P450. Insert shows CYP2B4 spectra with cytochrome b5 with and without benzphetamine (modified from Ref. 19). The dashed line in the insert is the same with saturating levels of either cytochrome b5 or benzphetamine.
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4. Jansson, I., and Schenkman, J. B. (1987) Drug Metab. Dispos. 15, 344–348. 5. Schenkman, J. B., Voznesensky, A. I., and Jansson, I. (1994) Arch. Biochem. Biophys. 314, 234–241. 6. Omata, Y., Robinson, R. C., Gelboin, H. V., Pincus, M. R., and Friedman, F. K. (1994) FEBS Lett. 346, 241–245. 7. Hackett, C. S., and Strittmatter, P. (1984) J. Biol. Chem. 259, 3275–3282. 8. Lambeth, J. D., Geren, L. M., and Millett, F. (1984) J. Biol. Chem. 259, 10025–10029.
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BENZPHETAMINE INCREASES CYP2B4 BINDING TO b5 IN MICROSOMES 9. Waldmeyer, B., and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5184–5190. 10. Nisimoto, Y., and Lambeth, J. D. (1985) Arch. Biochem. Biophys. 241, 386–396. 11. Tamburini, P. P., MacFarquar, S., and Schenkman, J. B. (1986) Biochem. Biophys. Res. Commun. 134, 519–526. 12. Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. USA 84, 11–15. 13. Nisimoto, Y., and Otsuka-Murakami, H. (1988) Biochemistry 27, 5869–5876. 14. Voznesensky, A. I., and Schenkman, J. B. (1994) J. Biol. Chem. 269, 15724–15731.
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15. Jansson, I., Mole, J., and Schenkman, J. B. (1995) Arch. Biochem. Biophys. 316, 275–284. 16. Laemmli, U. K. (1970) Nature 227, 680–685. 17. Favreau, L. V., and Schenkman, J. B. (1988) Diabetes 37, 577– 584. 18. Polson, A., von Wechmar, M. B., and van Regenmortel, M. H. V. (1980) Immunol. Commun. 9, 475–493. 19. Tamburini, P. P., White, R. E., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 4007–4015. 20. Jansson, I., Tamburini, P. P., Favreau, L. V., and Schenkman, J. B. (1985) Drug Metab. Dispos. 13, 453–458. 21. Chiang, J. Y. L. (1981) Arch. Biochem. Biophys. 211, 662–673. 22. Mayuzumi, H., Shimizu, T., Sambongi, C., Hiroya, K., and Hatano, M. (1994) Arch. Biochem. Biophys. 310, 367–372.
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