ARCHIVES
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 293, No. 2, March, pp. 219-223, 1992
Baculovirus-Mediated Expression and Functional Characterization of Human NADPH-P450 Oxidoreductase Shinji Tamura, Kenneth R. Korzekwa, Shioko Kimura, Harry V. Gelboin, and Frank J. Gonzalez? Laboratory
of Molecular
Carcinogenesis, National
Cancer Institute,
National
Institutes
of Health, Bethesda, Maryland
20892
Received July 11, 1991, and in revised form October 25, 1991
Human NADPH-PI60 oxidoreductase (OR) is an intrinsically membrane-bound flavoprotein that serves to transfer electrons from NADPH to cytochrome P460. OR is also involved in the metabolic activation of chemotherapeutic alhylating agents. The human OR cDNA was engineered into baculovirus and the recombinant virus was used to infect Spodoptera frugiperda (H’S) cells. Approximately 3.3% of total protein of infected cells was human OR. The enzyme was purified by ion exchange and afRnity chromatography to a specific activity of 20 units/mg protein. Baculovirus-expressed OR displayed an absolute spectrum typical of the protein purified from tissue sources. The purified enzyme was able to support P450 activity in a reconstituted lipid vesicle system where maximal P460 activity was achieved at an OR/ P460 ratio of 2. When recombinant OR and P450 DNAcontaining baculoviruses were used to coinfect Sf3 cells, the OR/P460 ratio needed to achieve half maximal P460 catalytic activity was less than 0.6. These studies demonstrate the utility of baculovirus to analyze the functional and structural relationship of OR and P450. 0 1892 Academic Press, Inc.
NADPH-P450 oxidoreductase (OR)2 is an intrinsically membrane-bound enzyme responsible for transferring electrons from NADPH to various forms of cytochrome P450. It contains 1 mol each of noncovalently associated FMN and FAD (1) and is anchored to the endoplasmic reticulum lipid bilayer by a hydrophobic amino terminal ’ To whom correspondence should be addressed at Building 37, Room 3324, National Institutes of Health, Bethesda, MD 20892. Fax: (301) 496-8419. * Abbreviations used: OR, NADPH-P450 oxidoreductase; VCR, a recombinant baculovirus containing the human NADPH-P450 oxidoreductase cDNA, vCYPSA1, a recombinant baculovirus containing the CYP2Al cDNA, FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; SDS, sodium dodecyl sulfate. ooo3-9861/92
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction
in any form reserved.
tail (2). The human OR cDNA has been cloned, sequenced, and expressed using vaccinia virus (3). The OR cDNA has also been cloned from rat (4,5), rabbit (6), and yeast (7) and the rat cDNA was expressed using bacteria (8,9) and yeast (5). In the present report, we developed a recombinant baculovirus containing human OR cDNA. Expressed OR was characterized for its ability to donate electrons to P450. OR was efficiently expressed using baculovirus and had enzymatic characteristics similar to that purified from mammalian sources. MATERIALS
AND METHODS
Con.stru&on of recombinant baculovirus. Recombinant baculovirus was constructed as detailedearlier (10,ll). Briefly, the OR cDNA insert was removed from a pUC9 vector using EcoRI and made blunt ended with the Klenow fragment of DNA polymerase I. The insert was ligated to the transfer plasmid pAc373 that had been digested with BamHI and made blunt ended. The pAc373 plasmid containing the OR cDNA was cotransfected with baculovirus DNA onto Sf9 cells. The recombinant viruses were selected and purified by five rounds of plaque purification OR cDNA probe. The virus titer was deterusing a 32P-nick-translated mined as described previously (11). Recombinant virua was used to infect Spodoptera frugipenfo (Sf9) cells at a multiplicity of infection of 2 plaque forming units/cell. Infections were carried out either in spinner flasks or in 150-mm plastic dishes; the latter generally yielded twofold higher levels of expression. Purification of OR. In order to purify baculovirus-expressed OR, infections were carried out in spinner flasks. Seventy-two hours after infection, cells were washed twice with isotonic saline in sodium phosphate buffer (pH 7.2) and lysed by homogenization in the same buffer. The homogenate was centrifuged at 1500g for 15 min and the resultant supematant was centrifuged at 100,OOOgfor 60 min. All operations were performed at 4°C. The microsomal pellet was resuspended in 10 mM Tris/HCl (pH 7.4) containing 20% glycerol and 0.1 mM EDTA and proteins were made soluble by addition of Emulgen 913 to a final concentration of 1%. The solution was centrifuged at 100,OOOg and the supematant was used to purify OR according to the protocol of Yasukochi and Masters (12) using DEAE-Sepharose and 2’,5’-ADP-Sepharose chromatography. OR activities were monitored by reduction of cytochrome c at 25’C and expressed as 1 rmol of cytochrome c reduced/ min = 1 unit. The visible spectrum of purified OR was recorded using an Aminco DW-2000 spectrophotometer under aerobic conditions. The complete oxidation of OR was obtained by addition of sodium ferricy219
220
TAMURA TABLE Purification Protein (md Cell lysate 15OOg-100,OOOg fraction Soluble fraction DEAE column fraction ADP column fraction
ET AL. I
of Baculovirus-Expressed
OR
OR activities (units)
1986 700 312 57.8 5.1
anide (13). The contents of FMN and FAD in the purified OR were determined as described by Vermilion and Coon (14). SDS-polyacrylamide gel electrophoresis (15) and Western immunoblotting (16) were carried out as described earlier using rabbit anti-rat CYPPAl (17) or rabbit anti-rat OR serum (3). Immunoblots were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG. CYP2Al was purified from 3-methylcholanthrene-treated rate as previously described (17). Baculovirus-expressed purified OR was reconstituted with 40 pmol of purified rat liver P450 CYP2Al and 10 fig of dilauroylphosphatidylcholine and testosterone 7cy-hydroxylase activities were measured as described earlier (18). FMN, FAD, and cytochrome c were obtained from Sigma, and 2’,5’-ADP-Sepharose was purchased from Pharmacia.
RESULTS
Expression of NADPH-P450 oxidoreductase. The human NADPH-P450 oxidoreductase cDNA was inserted into baculovirus using the vector pAc373. Infection of St8 cells with the recombinant virus, designated vOR, resulted in the production of catalytically active enzyme. Bacu-
Specific activities (units/mg protein)
695 455 260 240 99.5
0.35 0.65 1.22 4.16 19.5
100 65 40 35 14
lovirus-expressed OR was purified over 50-fold to a specific catalytic activity of 19.5 units/mg protein by ionexchange and affinity chromatography (Table I). The purified preparation exhibited a major protein of relative M, of 78,000 with two minor polypeptides of smaller molecular weight as assessed by SDS-polyacrylamide gel electrophoresis (Fig. 1). These minor polypeptides also reacted with rabbit anti-rat OR serum in Western immunoblotting (data not shown). Western immunoblots analysis of total cell lysates, however, revealed that only a single protein corresponding to the major 78,000-Da band reacted with the anti-OR antiserum. Thus, the minor polypeptides are probably degradation products of OR produced during the course of purification. Spectral analysis of the purified OR revealed a mixture of semiquinone and oxidized forms (Fig. 2). By addition of ferricyanide, purified OR was fully oxidized and exhibited a visible spectrum typical of the flavoprotein purified from mammalian liver (12) with peaks at 380 and 455 nm. Using an extinction coefficient of 455 nm = 21.2 mM+
0.14
t
0.m I 350
4m
so0
450 wavelength
FIG. 1. SDS-polyacrylamide gel electrophoresis of total cell lysate protein and purified OR from Sf9 cells infected with vOR. The electrophoresis was carried out using precast ll-23% gels (PhorCast, Amersham). Molecular weight standards were 14,300, 18,400, 29,000,43,000 and 97,400 Da.
Yield (%o)
050
I 600
(nm)
FIG. 2. Visible spectrum of purified and oxidized OR. Spectrum A shows purified OR (0.58 mg protein/ml) and spectrum B was recorded after complete oxidation by the addition of 7 nmol/ml of ferricyanide (13). Spectra were recorded with an Aminco DW-2000 spectrophotometer under aerobic conditions.
BACULOVIRUS-EXPRESSED
0
1 .o
2.0
3.0
NADPH-P450
4.0
OR / P450
FIG. 3. Reconstituted testosterone 7a-hydroxylase activities as function of the ORfCYPPAl ratio. Reconstituted reactions contained 40 pmol of purified rat liver CYPBAI, various amounts of purified OR, 10 bg of dilauroylphosphatidylcholine and 250 nmol of testosterone. The level of 7o-hydroxytestosterone was measured using a Hewlet-Packard HPLC.
cm-’ for the oxidized form of OR (14), the specific content of the purified enzyme was calculated to be 10.4 nmol/ mg protein. The contents of FMN and FAD per nanomole of OR, as determined fluometrically, were 1.0 and 0.8 nmol, respectively. The activity of OR in cells infected with 2 pfu/cell of vOR in monolayer culture was 0.8 units/mg of cell lysate protein. Baculovirus-expressed OR represented about 3.3% of total cell protein as calculated from the data in Table 1 and Fig. 2. This is about 16-fold higher expression than that obtained from vaccinia virus (3) and 20- to 30fold higher than that obtained from yeast (5). Function& analysis of baculovirus-expressed OR. To determine whether expressed OR was able to support P450 oxidation activity, reconstitution studies were carried out. The OR was able to support CYP2Al-mediated testosterone ‘la-hydroxylase activity with maximal substrate turnover of 22 min-l achieved at an OR/P450 ratio of about 2 (Fig. 3). Functional expression of OR was also demonstrated by coinfection experiments in which vOR and vCYP2Al (10, 11) were both simultaneously introduced into St9 cells. A constant number of vCYP2Al was mixed with different amounts of vOR and used to infect the insect cells. The infected cells were incubated with Grace’s medium containing 20% fetal calf serum and 4 pg/ml of hemin as described previously (11). The levels of CYPBAl and OR produced with increasing amounts of vOR were determined by CO-reduced difference spectra and cytochrome c reduction, respectively (Fig. 4). Interestingly, as OR levels increased, CYP2Al decreased. Analysis of Western immunoblots revealed that the level of CYP2Al apopro-
221
OXIDOREDUCTASE
tein remained constant at increasing vOR/vCYPBAl ratios (Fig. 5). Specific contents of spectrally active P450 in the total lysates of coinfected cells using 1.5 pfu/cell of vCYP2Al and 0.1, 0.4, and 0.8 pfu/cell of wild type baculovirus were 0.50, 0.50, and 0.47 nmol/mg protein, respectively. These data suggestthat coinfection with vOR reduced the levels of spectrally active P450. Levels of testosterone 7a-hydroxylase activity were measured in total lysates of coinfected cells expressing different levels of OR and CYPBAl (Fig. 6). The maximal substrate turnover of 32 min-’ was obtained at an OR/ P450 ratio of about 2; however, half maximal activity was observed at a ratio of 0.3 in the in situ-reconstituted system.as compared to 0.9 in the in vitro-reconstituted system using the purified enzyme. Thus, in the in situreconstituted system, OR and P450 appear to be more tightly linked to each other than in the in vitroreconstituted system. This might be due to differing orientations of the two enzymes in the artificial lipid vesicles and the fact that only those on the outer surface of the vesicle can interact with NADPH and be active. Testosterone 7cy-hydroxylase activities were also measured in a mixture of lysates of cells which were separately infected with vOR and vCYP2Al. Turnover numbers for the substrate in the mixture having molar ratios of OR to P450 of 0, 0.1, 0.3, 0.6, 4.1, and 16 were 0.5, 0.6, 0.7, 0.9,2.4 and 1.1 min-‘, respectively. These turnover numbers were considerably lower than those derived from the lysates of coinfected cells. These data indicate that the data shown in Fig. 6 are not the result of mixing of lysate
^OZ ‘1
a 0 z m 0.1 % f
00 0
0.2
0.4 vOR added
0.6
0.8
1.0
(pfu I cell)
FIG. 4. CYP2Al holoenzyme (0) and OR contents (0) in cells coinfected with vCYP2Al and vOR. Cells were infected with 1.5 pfu/cell of vCYP2Al and the indicated pfu of vOR, and incubated for 72 h at 27°C. P450 contents were determined as described by Omura and Sato (19). OR content was determined by cytochrome c reduction using a value for specific enzymatic activity of purified OR of 1.87 units./nmol OR.
222
TAMURA
P vCYPPA1
(1.5 pfulcell)
1 E’ 5 Bhhp8eo E 3 0 0’ 0’ 0’ 0’ 0’ 0’ VOR (PfUhll)
-OR -CYP2A 1
FIG. 5.
Western immunoblotting of CYPPAl and OR in cells coinfected with 1.5 pfu/cell of vCYP2Al and various pfu of vOR. One microgram of total cell lysate protein was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. The filter was incubated with a mixture of rabbit anti-rat CYP2Al serum (1~500 dilution) and rabbit anti-rat OR serum (1:250 dilution). The blots were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG and alkaline phosphatase substrate reagent.
from cells infected separately with vOR and vCYP2Al but result from coinfection of the same cells. Thus, in situ reconstitution of OR and P450 proteins by coinfection is necessary to obtain high catalytic rates of testosterone oxidation. DISCUSSION
Baculovirus as a vector to express OR offers advantages over other systems previously used to produce this membrane-bound flavoenzyme. This system results in production of OR at a level of about 3.3% of total cellular protein under optimal conditions in which the cells were infected as monolayers on plastic dishes. OR could also be produced from cells at levels that are about 16- to 30fold greater than those produced by expression using vaccinia virus (3) or Succhuromyces cereuisiae (5). Bacteria have also been successfully used to produce catalytically active OR (8,9); however, for optimal expression a signal peptide and a spacer sequence which adds an additional 14 amino acids to the amino terminus are necessary. Sf9 cells are not anchorage dependent and can be cultured in both spinner flasks and plastic dishes. Suspension culture in spinner flasks is suitable for producing large amounts of OR protein for purification. For example, we could obtain up to 700 units of OR in 2.5 liters of suspension culture. Baculovirus-expressed OR should prove a valuable source of enzyme for reconstitution studies with P450.
ET AL.
Monolayer cultures can be especially valuable for small scale studies such as titration of OR and P450 expression in coinfection experiments. This system produced high level expression of P450 and OR in cultures on ISO-mm dishes. Coinfection studies revealed that the in situ reconstitution system provided more efficient environments for electron transport than did the in vitro reconstitution system using purified enzymes. By means of this in situ system or even the in vitro-reconstituted system, site-directed mutagenesis studies on OR can be carried out to determine those amino acids responsible for its interaction with different forms of P450. In the coinfection study, OR levels increased concurrent with decreasesin spectrally active P450 levels, while P450 apoprotein levels remained constant. In earlier coinfection studies using the vaccinia virus system, P45Oprotein levels decreased as OR levels increased. This difference may result from the higher expression capacity of the baculovirus system as compared to the vaccinia system. The decrease of spectrally active P450 could be due either to lower incorporation of heme into P450 apoprotein or a destabilization of P450 holoprotein and loss of heme. Spectrally active P450 levels did not decrease by coinfection with wild type baculovirus, suggesting that expressed OR protein directly affected thelevels of P450 holoenzyme. Interestingly, OR is involved in heme degradation by providing electrons to the enzyme heme oxygenase (20). Thus, OR may decrease heme incorporation into P450 apoprotein by accelerating the breakdown of exogenously added hemin. However, supplying fresh hemin by frequent medium changes did not affect the levels of P450 holoenzyme. Another possibility for loss of P450 may be that OR initiates microsomal lipid peroxidation (21) which then results in degradation of P450. Indeed, the optical density at 420 nm increased as P450 levels decreased. It
2.0
1.0
3.0
OR I P450 FIG. 6. In situ reconstitution of CYP2Al and OR using baculovirus. Cellular contents of each enzyme were determined as described in the legend to Fig. 4. Testosterone ‘la-hydroxylase activities in the total lysates of coinfected cells were measured as described earlier (18).
BACULOVIRUS-EXPRESSED
NADPH-P450
OXIDOREDUCTASE
223
is also possible that coinfection with vOR might alter the intracellular distribution of P450 protein, leading to the decreased levels of P450 holoprotein. In any case, the mechanisms of P450 decrease require further investigation.
10. Summers,
REFERENCES
13. Vermilion, 2704.
J. L., and Coon, M. J. (1978) J. Biol. C&m. 253,2694-
14. Vermilion, 8819.
J. L., and Coon, M. J. (1978) J. Biol. Chem. 263,8812-
1. Iyanagi, T., and Mason, H. S. (1973) Biochemistry 12,2297-2308. 2. Black, S. P., and Coon, M. J. (1982) J. Biol. Chem. 257,5929-5938. 3. Yamano S., Aoyama, T., McBride, 0. W., Hardwick, J. P. Gelboin, H. V., and Gonzalez, F. J. (1989) Mol. Phurmacol. 35,83-88. 4. Porter, T. D., and Kasper, C. B. (1985) Proc. Natl. Acad. Sci. USA 82,973-977. 5. Murakami, H., Yabusaki, Y., and Ohkawa, H. (1986) DNA 5,1-10. 6. Katagiri, M., Murakami, Y., Yabusaki, Y., Sugiyama, T., Okamoto M. Yamano, T., and Ohkawa, H. (1986) J. B&hem. 100,945-954. Yabusaki, Y., Murakami, H., and Ohkawa, H. (1988) J. Biochem. 103,1004-1010. Porter, T. D., Wilson, T. E., and Kasper, C. B. (1987) Arch. B&hem. Biophys. 264, 353-367. Shen, A. L., Porter, T. D., Wilson, T. E., and Kasper, C. B. (1989) J. Biol. Chem. 264,7584-7589.
M. D., and Smith, D. E. (1987) Tex. Agric. Ezp. Stn. Bull.
B-155.
11, Gonzalez,
F. J., Kimura, S., Tamura, S., and Gelboin, H. V. (1991) in Methods in Enzymology 206,93-99. 12. Yasukochi, Y., and Masters, B. S. S. (1976) J. Biol. Chem. 257, 2702-2707.
15. Laemmli,
U. K. (1970) Nature 227, 680-685.
16. Towbin, H., Staelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76,4350-4355. 17. Nagata, K., Mataunaga, T., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1987) J. Bial. Chem. 262, 2787-2793. 18. Asseffa, A., Smith, S. J., Nagata, K., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1989) Arch. Biochem. Biophys. 274,481-490. 19. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2379-2385. 20. Schacter, B. A., Nelson, E. B., Marver, H. S., and Masters, B. S. S. (1972) J. Biol. Chem. 247, 3601-3607. 21. Pederson, T. C., and Aust, S. D. (1975) Biochem. Biophys. Acta. 385,232-241.
OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 293, No. 2, March, pp. 219-223, 1992
Baculovirus-Mediated Expression and Functional Characterization of Human NADPH-P450 Oxidoreductase Shinji Tamura, Kenneth R. Korzekwa, Shioko Kimura, Harry V. Gelboin, and Frank J. Gonzalez? Laboratory
of Molecular
Carcinogenesis, National
Cancer Institute,
National
Institutes
of Health, Bethesda, Maryland
20892
Received July 11, 1991, and in revised form October 25, 1991
Human NADPH-PI60 oxidoreductase (OR) is an intrinsically membrane-bound flavoprotein that serves to transfer electrons from NADPH to cytochrome P460. OR is also involved in the metabolic activation of chemotherapeutic alhylating agents. The human OR cDNA was engineered into baculovirus and the recombinant virus was used to infect Spodoptera frugiperda (H’S) cells. Approximately 3.3% of total protein of infected cells was human OR. The enzyme was purified by ion exchange and afRnity chromatography to a specific activity of 20 units/mg protein. Baculovirus-expressed OR displayed an absolute spectrum typical of the protein purified from tissue sources. The purified enzyme was able to support P450 activity in a reconstituted lipid vesicle system where maximal P460 activity was achieved at an OR/ P460 ratio of 2. When recombinant OR and P450 DNAcontaining baculoviruses were used to coinfect Sf3 cells, the OR/P460 ratio needed to achieve half maximal P460 catalytic activity was less than 0.6. These studies demonstrate the utility of baculovirus to analyze the functional and structural relationship of OR and P450. 0 1892 Academic Press, Inc.
NADPH-P450 oxidoreductase (OR)2 is an intrinsically membrane-bound enzyme responsible for transferring electrons from NADPH to various forms of cytochrome P450. It contains 1 mol each of noncovalently associated FMN and FAD (1) and is anchored to the endoplasmic reticulum lipid bilayer by a hydrophobic amino terminal ’ To whom correspondence should be addressed at Building 37, Room 3324, National Institutes of Health, Bethesda, MD 20892. Fax: (301) 496-8419. * Abbreviations used: OR, NADPH-P450 oxidoreductase; VCR, a recombinant baculovirus containing the human NADPH-P450 oxidoreductase cDNA, vCYPSA1, a recombinant baculovirus containing the CYP2Al cDNA, FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; SDS, sodium dodecyl sulfate. ooo3-9861/92
$3.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction
in any form reserved.
tail (2). The human OR cDNA has been cloned, sequenced, and expressed using vaccinia virus (3). The OR cDNA has also been cloned from rat (4,5), rabbit (6), and yeast (7) and the rat cDNA was expressed using bacteria (8,9) and yeast (5). In the present report, we developed a recombinant baculovirus containing human OR cDNA. Expressed OR was characterized for its ability to donate electrons to P450. OR was efficiently expressed using baculovirus and had enzymatic characteristics similar to that purified from mammalian sources. MATERIALS
AND METHODS
Con.stru&on of recombinant baculovirus. Recombinant baculovirus was constructed as detailedearlier (10,ll). Briefly, the OR cDNA insert was removed from a pUC9 vector using EcoRI and made blunt ended with the Klenow fragment of DNA polymerase I. The insert was ligated to the transfer plasmid pAc373 that had been digested with BamHI and made blunt ended. The pAc373 plasmid containing the OR cDNA was cotransfected with baculovirus DNA onto Sf9 cells. The recombinant viruses were selected and purified by five rounds of plaque purification OR cDNA probe. The virus titer was deterusing a 32P-nick-translated mined as described previously (11). Recombinant virua was used to infect Spodoptera frugipenfo (Sf9) cells at a multiplicity of infection of 2 plaque forming units/cell. Infections were carried out either in spinner flasks or in 150-mm plastic dishes; the latter generally yielded twofold higher levels of expression. Purification of OR. In order to purify baculovirus-expressed OR, infections were carried out in spinner flasks. Seventy-two hours after infection, cells were washed twice with isotonic saline in sodium phosphate buffer (pH 7.2) and lysed by homogenization in the same buffer. The homogenate was centrifuged at 1500g for 15 min and the resultant supematant was centrifuged at 100,OOOgfor 60 min. All operations were performed at 4°C. The microsomal pellet was resuspended in 10 mM Tris/HCl (pH 7.4) containing 20% glycerol and 0.1 mM EDTA and proteins were made soluble by addition of Emulgen 913 to a final concentration of 1%. The solution was centrifuged at 100,OOOg and the supematant was used to purify OR according to the protocol of Yasukochi and Masters (12) using DEAE-Sepharose and 2’,5’-ADP-Sepharose chromatography. OR activities were monitored by reduction of cytochrome c at 25’C and expressed as 1 rmol of cytochrome c reduced/ min = 1 unit. The visible spectrum of purified OR was recorded using an Aminco DW-2000 spectrophotometer under aerobic conditions. The complete oxidation of OR was obtained by addition of sodium ferricy219
220
TAMURA TABLE Purification Protein (md Cell lysate 15OOg-100,OOOg fraction Soluble fraction DEAE column fraction ADP column fraction
ET AL. I
of Baculovirus-Expressed
OR
OR activities (units)
1986 700 312 57.8 5.1
anide (13). The contents of FMN and FAD in the purified OR were determined as described by Vermilion and Coon (14). SDS-polyacrylamide gel electrophoresis (15) and Western immunoblotting (16) were carried out as described earlier using rabbit anti-rat CYPPAl (17) or rabbit anti-rat OR serum (3). Immunoblots were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG. CYP2Al was purified from 3-methylcholanthrene-treated rate as previously described (17). Baculovirus-expressed purified OR was reconstituted with 40 pmol of purified rat liver P450 CYP2Al and 10 fig of dilauroylphosphatidylcholine and testosterone 7cy-hydroxylase activities were measured as described earlier (18). FMN, FAD, and cytochrome c were obtained from Sigma, and 2’,5’-ADP-Sepharose was purchased from Pharmacia.
RESULTS
Expression of NADPH-P450 oxidoreductase. The human NADPH-P450 oxidoreductase cDNA was inserted into baculovirus using the vector pAc373. Infection of St8 cells with the recombinant virus, designated vOR, resulted in the production of catalytically active enzyme. Bacu-
Specific activities (units/mg protein)
695 455 260 240 99.5
0.35 0.65 1.22 4.16 19.5
100 65 40 35 14
lovirus-expressed OR was purified over 50-fold to a specific catalytic activity of 19.5 units/mg protein by ionexchange and affinity chromatography (Table I). The purified preparation exhibited a major protein of relative M, of 78,000 with two minor polypeptides of smaller molecular weight as assessed by SDS-polyacrylamide gel electrophoresis (Fig. 1). These minor polypeptides also reacted with rabbit anti-rat OR serum in Western immunoblotting (data not shown). Western immunoblots analysis of total cell lysates, however, revealed that only a single protein corresponding to the major 78,000-Da band reacted with the anti-OR antiserum. Thus, the minor polypeptides are probably degradation products of OR produced during the course of purification. Spectral analysis of the purified OR revealed a mixture of semiquinone and oxidized forms (Fig. 2). By addition of ferricyanide, purified OR was fully oxidized and exhibited a visible spectrum typical of the flavoprotein purified from mammalian liver (12) with peaks at 380 and 455 nm. Using an extinction coefficient of 455 nm = 21.2 mM+
0.14
t
0.m I 350
4m
so0
450 wavelength
FIG. 1. SDS-polyacrylamide gel electrophoresis of total cell lysate protein and purified OR from Sf9 cells infected with vOR. The electrophoresis was carried out using precast ll-23% gels (PhorCast, Amersham). Molecular weight standards were 14,300, 18,400, 29,000,43,000 and 97,400 Da.
Yield (%o)
050
I 600
(nm)
FIG. 2. Visible spectrum of purified and oxidized OR. Spectrum A shows purified OR (0.58 mg protein/ml) and spectrum B was recorded after complete oxidation by the addition of 7 nmol/ml of ferricyanide (13). Spectra were recorded with an Aminco DW-2000 spectrophotometer under aerobic conditions.
BACULOVIRUS-EXPRESSED
0
1 .o
2.0
3.0
NADPH-P450
4.0
OR / P450
FIG. 3. Reconstituted testosterone 7a-hydroxylase activities as function of the ORfCYPPAl ratio. Reconstituted reactions contained 40 pmol of purified rat liver CYPBAI, various amounts of purified OR, 10 bg of dilauroylphosphatidylcholine and 250 nmol of testosterone. The level of 7o-hydroxytestosterone was measured using a Hewlet-Packard HPLC.
cm-’ for the oxidized form of OR (14), the specific content of the purified enzyme was calculated to be 10.4 nmol/ mg protein. The contents of FMN and FAD per nanomole of OR, as determined fluometrically, were 1.0 and 0.8 nmol, respectively. The activity of OR in cells infected with 2 pfu/cell of vOR in monolayer culture was 0.8 units/mg of cell lysate protein. Baculovirus-expressed OR represented about 3.3% of total cell protein as calculated from the data in Table 1 and Fig. 2. This is about 16-fold higher expression than that obtained from vaccinia virus (3) and 20- to 30fold higher than that obtained from yeast (5). Function& analysis of baculovirus-expressed OR. To determine whether expressed OR was able to support P450 oxidation activity, reconstitution studies were carried out. The OR was able to support CYP2Al-mediated testosterone ‘la-hydroxylase activity with maximal substrate turnover of 22 min-l achieved at an OR/P450 ratio of about 2 (Fig. 3). Functional expression of OR was also demonstrated by coinfection experiments in which vOR and vCYP2Al (10, 11) were both simultaneously introduced into St9 cells. A constant number of vCYP2Al was mixed with different amounts of vOR and used to infect the insect cells. The infected cells were incubated with Grace’s medium containing 20% fetal calf serum and 4 pg/ml of hemin as described previously (11). The levels of CYPBAl and OR produced with increasing amounts of vOR were determined by CO-reduced difference spectra and cytochrome c reduction, respectively (Fig. 4). Interestingly, as OR levels increased, CYP2Al decreased. Analysis of Western immunoblots revealed that the level of CYP2Al apopro-
221
OXIDOREDUCTASE
tein remained constant at increasing vOR/vCYPBAl ratios (Fig. 5). Specific contents of spectrally active P450 in the total lysates of coinfected cells using 1.5 pfu/cell of vCYP2Al and 0.1, 0.4, and 0.8 pfu/cell of wild type baculovirus were 0.50, 0.50, and 0.47 nmol/mg protein, respectively. These data suggestthat coinfection with vOR reduced the levels of spectrally active P450. Levels of testosterone 7a-hydroxylase activity were measured in total lysates of coinfected cells expressing different levels of OR and CYPBAl (Fig. 6). The maximal substrate turnover of 32 min-’ was obtained at an OR/ P450 ratio of about 2; however, half maximal activity was observed at a ratio of 0.3 in the in situ-reconstituted system.as compared to 0.9 in the in vitro-reconstituted system using the purified enzyme. Thus, in the in situreconstituted system, OR and P450 appear to be more tightly linked to each other than in the in vitroreconstituted system. This might be due to differing orientations of the two enzymes in the artificial lipid vesicles and the fact that only those on the outer surface of the vesicle can interact with NADPH and be active. Testosterone 7cy-hydroxylase activities were also measured in a mixture of lysates of cells which were separately infected with vOR and vCYP2Al. Turnover numbers for the substrate in the mixture having molar ratios of OR to P450 of 0, 0.1, 0.3, 0.6, 4.1, and 16 were 0.5, 0.6, 0.7, 0.9,2.4 and 1.1 min-‘, respectively. These turnover numbers were considerably lower than those derived from the lysates of coinfected cells. These data indicate that the data shown in Fig. 6 are not the result of mixing of lysate
^OZ ‘1
a 0 z m 0.1 % f
00 0
0.2
0.4 vOR added
0.6
0.8
1.0
(pfu I cell)
FIG. 4. CYP2Al holoenzyme (0) and OR contents (0) in cells coinfected with vCYP2Al and vOR. Cells were infected with 1.5 pfu/cell of vCYP2Al and the indicated pfu of vOR, and incubated for 72 h at 27°C. P450 contents were determined as described by Omura and Sato (19). OR content was determined by cytochrome c reduction using a value for specific enzymatic activity of purified OR of 1.87 units./nmol OR.
222
TAMURA
P vCYPPA1
(1.5 pfulcell)
1 E’ 5 Bhhp8eo E 3 0 0’ 0’ 0’ 0’ 0’ 0’ VOR (PfUhll)
-OR -CYP2A 1
FIG. 5.
Western immunoblotting of CYPPAl and OR in cells coinfected with 1.5 pfu/cell of vCYP2Al and various pfu of vOR. One microgram of total cell lysate protein was subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. The filter was incubated with a mixture of rabbit anti-rat CYP2Al serum (1~500 dilution) and rabbit anti-rat OR serum (1:250 dilution). The blots were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG and alkaline phosphatase substrate reagent.
from cells infected separately with vOR and vCYP2Al but result from coinfection of the same cells. Thus, in situ reconstitution of OR and P450 proteins by coinfection is necessary to obtain high catalytic rates of testosterone oxidation. DISCUSSION
Baculovirus as a vector to express OR offers advantages over other systems previously used to produce this membrane-bound flavoenzyme. This system results in production of OR at a level of about 3.3% of total cellular protein under optimal conditions in which the cells were infected as monolayers on plastic dishes. OR could also be produced from cells at levels that are about 16- to 30fold greater than those produced by expression using vaccinia virus (3) or Succhuromyces cereuisiae (5). Bacteria have also been successfully used to produce catalytically active OR (8,9); however, for optimal expression a signal peptide and a spacer sequence which adds an additional 14 amino acids to the amino terminus are necessary. Sf9 cells are not anchorage dependent and can be cultured in both spinner flasks and plastic dishes. Suspension culture in spinner flasks is suitable for producing large amounts of OR protein for purification. For example, we could obtain up to 700 units of OR in 2.5 liters of suspension culture. Baculovirus-expressed OR should prove a valuable source of enzyme for reconstitution studies with P450.
ET AL.
Monolayer cultures can be especially valuable for small scale studies such as titration of OR and P450 expression in coinfection experiments. This system produced high level expression of P450 and OR in cultures on ISO-mm dishes. Coinfection studies revealed that the in situ reconstitution system provided more efficient environments for electron transport than did the in vitro reconstitution system using purified enzymes. By means of this in situ system or even the in vitro-reconstituted system, site-directed mutagenesis studies on OR can be carried out to determine those amino acids responsible for its interaction with different forms of P450. In the coinfection study, OR levels increased concurrent with decreasesin spectrally active P450 levels, while P450 apoprotein levels remained constant. In earlier coinfection studies using the vaccinia virus system, P45Oprotein levels decreased as OR levels increased. This difference may result from the higher expression capacity of the baculovirus system as compared to the vaccinia system. The decrease of spectrally active P450 could be due either to lower incorporation of heme into P450 apoprotein or a destabilization of P450 holoprotein and loss of heme. Spectrally active P450 levels did not decrease by coinfection with wild type baculovirus, suggesting that expressed OR protein directly affected thelevels of P450 holoenzyme. Interestingly, OR is involved in heme degradation by providing electrons to the enzyme heme oxygenase (20). Thus, OR may decrease heme incorporation into P450 apoprotein by accelerating the breakdown of exogenously added hemin. However, supplying fresh hemin by frequent medium changes did not affect the levels of P450 holoenzyme. Another possibility for loss of P450 may be that OR initiates microsomal lipid peroxidation (21) which then results in degradation of P450. Indeed, the optical density at 420 nm increased as P450 levels decreased. It
2.0
1.0
3.0
OR I P450 FIG. 6. In situ reconstitution of CYP2Al and OR using baculovirus. Cellular contents of each enzyme were determined as described in the legend to Fig. 4. Testosterone ‘la-hydroxylase activities in the total lysates of coinfected cells were measured as described earlier (18).
BACULOVIRUS-EXPRESSED
NADPH-P450
OXIDOREDUCTASE
223
is also possible that coinfection with vOR might alter the intracellular distribution of P450 protein, leading to the decreased levels of P450 holoprotein. In any case, the mechanisms of P450 decrease require further investigation.
10. Summers,
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