- Email: [email protected]
Partial purification of cytochromes P-450 and P-448 from rat liver microsomes
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Partial P u r i f i c a t i o n of Cytochromes P-450 and P-448 from Rat Liver Microsomes Anthony Y. H. Lu and Wayne Levin Department of Biochemistry and Drug Metabolism Hoffmann-La Roche Inc., Nutley, New Jersey 07110
Received D e c e m b e r 30, 1971
SUMMARY: Cytochromes P-450 (from rats treated with phenobarbital) and P-448 (from rats treated with 3-methylcholanthrene) were solubilized and p a r t i a l l y purified from rat l i v e r microsomes. The f i n a l preparation had a specific a c t i v i t y (nmoles per mg protein) of 5.5 for cytochrome P-450 and 5.3 for cytochrome P-448. The p a r t i a l l y purified cytochromes P-450 and P-448 exhibited d i f f e r e n t substrate s p e c i f i c i t i e s for benzphetamine and 3,4-benzpyrene.
Numerous attempts have been made in the past decade to s o l u b i l i z e and purify l i v e r microsomal cytochrome P-450 ( I ) , the terminal oxidase of the mixed-function oxidase system which metabolize~ a variety of drugs, steroids, f a t t y acids and other foreign compounds (2,3).
Earlier attempts to s o l u b i l i z e
cytochrome P-450 resulted in the extensive formation of cytochrome P-420 (1,4). In recent years, l i v e r microsomal cytochrome P-450 has been solubilized and p a r t i a l l y purified (5-9), but most of these preparations do not catalyze the hydroxylation of substrates. P-450 preparations which s t i l l
To our knowledge, the only solubilized cytochrome retain c a t a l y t i c a c t i v i t y are those of Lu and
Coon (7), and of Fujita et al (8).
The former preparation was capable of
hydroxylating a variety of substrates such as f a t t y acids, alkanes and drugs (10-12), while the l a t t e r was only active in aniline hydroxylation.
Recently
we have b r i e f l y described a procedure for the s o l u b i l i z a t i o n of cytochromes P-450 from rats treated with phenobarbital (PB), and P-448 from rats treated with 3-methylcholanthrene (3-MC) (13).
These preparations have been shown
to be active in the hydroxylation of a number of drugs and steroids (14-16). Studies also indicate that l i t t l e ,
i f any, cytochrome P-448 is present in 1334
Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PB-treated rats and cytochrome P-450 in 3-MC-treated rats (16,17).
In the
present paper, the partial p u r i f i c a t i o n of cytochrome P-450 and P-448 is described.
METHODS: Microsomes:
Immature male Long-Evans rat- (50-55 gm) were treated
with PB or 3-MC as previously described (18).
Liver microsomes were prepared
in O.05M T r i s , pH 7.4, containing 1.15% KCI, washed with 1.15% KCl containing I0 mM EDTA, suspended in 0.25 M sucrose to a final protein concentration of 30-40 mg/ml and stored under N2 for 2-3 days prior to use. Solubilization ~nd F i r s t Ammonium Sulfate Fractionation: Cytochromes P-450 (from rats treated with PB) and P-448 (from rats treated with 3-MC) were solubilized from l i v e r microsomes by a s l i g h t modification of the method described previously (13).
In a typical preparation, each 800-1000 mg of
microsomal protein was diluted to 61 ml in a mixture containing 14 ml of glycerol, 7 ml of IM potassium phosphate (pH 7.7), 0.7 ml of O.IM d i t h i o t h r e i t o l (DTT), 0.7 ml of O.IM EDTA and 0.25 M sucrose and each mixture was sonicated as previously described (13).
Eight to ten ml of 10% sodium cholate was added
bringing the final concentration to 1 mg cholate per mg protein.
Each mix-
ture was s t i r r e d in ice for 20 min and centrifuted at 105,000 x g for 1 hour. After centrifugation, fractionation.
the individual preparations were pooled f o r further
Solid ammonium sulfate was added to the pooled supernatant
fraction to 40% saturation and then to 50% saturation.
The resulting pre-
c i p i t a t e from 40-50% saturation was dissolved in O.05M potassium phosphate buffer, pH 7.7, and centrifuged at 160,000 x g for 1 hour to remove insoluble material.
The supernatant fraction was dialyzed overnight
against O.02M
potassium phosphate buffer, pH 7.7, containing 20% glycerol, 10 -4 M DTT, 10"4 M EDTA and 0,1% cholate and centrifuged at 35~000 x g for 20 min. Second Ammonium Sulfate Fractionation:
The dialyzed sample was d i l u t -
ed to 3-4 mg protein/ml with O.02M potassium phosphate buffer, pH 7.7, contain-
1335
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ing 20% glycerol, lO-4 M DTT, lO"4 M EDTA and 0.2% cholate.
Solid ammonium
sulfate was added to 43% saturation and then to 50% saturation.
The resulting
precipitate from 43-50% saturation was dissolved in O.O05M potassium phosphate buffer, pH 7.7, containing 20% glycerol, lO "4 M DTT, I0-4 M EDTA and 0.1% cholate, dialyzed overnight against the same buffer mixture, and centrifuged at 35,000 x g for 20 min to remove any insoluble material. Calcium phosphate Gel Fractionation:
The dialyzed sample was diluted
to a protein concentration of 8 mg per ml with the dialyzing buffer.
Calcium
phosphate gel (Bio-Rad Laboratories) was added to a gel to protein ratio of 3. The gel was extracted twice with O.l M potassium phosphate, buffer pH 7.7, and then with 0.25 M potassium phosphate buffer, pH 7.7.
The 0.25 M phosphate buffer ex-
tract was concentrated to a protein concentration of approximately lO mg per ml by membrane u l t r a f i l t r a t i o n using a Diaflo XM-IOOAmembrane, dialyzed against O.Ol M potassium phosphate, pH 7.7, containing 20% glycerol, lO-4M DTT, lO -4 M EDTA and 0.05% cholate, and centrifuged.
The supernatant fraction was stored at
-20 ° under N2. RESULTS: The steps in the purification procedure for cytochromes P-450 and P-448 are summarized in Table I.
The final preparation resulted
in a 2.5 and 3.3-fold purification for cytochromes P-450 and P-448 respectively.
The final specific a c t i v i t y of cytochromes P-450 and P-448 (5.3-5.5
nmoles/mg protein) in this study is s i g n i f i c a n t l y higher than has been reported previously using rat l i v e r microsomes (8).
While the preparations
after ammonium sulfate fractionation remained soluble, as judged by centrifugation at 160,000 x g for 60 min, the final preparation sedimented upon high speed centrifugation.
However, the red transparent pellet would readily redissolve
in the centrifuge tube upon standing in the cold.
The cause of this phenomenon
is not known at the present time. We have recently reported that the solubilized cytochrome P-450 and P-448 fractions exhibit different specif ici t y with respect to a number of substrates (13-16).
Similar results have now been obtained with these purified 1336
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 Purification of Cytochromes P-450 and P-448 from Rat Liver Microsomes
Volume (ml)
Protein (g)
247
7.41
16.2
2.2
I00
I s t Ammonium sulfate (40-50%)
65
1.08
3.5
3.2
22
2nd Ammonium sulfate (43-50%)
32
0.29
1.2
4.1
8
Calcium phosphate gel
12
0.II
0.6
5.5
4
315
8.01
12.7
1.6
I00
I s t Ammonium sulfate (40-50%)
82
1.20
3.6
3.0
28
2nd Ammonium sulfate (43-50%)
37
0.39
1.6
4.1
13
Calcium phosphate gel
14
0.15
0.8
5.3
6
Fraction
P-450 or P-448 (pmoles)
Specific A c t i v i t y (nmoles P-450 or P-448/mgprotein)
Yield (%)
A) Cytochrome P-450 from PB-treated rats Microsomes
B) Cytochrome P-448 from 3-MC-treated rats Microsomes
Cytochromes P-450 and P-448 were determined by the method of Omura and Sato (I) using an extinction coefficient of 91 mM-I cm-I for A450_490. Protein was determined by the method of Lowry et al (19).
cytochrome P-450 and P-448 fractions assayed in the presence of the reductase and l i p i d fractions.
Thus the cytochrome P-450 fraction was active for benz-
phetamine N-demethylation, but rather inactive for 3,4-benzpyrene hydroxylation whereas the cytochrome P-448 fraction showed high 3,4-benzpyrene hydroxylase a c t i v i t y but low benzphetamine N-demethylase a c t i v i t y .
These results are con-
sistent with the view that cytochrome P-450 is c a t a l y t i c a l l y d i f f e r e n t from cytochrome P-448.
1337
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
i
I
..
i
I
/
i
I
i
\
/ /
',
0,4
/
x
i
i
0.5 z
g g <
/ i
0.2 /
/
0.1
U I 420
I
I 440
t
WAVELENGTH
Figure I :
I 460
I
A 480
(m~)
Carbon monoxide difference spectra of p a r t i a l l y
p u r i f i e d cytochromes P-450 ( s o l i d l i n e , 0.46 mg protein/ml) and P-448 (dashed l i n e , 0.52 mg protein/ml).
The reduced CO-difference spectra of the p u r i f i e d cytochromes P-450 and P-448 is shown in Figure I .
I t can be seen that the f i n a l preparations contain-
ed i n s i g n i f i c a n t amounts (less than 5%) of cytochrome P-420.
The heme con-
centration of the f i n a l preparations was determined by the pyridine hemochromogen method ( I ) to be 5.48 and 4.65 nmoles per mg protein for cytochromes P-450 and P-448 respectively.
Thus, the e x t i n c t i o n c o e f f i c i e n t f o r A450_490 or A448_490
of the reduced hemoprotein CO complex was calculated as 91 mM-I cm-I f o r cytochrome P-450 and 103 mM-I cm-I for cytochrome P-448.
These values are in close
agreement with those reported by Omura and Sato ( I ) , and Greene et al (20) using microsomal suspensions, but d i f f e r e n t from those reported by Fujita et al (8) for t h e i r s o l u b i l i z e d preparation of cytochromes P-450 (58 mM-I cm- I ) and P-448 (78 mM-I cm-l). 1338
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Fujita et al (8) have reported that their solubilized cytochrome P-448 (referred to as cytochrome PI-450) did not give a type I difference spectra and therefore suggested that cytochrome P-448 may not have a type I binding site.
In our preliminary studies, however, both the purified cytochromes P-450
and P-448 gave a typical type I spectra with benzphetamine. Thus, i t appears that our cytochrome P-450 and P-448 preparations are not only cataytically active in hydroxylation, but also retain the properties of the native microsomal bound forms. Further studies on the physical, spectral and catalytic properties of the purified cytochrome P-450 and P-448 preparations are now in progress in our laboratory. ACKNOWLEDGMENTS: We are grateful to Drs. R.Kuntzman and A. H. Conney for their helpful criticism in the course of the parparation of this manuscript and to S. West and B. Ryan for their able technical assistance.
We also wish
to thank Mrs. L. Rubino for preparing the manuscript. References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Omura, T., and Sato, R., J. Biol. Chem. 239, 2370 (1964). Conney, A. H., Pharmacol. Rev. 19, 317 ( ~ 7 ) . Kuntzman, R., Ann. Rev. PharmacoT. 9, 21 (1969). Orrenius, S., J. Cell Biol. 26, 713-(1965). MacLennan, D. H., Tzagoloff, A., and McConnel, D. G., Biochim. Biophys. Acta 131, 59 (1967). Miyaka, Y., Gaylor, J. L., and Mason, H. S., J. Biol. Chem. 243, 5788 (1968). Lu, A. Y. H., and Coon, M. J., J. Biol. Chem. 243, 1331 (1968). Fujita, T., and Mannering, G. J., Chem.-Biol. Interactions 3, 264 (1971). Mitani, F., Alvares, A. P., Sassa, S., and Kappas, Ao, Mol. Pharmacol. 7, 280 (1971). Lu, A. Y. H., Junk, K. W., and Coon, M. J., J. Biol. Chem. 244, 3714 (1969). Lu, A. Y. H., Strobel, H. W., and Coon, M. J., Biochem. B i o ~ s . Res. Comm. 36, 545 (1969). Lu, A. Y. H., Strobel, H. W., and Coon, M. J . , M o l . Pharmacol. 6, 213 (1970). Lu, A. Y. H., Kuntzman, R., West, S., and Conney, A. H., Biochem. Biophys. Res. Comm. 42, 1200 (1971). Kuntzman, R., Lu, A. Y. H., West, S., Jacobson, M., and Conney, A. H., ChemBiol. Interactions 3, 264 (1971). Lu, A. Y. H., Kuntzman, R., West, S., Jacobson, M., and Conney, A. H., Pharmacologist L3, 222 (1971). Lu, A. Y. H., Kuntzman, R., West, S., Jacobson, M., and Conney, A. H., J. Biol. Chem. (1972), in press. Sladek, N. E., and Mannering, G. J., Mol. Pharmacol. 5, 186 (1969). Levin, W., Alvares, A. Jacobson, M., and Kuntzman, R., Biochem. Pharmacol. 18, 883 (1969). Lowry, O. H., Rosenbough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Greene, F. E., Stripp, B., and G i l l e t t e , J. R., Pharmacology 5, 43 (1971). 1339
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Partial P u r i f i c a t i o n of Cytochromes P-450 and P-448 from Rat Liver Microsomes Anthony Y. H. Lu and Wayne Levin Department of Biochemistry and Drug Metabolism Hoffmann-La Roche Inc., Nutley, New Jersey 07110
Received D e c e m b e r 30, 1971
SUMMARY: Cytochromes P-450 (from rats treated with phenobarbital) and P-448 (from rats treated with 3-methylcholanthrene) were solubilized and p a r t i a l l y purified from rat l i v e r microsomes. The f i n a l preparation had a specific a c t i v i t y (nmoles per mg protein) of 5.5 for cytochrome P-450 and 5.3 for cytochrome P-448. The p a r t i a l l y purified cytochromes P-450 and P-448 exhibited d i f f e r e n t substrate s p e c i f i c i t i e s for benzphetamine and 3,4-benzpyrene.
Numerous attempts have been made in the past decade to s o l u b i l i z e and purify l i v e r microsomal cytochrome P-450 ( I ) , the terminal oxidase of the mixed-function oxidase system which metabolize~ a variety of drugs, steroids, f a t t y acids and other foreign compounds (2,3).
Earlier attempts to s o l u b i l i z e
cytochrome P-450 resulted in the extensive formation of cytochrome P-420 (1,4). In recent years, l i v e r microsomal cytochrome P-450 has been solubilized and p a r t i a l l y purified (5-9), but most of these preparations do not catalyze the hydroxylation of substrates. P-450 preparations which s t i l l
To our knowledge, the only solubilized cytochrome retain c a t a l y t i c a c t i v i t y are those of Lu and
Coon (7), and of Fujita et al (8).
The former preparation was capable of
hydroxylating a variety of substrates such as f a t t y acids, alkanes and drugs (10-12), while the l a t t e r was only active in aniline hydroxylation.
Recently
we have b r i e f l y described a procedure for the s o l u b i l i z a t i o n of cytochromes P-450 from rats treated with phenobarbital (PB), and P-448 from rats treated with 3-methylcholanthrene (3-MC) (13).
These preparations have been shown
to be active in the hydroxylation of a number of drugs and steroids (14-16). Studies also indicate that l i t t l e ,
i f any, cytochrome P-448 is present in 1334
Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PB-treated rats and cytochrome P-450 in 3-MC-treated rats (16,17).
In the
present paper, the partial p u r i f i c a t i o n of cytochrome P-450 and P-448 is described.
METHODS: Microsomes:
Immature male Long-Evans rat- (50-55 gm) were treated
with PB or 3-MC as previously described (18).
Liver microsomes were prepared
in O.05M T r i s , pH 7.4, containing 1.15% KCI, washed with 1.15% KCl containing I0 mM EDTA, suspended in 0.25 M sucrose to a final protein concentration of 30-40 mg/ml and stored under N2 for 2-3 days prior to use. Solubilization ~nd F i r s t Ammonium Sulfate Fractionation: Cytochromes P-450 (from rats treated with PB) and P-448 (from rats treated with 3-MC) were solubilized from l i v e r microsomes by a s l i g h t modification of the method described previously (13).
In a typical preparation, each 800-1000 mg of
microsomal protein was diluted to 61 ml in a mixture containing 14 ml of glycerol, 7 ml of IM potassium phosphate (pH 7.7), 0.7 ml of O.IM d i t h i o t h r e i t o l (DTT), 0.7 ml of O.IM EDTA and 0.25 M sucrose and each mixture was sonicated as previously described (13).
Eight to ten ml of 10% sodium cholate was added
bringing the final concentration to 1 mg cholate per mg protein.
Each mix-
ture was s t i r r e d in ice for 20 min and centrifuted at 105,000 x g for 1 hour. After centrifugation, fractionation.
the individual preparations were pooled f o r further
Solid ammonium sulfate was added to the pooled supernatant
fraction to 40% saturation and then to 50% saturation.
The resulting pre-
c i p i t a t e from 40-50% saturation was dissolved in O.05M potassium phosphate buffer, pH 7.7, and centrifuged at 160,000 x g for 1 hour to remove insoluble material.
The supernatant fraction was dialyzed overnight
against O.02M
potassium phosphate buffer, pH 7.7, containing 20% glycerol, 10 -4 M DTT, 10"4 M EDTA and 0,1% cholate and centrifuged at 35~000 x g for 20 min. Second Ammonium Sulfate Fractionation:
The dialyzed sample was d i l u t -
ed to 3-4 mg protein/ml with O.02M potassium phosphate buffer, pH 7.7, contain-
1335
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ing 20% glycerol, lO-4 M DTT, lO"4 M EDTA and 0.2% cholate.
Solid ammonium
sulfate was added to 43% saturation and then to 50% saturation.
The resulting
precipitate from 43-50% saturation was dissolved in O.O05M potassium phosphate buffer, pH 7.7, containing 20% glycerol, lO "4 M DTT, I0-4 M EDTA and 0.1% cholate, dialyzed overnight against the same buffer mixture, and centrifuged at 35,000 x g for 20 min to remove any insoluble material. Calcium phosphate Gel Fractionation:
The dialyzed sample was diluted
to a protein concentration of 8 mg per ml with the dialyzing buffer.
Calcium
phosphate gel (Bio-Rad Laboratories) was added to a gel to protein ratio of 3. The gel was extracted twice with O.l M potassium phosphate, buffer pH 7.7, and then with 0.25 M potassium phosphate buffer, pH 7.7.
The 0.25 M phosphate buffer ex-
tract was concentrated to a protein concentration of approximately lO mg per ml by membrane u l t r a f i l t r a t i o n using a Diaflo XM-IOOAmembrane, dialyzed against O.Ol M potassium phosphate, pH 7.7, containing 20% glycerol, lO-4M DTT, lO -4 M EDTA and 0.05% cholate, and centrifuged.
The supernatant fraction was stored at
-20 ° under N2. RESULTS: The steps in the purification procedure for cytochromes P-450 and P-448 are summarized in Table I.
The final preparation resulted
in a 2.5 and 3.3-fold purification for cytochromes P-450 and P-448 respectively.
The final specific a c t i v i t y of cytochromes P-450 and P-448 (5.3-5.5
nmoles/mg protein) in this study is s i g n i f i c a n t l y higher than has been reported previously using rat l i v e r microsomes (8).
While the preparations
after ammonium sulfate fractionation remained soluble, as judged by centrifugation at 160,000 x g for 60 min, the final preparation sedimented upon high speed centrifugation.
However, the red transparent pellet would readily redissolve
in the centrifuge tube upon standing in the cold.
The cause of this phenomenon
is not known at the present time. We have recently reported that the solubilized cytochrome P-450 and P-448 fractions exhibit different specif ici t y with respect to a number of substrates (13-16).
Similar results have now been obtained with these purified 1336
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 Purification of Cytochromes P-450 and P-448 from Rat Liver Microsomes
Volume (ml)
Protein (g)
247
7.41
16.2
2.2
I00
I s t Ammonium sulfate (40-50%)
65
1.08
3.5
3.2
22
2nd Ammonium sulfate (43-50%)
32
0.29
1.2
4.1
8
Calcium phosphate gel
12
0.II
0.6
5.5
4
315
8.01
12.7
1.6
I00
I s t Ammonium sulfate (40-50%)
82
1.20
3.6
3.0
28
2nd Ammonium sulfate (43-50%)
37
0.39
1.6
4.1
13
Calcium phosphate gel
14
0.15
0.8
5.3
6
Fraction
P-450 or P-448 (pmoles)
Specific A c t i v i t y (nmoles P-450 or P-448/mgprotein)
Yield (%)
A) Cytochrome P-450 from PB-treated rats Microsomes
B) Cytochrome P-448 from 3-MC-treated rats Microsomes
Cytochromes P-450 and P-448 were determined by the method of Omura and Sato (I) using an extinction coefficient of 91 mM-I cm-I for A450_490. Protein was determined by the method of Lowry et al (19).
cytochrome P-450 and P-448 fractions assayed in the presence of the reductase and l i p i d fractions.
Thus the cytochrome P-450 fraction was active for benz-
phetamine N-demethylation, but rather inactive for 3,4-benzpyrene hydroxylation whereas the cytochrome P-448 fraction showed high 3,4-benzpyrene hydroxylase a c t i v i t y but low benzphetamine N-demethylase a c t i v i t y .
These results are con-
sistent with the view that cytochrome P-450 is c a t a l y t i c a l l y d i f f e r e n t from cytochrome P-448.
1337
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
I
i
I
..
i
I
/
i
I
i
\
/ /
',
0,4
/
x
i
i
0.5 z
g g <
/ i
0.2 /
/
0.1
U I 420
I
I 440
t
WAVELENGTH
Figure I :
I 460
I
A 480
(m~)
Carbon monoxide difference spectra of p a r t i a l l y
p u r i f i e d cytochromes P-450 ( s o l i d l i n e , 0.46 mg protein/ml) and P-448 (dashed l i n e , 0.52 mg protein/ml).
The reduced CO-difference spectra of the p u r i f i e d cytochromes P-450 and P-448 is shown in Figure I .
I t can be seen that the f i n a l preparations contain-
ed i n s i g n i f i c a n t amounts (less than 5%) of cytochrome P-420.
The heme con-
centration of the f i n a l preparations was determined by the pyridine hemochromogen method ( I ) to be 5.48 and 4.65 nmoles per mg protein for cytochromes P-450 and P-448 respectively.
Thus, the e x t i n c t i o n c o e f f i c i e n t f o r A450_490 or A448_490
of the reduced hemoprotein CO complex was calculated as 91 mM-I cm-I f o r cytochrome P-450 and 103 mM-I cm-I for cytochrome P-448.
These values are in close
agreement with those reported by Omura and Sato ( I ) , and Greene et al (20) using microsomal suspensions, but d i f f e r e n t from those reported by Fujita et al (8) for t h e i r s o l u b i l i z e d preparation of cytochromes P-450 (58 mM-I cm- I ) and P-448 (78 mM-I cm-l). 1338
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
Fujita et al (8) have reported that their solubilized cytochrome P-448 (referred to as cytochrome PI-450) did not give a type I difference spectra and therefore suggested that cytochrome P-448 may not have a type I binding site.
In our preliminary studies, however, both the purified cytochromes P-450
and P-448 gave a typical type I spectra with benzphetamine. Thus, i t appears that our cytochrome P-450 and P-448 preparations are not only cataytically active in hydroxylation, but also retain the properties of the native microsomal bound forms. Further studies on the physical, spectral and catalytic properties of the purified cytochrome P-450 and P-448 preparations are now in progress in our laboratory. ACKNOWLEDGMENTS: We are grateful to Drs. R.Kuntzman and A. H. Conney for their helpful criticism in the course of the parparation of this manuscript and to S. West and B. Ryan for their able technical assistance.
We also wish
to thank Mrs. L. Rubino for preparing the manuscript. References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Omura, T., and Sato, R., J. Biol. Chem. 239, 2370 (1964). Conney, A. H., Pharmacol. Rev. 19, 317 ( ~ 7 ) . Kuntzman, R., Ann. Rev. PharmacoT. 9, 21 (1969). Orrenius, S., J. Cell Biol. 26, 713-(1965). MacLennan, D. H., Tzagoloff, A., and McConnel, D. G., Biochim. Biophys. Acta 131, 59 (1967). Miyaka, Y., Gaylor, J. L., and Mason, H. S., J. Biol. Chem. 243, 5788 (1968). Lu, A. Y. H., and Coon, M. J., J. Biol. Chem. 243, 1331 (1968). Fujita, T., and Mannering, G. J., Chem.-Biol. Interactions 3, 264 (1971). Mitani, F., Alvares, A. P., Sassa, S., and Kappas, Ao, Mol. Pharmacol. 7, 280 (1971). Lu, A. Y. H., Junk, K. W., and Coon, M. J., J. Biol. Chem. 244, 3714 (1969). Lu, A. Y. H., Strobel, H. W., and Coon, M. J., Biochem. B i o ~ s . Res. Comm. 36, 545 (1969). Lu, A. Y. H., Strobel, H. W., and Coon, M. J . , M o l . Pharmacol. 6, 213 (1970). Lu, A. Y. H., Kuntzman, R., West, S., and Conney, A. H., Biochem. Biophys. Res. Comm. 42, 1200 (1971). Kuntzman, R., Lu, A. Y. H., West, S., Jacobson, M., and Conney, A. H., ChemBiol. Interactions 3, 264 (1971). Lu, A. Y. H., Kuntzman, R., West, S., Jacobson, M., and Conney, A. H., Pharmacologist L3, 222 (1971). Lu, A. Y. H., Kuntzman, R., West, S., Jacobson, M., and Conney, A. H., J. Biol. Chem. (1972), in press. Sladek, N. E., and Mannering, G. J., Mol. Pharmacol. 5, 186 (1969). Levin, W., Alvares, A. Jacobson, M., and Kuntzman, R., Biochem. Pharmacol. 18, 883 (1969). Lowry, O. H., Rosenbough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193, 265 (1951). Greene, F. E., Stripp, B., and G i l l e t t e , J. R., Pharmacology 5, 43 (1971). 1339