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and rabbit P450s. Of the three P450s, only P450IIC8 metabolizes the model monooxygenase substrate aminopyrine to an appreciable extent. Acknowledgments This work was supported by Research Grants AA-08139 and AA-07842. The authors would like to thank Ms. Barbara Bloswick and Mrs. Michelle Mouck for skilled technical assistance.

[57] C o n s t i t u t i v e a n d I n d u c i b l e F o r m s o f C y t o c h r o m e P 4 5 0 from Hepatic Mitochondria

By RASS M. SHAYIQ, SANKAR ADDYA, and NARAYAN G. AVADHANI Introduction Until recently it was believed that hepatic mitochondria contain one or two forms of cytochrome P450 involved in the side-chain hydroxylation of C27 sterols. Although the ability of isolated mitochondria and mitochondrial extracts from the hepatic tissue to carry out C-25 hydroxylation of vitamin D 3 and C-26 hydroxylation of cholesterol were reported in the early 1970s by three different groups, ~-4 it was not clear whether the same P450 forms were involved in these hydroxylations. Purification of P450 enzymes exhibiting high specificity either for the bile acid precursors or for vitamin D 3 substrates coupled with the isolation of monoclonal antibodies with discriminating specificities 5-7 suggested that the two hydroxylation activities described above might be carded out by two distinct P450 isoforms. A recent study in our laboratory 8 on the transient expression of a eDNA in COS cells revealed that a single P450 designated as CYP26/25 can catalyze both cholesterol 26-hydroxylase and vitamin D 3 25-hydroxylase activities. In addition to this constitutive form, studies t S. Taniguchi, N. Hoshita, and K. Okuda, Eur. J. Biochem. 40, 607 (1973). 2 I. Bjorkhem and J. Gustafsson, J. Biol. Chem. 249, 2528 (1974). 3 I. Bjorkhem and I. Holmberg, J. Biol. Chem. 253, 842 (1978). 4 y. Atsuta and K. Okuda, J. Biol. Chem. 256, 9144 (1981). 5 0 . Masumoto, Y. Ohyama, and K. Okuda, J. Biol. Chem. 263, 14256 (1988). 6 H. Dahlback, Biochem. Biophys. Res. Commun. 157, 30 (1988). 7 K. WikvaU, J. Biol. Chem. 259, 3800 (1983). P. Su, H. Rennert, R. Shayiq, R. Yamamoto, Y.-M. Zheng, S. Addya, J. Strauss, and N. Avadhani, DNA Cell. Biol. 9, 657 (1990).

METHODS IN ENZYMOLOGY,VOL. 206

Copyright © 1991by Academic Press, Inc. All fightsof reproduction in any form reserved.

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reported from our laboratory 9-~1 demonstrated the occurrence of four distinct hepatic mitochondrial P450 forms inducible with xenobiotic agents like phenobarbital (PB) and/3-naphthoflavone (fl-NF). Here we describe the purification and biochemical as well as immunochemical characterization of these various mitochondrial P450 isoforms.

Isolation of Mitochondria Because of the generally low P450 content of hepatic mitochondria even under the fully induced state, it is important to start with 5 to 8 g of mitochondria for purification. Second, because of 5- to 8-fold higher P450 content of the microsomal fraction, it is imperative to use mitochondria devoid of significant microsomal contamination. The following procedure has been found successful for the isolation and purification of both inducible and constitutive isoforms of mitochondrial P450 from rat liver:

Reagents Tris-buffered saline: 10 mM Tris-HCl (pH 7.5), 150 mM NaC1 Isolation buffer: 2 mM HEPES (pH 7.4), 70 mM sucrose, 220 mM o-mannitol, 2 mM EDTA 2% Digitonin (twice crystallized): 2% (w/v) in isolation buffer Procedure. Sprague-Dawley rats (150-200 g weight) are treated with PB (75 mg/kg/day for 5 days) or/3-NF (40 mg/kg/day for 4 days) by intraperitoneal injections as described before. 9,1° All the following steps are carried out at 0o-4 °. Livers from 40 treated or untreated rats (350-400 g wet weight) are washed free of blood clots, perfused with ice-cold Trisbuffered saline, and rinsed once with ice-cold mitochondrial isolation buffer. The livers are minced into small pieces with a pair of scissors and homogenized in 450-500 ml of isolation buffer containing 0.5 mg/ml bovine serum albumin (BSA) with 5-6 strokes using a loose-fitting PotterElvehjem glass homogenizer with an electrically driven Teflon pestle (0.15 mm clearance). The homogenate is diluted with the isolation buffer containing 0.5 mg/ ml BSA to yield a 15% suspension and centrifuged in Sorvall GSA3 rotor (500-ml plastic bottles) for 10 min at 1000 g. The supernatant from this run is centrifuged once more at the same speed for 10 min. The postnuclear supernatant is centrifuged at 10,000 g for 15 min to pellet mitochondria. The crude mitochondria are washed 3 times with the isolation buffer 9 H. Raza and N. Avadhani, J. Biol. Chem. 263, 9533 (1988). 1oR. Shayiq and N. Avadhani, Biochemistry 28, 7546 (1989). ll R. Shayiq and N. Avadhani, Biochemistry 29, 866 (1990).

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(300-400 ml for each wash) to reduce the microsomal contamination. The mitochondria are suspended in the isolation buffer to a protein concentration of 40-50 mg/ml (based on the differential absorbance at 280 and 310 nm described before, Ref. 12). An appropriate volume of 2% digitonin solution is added to the mitochondrial suspension to a final concentration of 75/zg/mg protein, and the mixture is shaken gently on ice for 5 min. This treatment is a critical step in removing the microsomal and peroxisomal contaminants. Resultant mitoplasts (mitochondria devoid of the outer membrane) are pelleted by centrifugation at 12,000 g for 10 min, washed 3 times with the isolation buffer, and the final pellet used for P450 purification. Purification of P450 To ensure optimal yield and reproducible enzyme activity, it is important to use freshly isolated mitoplasts for solubilization and purification of P450. Second, a number of hepatic mitochondrial P450 forms, such as P450mtl, P450mt3, and the constitutive forms of P450, are extremely hydrophobic, while others are relatively more hydrophilic. Thus, a careful use of detergent concentration for column elution or differential elution with buffers containing varying concentrations of the detergent is needed for optimal recovery. Generally, the purification involves five discrete steps including P450 solubilization by cholate, fractionation with polyethylene glycol (PEG) 8000, and column chromatography on to-octylamine agarose, DEAE-Sephacel, and hydroxylapatite.

Reagents Buffer A: 100 mM potassium phosphate (pH 7.4), 20% glycerol, 1.0 mM EDTA, and 1.0 mM dithiothreitol (DTT) Buffer B: 10 mM potassium phosphate (pH 7.7), 20% glycerol, 0.2% cholate, 0.05-0.2% Lubrol PX as specified Buffer C: 5 mM potassium phosphate (pH 7.5), 20% glycerol, 0.2% cholate, 0.1 mM EDTA, and 0.05% Lubrol PX Buffer D: 50 mM potassium phosphate (pH 7.5), 20% glycerol, 1 mM EDTA, and 1 mM DTT Procedure. A suspension of mitoplasts in buffer A (25 mg/ml) is pulse sonicated for 3 min (30-see pulse followed by 60 sec of standing on ice) at setting 6 of a Branson Sonifier. Sodium cholate [20% (w/v) solution] is added to a final concentration of 0.8%, and the mixture is stirred for 30 min at 4° to solubilize the P450. Solid PEG is added to the solution to a 12S. Clarke, J. Biol. Chem. 251, 950 (1976).

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TABLE I DETERGENTAND SALT CONCENTRATIONSFOR ELUTIONOF RAT HEPATIC MITOCHONDRIALCYTOCHROMEP450sa Concentration

Cytochrome P450 form

Lubrol PX (%) (OAA column step gradient)

mtl rot2 rot3 rot4 CYP26/25

0.3 0.06 0.3 0.06 0.4

NaCI (mM) (DEAE column linear gradient) 60-70 70-80 55-65 60-70 60-70

(0.2%) (0.1%) (0.2%) (0. I%) (0.2%)

K2HPO4 (raM) (HTP column step gradient) 40 60 30 40 60

From to-octylamine-agarose (OAA), DEAE-Sephacel (DEAE), and hydroxylapatite (HTP) columns. Values in parentheses are the Lubrol PX concentrations used in DEAE column chromatography.

concentration of 15% (w/v) and stirred at 4 ° for 1 hr. The precipitate is collected by centrifugation at 100,000 g for 1 hr and dissolved in buffer A containing 0.5% cholate by homogenization with a glass homogenizer followed by stirring for 30 min at 4°. The resulting solution, adjusted to a protein concentration of 10 mg/ ml, is layered on a 2.5 x 40 cm to-octylamine-agarose column (Sigma Chemical Co., St. Louis, MO), preequilibrated with buffer A containing 0.4% cholate. The column is washed with equilibration buffer until the A2s0 of the eluate is reduced to less than 0.05. The P450, appearing as a brownish-red band, 3-4 cm from the top of the column, is eluted at a flow rate of 40 ml/hr with buffer A containing 0.06 to 0.4% Lubrol PX depending on the nature of P450 to be purified (see Table I). Fractions rich in P450 ( > 0 . 1 0 D at 417 nm) are pooled, concentrated to 10-20 ml under N2 in an ultrafiltration system using a 20 K molecular weight cutoff filter, and dialyzed against buffer B overnight with three changes. The resultant fraction is applied to a 2.5 x 40 cm DEAE-Sephacel column preequilibrated with 5 column volumes of buffer B containing Lubrol PX. The column is washed with 5 column volumes (500 ml) of Lubrol-containing buffer B, and the P450 is eluted with 10 column volumes of a 0 to 0.25 M NaCI gradient in Lubrol-containing buffer B. The concentration of Lubrol used for the DEAE chromatography ranges from 0.1 to 0.2% depending on the type of P450 being purified (see Table I). Fractions rich in P450 as judged by absorbance at 417 nm are checked for relative purity by electrophoresis on a sodium dodecyl sulfate (SDS)-polyacrylamide minigel system. Some P450 isoforms such as P450mt4 do not even

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require the hydroxyapatite step as they purify to over 80% purity at the DEAE step. The pooled fractions are dialyzed against buffer C overnight with one change of buffer and applied to a 1 × 20 cm hydroxylapatite (BioGel HTP from Bio-Rad Laboratories, Richmond, CA) column preequilibrated with buffer B containing 0.05% Lubrol PX. The column is washed with 150 ml of buffer B containing 0.05% Lubrol PX and eluted with a step gradient of 100 ml each of 30, 40, 50, and 60 mM potassium phosphate in buffer B containing 0.05% Lubrol PX, at a flow rate of 20 ml/hr. Fractions of 3 ml each are collected and the optical density at 417 nm determined. Aliquots from 417 nm absorbing fractions are subjected to electrophoretic analysis using a polyacrylamide-SDS minigel system, and fractions containing over 80% pure proteins are pooled. The detergent from the pooled fraction is removed by stirring with Bio-Beads SM-2 from Bio-Rad Laboratories (I g beads/mg protein) for 2 hr at 4°, concentrated as above, and dialyzed against buffer D. The purified fractions are stored in 50- to 100-/zl aliquots at - 8 0 ° until use. Characteristics of Various P450 Forms The purification procedure described above generally applies to many of the P450s from hepatic mitochondria. Nevertheless, the detergent concentration used for various chromatographic elution varies depending on the isoform (see Table I) that needs to be purified. For example, the two PB-inducible forms designated as P450mt3 and P450mt4 could be quantitatively separated at the oJ-octylamine-agarose step. P450mt4, which is more hydrophilic, is first eluted from the column with a buffer containing 0.06% Lubrol, and the more hydrophobic P450mt3 is next eluted with a buffer containing 0.3% Lubrol PX. Similarly, the P450 isoforms purified from both induced and uninduced livers appear to exhibit a wide range of isoelectric pH and, therefore, elute at different salt concentrations from both the DEAE-Sephacel and hydroxylapatite columns as indicated in Table I. A unique feature of mitochondrial P450 isoforms thus far purified is their ability to accept electrons from the mitochondrial type of electrontransfer proteins involving a flavoprotein and flavoprotein reductase. 13 In fact this property has often been used as an important criterion for assigning mitochondrial identity for various P450 isoforms. All the five isoforms purified in our laboratory fulfill this criterion in that they are reconstix3R. Sato, Y. Atsuta, Y. Imai, S. Taniguchi, and K. Okuda, Proc. 74, 5477 (1977).

Natl. Acad. Sci. U.S.A.

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tutible, in most cases, exclusively with the mitochondrial type of electroncarder proteins. Another important feature of a large majority of proteins (including P450) destined to the mitochondrial matrix and inner membrane compartments is that they are synthesized as precursors with N-terminal extensions.14 These precursors are transported into mitochondria and processed into mature proteins under in vitro conditions. All of the mitochondrial P450 forms purified in our laboratory also exhibit this important characteristic, suggesting that they are indeed of mitochondrial location (Refs. 1I, 15, and R. M. Shayiq, S. Addya, and N. G. Avadhani, unpublished results, 1990). The constitutive form of P450 (CYP26/25) which catalyzes the 25hydroxylation of vitamin D3 and 26-hydroxylation of cholesterol exhibits an N-terminal sequence of Ala-Ile-Pro-Ala-Ala-Leu-Arg-Asp-His-Glu.8 CYP26/25 represents a distinct molecular species as indicated by its substrate specificity and reaction to a discriminating monoclonal antibody (Ref. 8 and R. M. Shayiq, S. Addya, and N. G. Avadhani, unpublished results, 1990). Although one of the PB-inducible forms, designated as P450mt3, exhibits an N-terminal sequence nearly similar to CYP26/25, it lacks significant 25-hydroxylation activity and shows high levels of aflatoxin hydroxylation and d-benzphetamine demethylase and aminoantipyrine demethylase activities that are not detected in the constitutive form (Table II). Similarly,/3-NF-inducible P450mtl catalyzes the metabolism of almost all of the xenobiotic substrates, though at a considerably lower level as compared to P450mt3. Further, P450mtl exhibits cholesterol 26-hydroxylase activity which is not detected in P450mt3 (Table II). Thus, there are three functionally different forms of P450 in rat hepatic mitochondria with close immunochemical and possibly structural similarities (Tables III and IV). The second species of PB-induced form, designated as P450mt4, resembles the similarly induced microsomal P450b/e with respect to N-terminal sequence and reactivity to polyclonal antibodies (Tables III and IV). This form, however, is a true mitochondrial form since it can accept electrons exclusively through the ferredoxin/ferredoxin reductase electron carrier system. 11 Furthermore, results of immunoblot analysis 11 show that it is induced over 20-fold in male and marginally in female livers, suggesting that this mitochondrial form is induced in a sex-dominant fashion. Although both of the PB-inducible types can metabolize aflatoxin B~, P450mt3 yields higher levels of DNA reactive epoxides 1° compared to 14G. Schatz and R. Butow, Cell (Cambridge, Mass.) 32, 316 (1983). 15 B. Niranjan, H. Raza, R. Shayiq, C. Jefcoate, and N. Avadhani, J. Biol. Chem. 263, 575 (1988).

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T A B L E II METABOLISM OF VARIOUS XENOBIOTIC AND PHYSIOLOGICAL SUBSTRATES BY PURIFIED RAT HEPATIC M1TOCHONDRIAL CYTOCHROME eg50s Activity (nmol product/min/nmol of P450) Substrate (type of reaction) d-Benzphetamine (demethylation) Dimethyl aminoantipyrine (demethylation) Aflatoxin (hydroxylation) Benzo[a]pyrene (hydroxylation) Vitamin D3 (25-hydroxylation) l a - O H Vitamin D3 (25-hydroxylation) Cholesterol (26-hydroxylation)

P450mtl

P450mt2

P450mt3

P450mt4

CYP26/25

17.9

--~

69.3

69.1

N.D.C

14.8

--

54.6

58.1

N.D.

1.96

--

4.42

3.69

0.64

0.22

0.42

--

--

N.D.

0.02

0.04

0.05

0.08

0.229

.

.

.

+b

.

__

1.28 __

__

+

a Not done. b Cholesterol 26-hydroxylase was assayed using 5 nmol (106 dpm) of [3H]cholesterol? The values ( + ) correspond to 3.1 to 4.3% product formed in 30 min/40 pmol P450. c N.D., Not detected.

T A B L E III MOLECULAR CHARACTERISTICS OF RAT HEPATIC MITOCHONDRIAL P450 Antibody cross-reactivity

P450 form

Molecular weight a

Inducer

Poly. Ab b P450b

mtl mt2 mt3 rot4 CYP26/25

52,000 54,000 52,000 50,000 52,000

/3-NF /3-NF PB PB --

. + -

Poly. Ab ¢ P450c .

Poly. Ab P450mt3 .

+ -

Monoclonal Ab CYP26/25

Sex specificity a

+

None c~ None c~ 9 -> c~

. + +

a Molecular weight determined by SDS-polyacrylamide gel electrophoresis. b Polyclonal antibody (poly. Ab) to a phenobarbital-induced microsomal P450b. c Polyclonal antibody to B-naphthoflavone-induced microsomal P450c. d When amount of P450 is present at more than 10 times higher levels in one sex.

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TABLE IV NH2-TERMINALAMINOACID SEQUENCESOF RAT HEPATIC MITOCHONDRIALCYTOCHROMEP450 ISOFORMS

N-terminal residues P450 form

1

2

3

4

5

6

7

8

9

CYP26/25 mtl mt3 mt2 mt4~

A A

10

I

P

A

A

L

R

D

H

E

I

G

A

T

L

T

D

L

G

A P

I F

P P

A A

A F

L T

A P

T A

A T

T

M

G

P

S

I

L

L

L

A

L

(T)

(P)

a Residues in parentheses are variable amino acids detected at these positions for rot4. microsomal P450b/e, suggesting that these mitochondrial forms may have significant roles in hepatic detoxification of carcinogens. Of the two /3-NF-inducible types, the form designated as P450mt2 shows immunochemical similarity with similarly induced microsomal P450C. 9 Furthermore, as observed with the PB-inducible P450mt4, this isoform is also predominantly induced in male rat livers (R. M. Shayiq, H. Raza, and N. G. Avadhani, unpublished results, 1988). P450mt2, however, differs from the microsomal P450C with respect to its isoelectric pH, N-terminal sequence, and peptide fingerprint. 9 The second form, on the other hand, designated as P450mtl, shows no immunological similarity to any of the microsomal forms (Table III). The N-terminal sequence of P450mtl, however, shows nearly 70% positional identity with those of CYP26/25 and P450mt3, suggesting that it may be structurally closer to them (Table IV). In summary, hepatic mitochondria contain multiple species of both constitutive and inducible types of P450. Although the precise biological functions of many of these forms remain to be established, it appears that they exhibit overlapping as well as unique specificity for the metabolism of structurally different sterols and carcinogenic chemicals. Acknowledgments Research in the authors' laboratorywas funded by National Institutesof Health Grants CA-22762 and GM-34883 to N.G.A.

Constitutive and inducible forms of cytochrome P450 from hepatic mitochondria.

[57] P450s FROMHEPATICMITOCHONDRIA 587 and rabbit P450s. Of the three P450s, only P450IIC8 metabolizes the model monooxygenase substrate aminopyrin...
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