identification of cross-linked cytochrome p-450 in rat liver microsomes by enzyme-immunoassay

Vol. 108, No. 2, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

September 30, 1982 Pages 700-707

IDENTIFICATION OF CROSS-LINKED CYTOCHROME P-450 IN RAT LIVER MICROSOMES BY ENZYME-IMMUNOASSAY

Leonard S. Baskin and Chung S. Yang

Department of Biochemistry, UMDNJ-New Jersey Medical School Newark, New Jersey 07103

Received August 10, 1982

SUMMARY: The topography of microsomal proteins was studied by 2-dimensional gel- electrophoresis. The second dimension was run in the presence of P-mercaptoethanol, thus allowing detection of proteins previously cross-linked bydisulfide bonds as off-diagonal spots. With hepatic microsomes from phenobarbital pretreated rats, several off-diagonal spots were seen. The most intense spot, with a molecular weight of 52,000, was derived from a dimer of this protein. It was identified as cytochrome P-450 (P-450) by a double antibody enzyme- immunoassay. The dimer is probably formed by oxidation of sulfhydryl groups of P- 450 molecules during the preparation of microsomes. P-450 can also be cross- linked to form 105,000, 167,000, and 240,000 da1 oligomers by treating microsomes with dithiobis(succinimidy1 propionate) at 0°C. Cross-linking of P-450 to other proteins was also observed with one-dimensional gel-electrophoresis. The results suggest that the cross-linked proteins are close neighbors of P-450 in the membrane.

The endoplasmic reticulum contains a large number of functionally related

proteins, among them are P-4501, NADPH-P-450 reductase, epoxide hydrolase, UDP-

glucuronyltransferase, and the NADH-dependent fatty acid desaturase enzyme system

(1). The lateral topography of these proteins in the membrane is not clearly

understood. A chemical cross-linking approach ,has been used previously by

McIntosh and Freedman (2, 3) in studies with 6-naphthoflavone-induced rabbit

liver microsomes. With cupric-phenanthroline as the reagent, these authors

observed the association of two proteins with M, of 53 K and 57 K by a cross-

linkage other than a disulfide bond. Omura et al. (4) used glutaraldehyde to

cross-link proteins in 3-methylcholanthrene induced rat liver microsomes. As

analyzed with a P-450-specific antibody column, they observed some extent of

cross-linking between P-450 and NADPH-P-450 reductase. Previously, we have used

lAbbreviations used: P-450, cytochrome P-450; M,, molecular weight; K, molecular weight in thousands; DSP, dithiobis(succinimidy1 propionate); SDS, sodiumdodecyl sulfate; BSA, bovine serum albumin.

0006-291X/82/180700-08$01.00/0 Copyright 0 198.2 by Academic Press, Inc. All rights of reproduction in any form reserved. 700


a combined cross-linking and gel-electrophoresis approach to study the organiza-

tion of purified P-450 and NADPH-P-450 reductase in solution (5). When the cross-

linking approach was used to investigate the topography of P-450 in the microsomal

membrane, cross-linking between microsomal proteins was observed but the

identities of the cross-linked species were not established (6). In the present

work, the identification of P-450 as a component of cross-linked oligomers was

accomplished by a double antibody enzyme-immunoassay.

MATERIALS AND METHODS

Materials. DSP was obtained from Pierce. Freund's complete adjuvant (Difco) was obtained from VWR Scientific. BSA (radioimmunoassay grade), goat anti-rabbit IgG, horse radish peroxidase conjugate, and Tween 20 were obtained from Sigma. O- dianisidin was from Fluka. Nitrocellulose sheets were from BioeRad. Liver microsomes were prepared from phenobarbital-treated male Sprague-Oawley rats (7). The microsomes were suspended in 0.25 M sucrose at 30-40 mg protein/ml and stored at -86°C. P-450 was prepared as described previously (5).

Cross-Linking and Gel Electrophoresis. Cross-linking and two-dimensional electrophoresis were carried out as described (6) except that the cross-linking reactions were quenched by mixing 500 ul of sample with 75 ul freshly prepared 0.08 M glycine solution containing 0.6 M N-ethylmaleimide, 20% SDS and 17% ethanol. SDS-gel electrophoresis was carried out on 7.5% separation-3% stacking gels according to Laemmli (8) with some modifications (6).

Antiserum Preparation. Purified P-450 (2 mg/ml) in a 0.1 M phosphate-20% glycerol buffer (pH 7.4) was mixed vigorously with an equal volume of Freund's complete adjuvant and 0.1 ml was injected subcutaneously at each of 3 sites along the shaved flank of a male white New Zealand rabbit. Injections were made weekly. Ten days after the third injection, serum was obtained for immunoassays. Thereafter, booster injections were made monthly with Freund's complete adjuvant. Blood was drawn ten days later, clotted at room temperature, and centrifuged at 2000 x g for 30 min at 5°C. The serum was kept at 56°C for 30 min, cooled on ice, recentrifuged for 10 min, and stored frozen at -86°C.

Western Blotting and Enzyme Immunoassay. Proteins separated by gel electrophoresis were replica plated onto nitrocellulose paper by a diffusion transfer procedure (9). Two 9 x 14 cm gel slabs were soaked in 750 ml PBS2 (containing 1% Triton X-100) with shaking at room temperature for 1 h to remove SDS. The gel slabs were subsequently rinsed 3 times with distilled water and soaked in 750 ml PBS for 1 h. Each gel was then sandwiched between two sheets of nitrocellulose backed by filter paper and a porous polyethylene sheet on each side. The sandwich was immersed for 2 days in 4 changes of 2 L of PBS. The blotted nitrocellulose sheet was incubated (10) in 50 ml of 2% BSA in PBS-Tween3 for 1 h at 37", washed 3 times with 50 ml PBS-Tween and incubated for 2 h at room temperature in 50 ml of a PBS-Tween mixture containing 1% P-450-specific antiserum, 3% BSA, and 10% normal rabbit serum. It was washed 3 times with 50 ml PBS-Tween, incubated for 2 h at room temperature on a shaker in 50 ml of PBS-Tween (containing 50 ul goat anti-rabbit IgG peroxidase conjugate, 3% BSA, and 10% normal rabbit serum), and washed 3 times with 50 ml PBS-Tween. Then the nitrocellulose sheet was placed in 50 ml of 0.1 M NaH2PO4-citrate buffer, pH 7.4,

2PBS, phosphate buffer-saline: a solution containing 0.2 g KH2P04, 1.15 g Na2P04, 8 g NaCl, and 0.2 g KC1 in 1 L at pH 7.4.

3PBS-Tween, PBS containing 0.05% Tween 20.

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and reacted with 1.25 mg of 0-dianisidin and 125 pl of 0.3% H202 for 15-20 min at room temperature under dim light. The reaction was stopped by rinsing the nitrocellulose sheet with distilled water 3 times. The sheet was blotted dry with filter paper and stored wrapped in aluminum foil.

RESULTS

Cross-Linked Proteins Analyzed by Gel Electrophoresis. When phenobarbital-

induced rat liver microsomes were analyzed by 2-dimensional gel electrophoresis,

several off-diagonal spots were seen (Figure la). Because the second dimension

was run in the presence of 2-mercaptoethanol, these spots are believed to be

derived from oligomers cross-linked by disulfide bonds. The most intense off-

diagonal spot with a M, of 52 K (Spot 1) was derived from a protein oligomer of 105

K, apparently a dimer of the 52 K species. This cross-linked species was not

observed when the sulfhydryl reagent N-ethylmaleimide was included in the

homogenizing buffer during the preparation of liver microsomes (6). This and the

fact that the dimer was cleaved by 2-mercaptoethanol suggest that the dimer was

linked via a disulfide bond formed by oxidation of protein sulfhydryl groups. It

is not clear whether this process is enzymic or nonenzymic.

When the microsomes were treated with DSP, Spot 1 increased in intensity as

a result of the chemical cross-linking (Figure lb). Two additional spots in the

52 K region were also detected: Spot 2 derived from a 167 K oligomer and Spot 3

from a 240 K species. In addition, horizontal streaks in the 52 K region increased

appreciably, suggesting that the 52 K protein was also cross-linked with many

other proteins. Spot 4 (49 K) which appeared slightly below Spot 1 was shown to

be derived from a chemically cross-linked species of 100 K. In the 79 K region,

two faint spots also showed up as a result of chemical cross-linking, possibly to

proteins in the 52 K region. Spot 5 was derived from an oligomer of 130 K and Spot

6 from one of 180 K. Other faint spots were also observed but their identities

remain to be characterized.

Identification of P-450 by Enzyme-Immunoassay. In order to demonstrate that

P-450 was involved in the cross-linking, the proteins from the 2-dimensional gels

were transferred to nitrocellulose sheets and stained by an enzyme-immunoassay

procedure. With the diffusion blot transfer technique, the proteins were not

702


b

Fiqure 1. Two-dimensional gel electrophoresis of microsomes (160 ug protein per slab). Panel a, phenobarbital-induced microsomes. Panel b, the microsomes (4.8 mg protein/ml) were treated with 0.5 mM DSP at 0°C for 30 min.

completely transferred from the gel to the nitrocellulose, but the latter showed

the same protein pattern as the gel upon staining with amido black (data not

shown). The enzyme-immunoassay was specific for P-450. Among all the microsomal

proteins on the diagonal line, only the spot corresponding to P-450 was stained

(Figure 2). With this method, Spot 1 was identified as P-450. The amount of

cross-linked P-450 in Spot 1 increased as a result of cross-linking with DSP

(Figure 2b). Spots 2 and 3 were also identified as P-450. In addition, the

horizonal streaks were shown to contain P-450 (Figure 2b). Spot 4 (49 K) was not

stained by this procedure. It is interesting to note that the P-450 molecule

retained its specific antigenic activity even after being cross-linked and

denatured by SDS.

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a

b

Figure 2. Enzyme-immunoassay of cytochrome P-450 on nitrocellulose sheets. The c0nm.s for cross-linking and electrophoresis were the same as those described in Figure 1. Panel a, microsomes without DSP treatment. Panel b, microsomes cross-linked with DSP.

Cross-linked P-450 Analyzed by One-Dimensional Gel Electrophoresis. The

availability of a P-450 specific enzyme-immunoassay allowed us to identify cross-

linked P-450 after gel electrophoresis in the first dimension (Figure 3). The

left panel of Figure 3 is the stained nitrocellulose sheet after the diffusion

transfer. The gel was intentionally overloaded with microsomal proteins and the

protein bands broadened somewhat during the diffusion transfer. A complicated

multi-banded pattern was seen. On the other hand, only three bands (zones) were

stained by the enzyme-immunoassay (Figure 3, right panel). The lowest band in the

50-55 K region corresponded to monomeric P-450 and above which was a band of lOO-

111 K attributable to a dimer of P-450. P-450-containing oligomers also appeared

in the 130 to 180 K region as a result of cross-linking with DSP. These cross-

linked bands were not detected when 2-mercaptoethanol was included in the gel-

electrophoresis system. P-450-containing high molecular aggregates from the

cross-linked sample were also detected on the top of the gels (data not shown).

This confirms the results obtained by 2-dimensional gel-electrophoresis and

704


Figure 3. Amide black stain and enzyme-immunoassay of microsomal proteins on ~trocellulose sheets. Microsomal proteins (48 ug applied in each track) were subjected to electrophoresis on a 1.5 IIIII thick polyacrylamide slab gel and then transferred to two nitrocellulose sheets. One sheet was stained with amide black (Left Panel) and the other by the enzyme-irenunoassay (Right Panel). Tracks (numbered from left to right) 1, 3, and 5, proteins without cross-linking reagent. Tracks 2 and 6: microsomes cross-linked with 0.5 n+l DSP. Track 4, microsomes cross-linked with 0.25 mM DSP.

demonstrates directly that P-450 was present in cross-linked oligomers in

microsomes.

DISCUSSION

The present results demonstrate that during the preparation of rat liver

microsomes, P-450 can be oxidized to form a dimer. Although the dimer formation

was quite evident in liver microsomes from phenobarbital-pretreated rats, it was

not apparent with microsomes from 3-methylcholanthrene-treated or untreated rats

(6). A possible interpretation of these results is that the phenobarbital-induced

P-450 species has a specific sulfhydryl group that can be readily oxidized to form

a disulfide bond in dimer formation. The results in Figure lb indicate that P-450

dimers can also be formed via cross-linking with DSP. Spot 2 may be derived from

trimers of P-450, since no other spot can be seen as a possible cross-linking


pattern of this protein. Spot 3 may be derived from pentamers of P-450, but the

assignment is less certain. The identities of Spots 4, 5, and 6 remain to be

investigated. Because their molecular weights correspond to those of epoxide

hydrolase and NAOPH-P-450 reductase, antibodies specific to these enzymes may be

useful for the identification of these proteins.

With cupric phenanthroline as the cross-linking agent, McIntosh et al. (11)

observed cross-linked proteins in 8-naphthoflavone-induced rabbit liver micro-

somes. The reaction was not observed in microsomes from untreated or phenobar-

bital-induced rabbits. Oligomers larger than dimers were not seen. The assignment

of P-450 in the cross-linked oligomers was based on the inducibility of the

species. In the present work, it is demonstrated immunochemically that the P-450

molecules can be chemically cross-linked or oxidized to form dimers in the

microsomes. Chemical cross-linking can take place between proteins located

adjacent to each other or when two proteins come together by collision. At low

temperatures there is very little lateral mobility of membrane proteins (12) and

the latter type of cross-linking is believed to be insignificant (6, 13). Since

the cross-linking takes place at ice temperature in the membrane, we suggest that

the cross-linked proteins are located adjacent to each other. Based on this

argument,it appears that the most abundant nearest neighbor of P-450 is another

molecule of the same species. Other molecular species, including NADPH-P-450

reductase, may also be located close enough to P-450 to allow cross-linking

reactions to occur. Additional investigation is needed to further define the

enzyme topography of the endoplasmic reticulum membrane.

Acknowledgements: This work was supported by Grant CA-16788 from the National Cancer Institute and Grant IN-92 from the American Cancer Society. The authors acknowledge the excellent technical assistance of Mrs. Shu-Min Ning. They also thank Dr. Aaron Abramovitz for helpful discussions on immunochemical techniques.

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identification of cross-linked cytochrome p-450 in rat liver microsomes by enzyme-immunoassay

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