NADH-dependent cytochrome P-450 oxidase system in submicrosomal particles

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NADH-DEPENDENT CYTOCHROME P-450 OXIDASE SYSTEM IN SUBMICROSOMAL PARTICLES YOSHIYUKI ICHIKAWA and JOANN S. LOEHR Department of Biochemistry, University of Oregon Medical School, Portland, Oregon 97201

Received D e c e m b e r 18, 1971 SUMMARY: An NADH-dependent pathway of electron transfer has been observed in the cytochrome P-~50 oxidase systems of submicrosomal particles of rabbit hepatocytes. This NADH-mediated reduction of cytochrome P-450 proceeds in the absence of cytochrome b 5 and is not due to indirect reduction by superoxide. Smooth and rough endoplasmic reticulum appear to contain the NADH-dependent system as well as an NADPH-dependent system. INTRODUCTION:

It has been shown that the cytochrome P-450 mixed function oxl-

dase systems in rabbit liver microsomes can be reduced by either NADPH or NADH, although the lower affinity of NADPH-cytochrome c reductase for NADH makes NADH a less likely hydrogen donor at the normal physiological concentrations of NADH and NADPH (1,2).

Increased NADH-directed electron transfer in the presence of

NADPH was ascribed to the transhydrogenase activity of NADH-cytochrome b5 reductase for NADH and NADP + (i).

We have now been able to observe an NADH-

dependent flow of electrons to the cytochrome P-450 oxldase system in submicrosomal particles of rabbit hepatocytes which is mediated by an NADH-ferrlcyanide reductase in the absence of cytoehrome b 5.

This NADH-dependent system operates

at the physiological concentration of NADH (3) and is present in submlcrosomal particles f~om both smooth and rough endoplasmlc retlculum of rabbit hepatocyte MATERIALS AND METHODS:

Rough and smooth endoplasmic retlculum were prepared

from the livers of phenobarbital-treated male rabbits as described previously (1,4).

Contamination with mitochondria as estimated by succinie dehydrogenase

activity was less than 1%.

Endoplasmic reticulum was digested with trypsin

which had been treated with tosylphenylalanine chloromethyl ketone (TPCK) to inhibit contaminant chymotrypsin activity (5).

Submlcrosomal particles were

isolated by the method of Ichlkawa and Yamano (6).

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NADH- and NADPH-cytochrome e reductase activities were determined spectrophotometrically by the method of Hatefi and Rieske (7), in the presence or absence of 1.5 BM rotenone to determine rotenone sensitivity (8).

NADH-

and NADPH-ferricyanide reductase or NADH- and NADPH-cytochrome b 5 reductase activities were determined similarly using 6 mM ferricyanide or 5 ~M cytochrome b 5 as electron acceptor.

NADH- and NADPH-neotetrazolium reductase were

determined by the method of Dallner p~ al. (9). The oxidation of NADH and NADPH were observed as the decrease in absorbance at 340 nm using an extinction coefficient of 6.22 mM-icm -I (i0).

Optical measurements were performed

with a Cary 14 recording spectrophotometer using cuvettes of 1 cm path length and a cell compartment thermostatically controlled at 25 °. Superoxide dismutase was purified from bovine erythrocytes and assayed as described (ii) in the presence of bovine catalase to prevent reoxidation of reduced cytochrome c with hydrogen peroxide.

Cytochrome b 5 was purified

from rabbit liver microsomes by treatment with detergent (12) and trypsin (13). Accepted procedures were used in determining the concentrations of cytochrome b 5 (14), cytochrome P-450 and P-420 (15), and protein (16).

The cytochrome

b 5 content of submicrosomal particles was checked with NADH-cytochrome b 5 reductase, purified by the methods of Spatz and Strittmatter (12), and Takesue and Omura (17).

Trypsin (bovine pancreas, type i, twice crystallized) and

cytochrome c (bovine heart, type V) were purchased from Sigma.

Crystalline

phenylisocyanide was synthesized by the method of Prayer et al. (18). RESULTS AND DISCUSSION:

The properties of whole microsomes and submicrosomal

particles obtained after exposure to TPCK-treated trypsin in 20% glycerol are compared in Table I.

While the submicrosomal particles contain sizable

amounts of cytochrome P-450 and NADH-ferricyanide reductase, they have lost other microsomal pl~oteins such as cytochrome b 5 and NADPH-dependent reductase activities with electron acceptors such as cytochrome c, ferricyanide, and neotetrazolium.

The low NADH-cytochrome c reductase activity results from

the lack of cytochrome b 5.

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Table i. Comparison of electron transport components of smooth endoplasmic reticulum and its submicrosomal particles from livers of phenobarbital treated rabbits. Enzyme

Smooth endoplasmic Submicrosomal retlculum particles nmoles/min/mg protein (mean±S.E.)

No. of Expt.

NADPH-cytochrome c reductase

6

290 ± 30

NADPH-ferricyanide reductase

5

550 ± 30

0.5

± 0.i

NADPH-neotetrazolium reductase

5

245 ± 35

0.2

±

NADPH-eytochrome b 5 reductase

i0

<0.01

15.5 ± 3.2

0.i

<0.01

Rotenone-sensitive NADHcytochrome c reductase

7

Rotenone-insensitive NADHcytochrome c reductase

7

320 ± 50

0.4 ± 0.I

NADH-ferricyanide reductase

7

1541 ± 270

956 ± 40

NADH-neotetrazolium reductase

7

124

± 25

64

±

i0

132

± 34

25

± 5

NADH-cytochrome b 5 reductase

<0.42

<0.01

i0

nmoles / mg protein (mean S.E.) Cytochrome b 5

5

1.95 ± 0.01

Cytochrome P-450

i0

3.30 ± 0.01

Cytoehrome P-420

i0

<0.02

<0.03 7.70 ± 0.50 <0.15

The cytochrome P-450-phenylisocyanide complex of submicrosomal particles is rapidly reduced by NADH under both aerobic and anaerobic conditions, but not by NADPH under the same experimental conditions (Fig. I, A and B).

How-

ever, the complex is slowly reduced at high concentrations of NADPH, presumably due to the affinity of NADH-fer~icyanide reductase for NADPH.

The close

similarity of the anaerobic results to those obtained in the presence of oxygen argues against indirect reduction by superoxide, which could be formed by the NADH- dependent reductase system under aerobic conditions.

Ftmther-

more, addition of high concentrations of superoxide dismutase had no effect

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A

I

I

NADH

I

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I-

I

I

I

I

i

I

I

I

I

I

-8

-'~ Absorbancy at 455 nm=O.I-

NADH

T

i Absorboncy at 455nm=0.1

30sec

NADPH

-"-I 30 sec I ~

I

I

Fig, 1.

I

I

I

I

I

I

I

I

I

I

Kinetics of reduction of cytochrome P-450-phenylisocyanide complex. Solutions containing submicrosomal particles (2 mg protein/ml) in 0.i M potassium phosphate buffer (pH 7.4) and 7 x IO-SM phenylisocyanide were placed in two cuvettes. The time course of the change in absorbancy at 455 nm was determined upon addition of 1 x i0- M NADH or 1 x IO-5M NADPH to the sample cuvette. (A) aerobic conditions; (B) anaerobic conditions, 5 x IO-6M superoxlde dismutase added to both cuvettes.

on the NADH-mediated reduction of cytochrome P-450 in the presence of phenylisocyanide or carbon monoxide.

Much lower concentrations of superoxide

dismutase are sufficient to inhibit the superoxide-dependent reduction of fe~ricytochrome c by direct addition of superoxide anion or by superoxide produced in the reaction with xanthine oxidase (ii). Although the reduced form of cytochrome P-450 in microsomal membranes is readily oxidized upon exposure to oxygen, it is maintained in the reduced state by complexation of cytochrome P-450 with phenylisocyanide.

One possible

explanation for the ease of reduction of the cytochrome P-450-phenylisocyanide complex of submicrosomal particles by NADH and for the stability of the reduced complex even undem aerobic conditions is that complexation with phenylisocyanide increases the oxidation-reduction potential of cytochrome P-450, as has been observed on complexation with substrates (19).

In order to

clarify this point reduction experiments were carried out in the presence of carbon monoxide which binds reduced cytochrome P-450 by complex formation, but does not bind to the oxidized form.

Formation of the reduced cytochrome

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I

I

I

I

I

R

Fig. 2.

-.H

I

I

~ Absorboncy

/

I

I

30 sec I

I-,.I

I

I

I

Kinetics of formation of cytochrome P-450-C0 complex upon NADH reduction. Solutions of submlcrosomal particles (2 mg protein/ml) in 0.i M potassium phosphate buffer (pH 7.4) were placed in a reference cuvette and a Thunberg sample cuvette. The sample cuvette was flushed with CO under strict anaerobic conditions. The time course of the change in absorbancy at 450 nm was determined upon addition of 1 x 10 -5 NADH to the sample cuvette.

P-450-C0 complex upon addition of NADH occurred to the same extent as in the presence of phenylisocyanide (Fig. 2).

Again NADPH had no effect.

The

similarity of the reduction reactions with both free and complexed forms of oxidized cytochrome P-450 indicates that the NADH-dependent pathway is not due to an increase in the oxidatlon-reductlon potential of cytoch~ome P-~50 upon phenylisocyanide binding. Estabrook et al. have evidence for electron flow in the presence of substrate from NADH to cytochrome P-450 of liver mlcrosomes via NADHcytochrome b 5 reductase and cytochrome b 5 (20).

The NADH-dependent cyto-

chrome P-~50 oxidase system in submicrosomal particles operates in the absence of cytochrome b 5.

However, it is possible that the submicrosomal system

could contain an NADH-cytochrome P-450 reductase different from NADHeyTochrome h5 reductase and/or cytochrome P-450 molecules different from those in the NADPH-dependent system.

The extent of NADH-dependent reduction

of cytochrome P-450 in submicrosomal particles is not increased by addition of detergent- or lysosomal enzyme-treated NADH-cytochrome b 5 reductase, indicating that other microsomal factors are limiting and/or that the NADH-

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cytochrome b 5 reductase was altered by treatment during purification.

Addi-

tion of either trypsin treated or detergent-treated cytochrome b 5 under anaerobic conditions also had no effect on the rate of formation of reduced cytochrome P-450 complex by the NADH-dependent system of submicrosomal particles. Although it is possible that trypsin treatment of whole microsomes has caused geometric changes of enzyme components in the microsomal electron transport systems, it seems unlikely from the fact that submicrosomal particles from rough or smooth endoplasmic reticulum show the same rapid rate of reduction of 25-45% of cytochrome P-450 by 1 x 10-SM NADH as intact smooth endoplasmlc reticulum under the same conditions.

Additional evidence for two independent

pathways of cytochrome P-450 oxidase systems in intact endoplasmlc reticulum is the selective inhibition by NADP + of the NADPH-dependent system, but not the NADH-dependent system.

In contrast to liver microsomes, NADH-dependent

cytochrome P-450 oxidase system was not observed with adrenal cortical microsomes. A hypothetical scheme for the microsomal electron transport systems based on the results described in this paper is shown in Figure 3. Ferricyanide Cyto~rome c NADPH

>

NADPH-ferricyanide reductase (NADPH~ cytochrome c r e d u c t a s e )

Neotetrazolium

l !

I

NADH.

NADH-ferricyanide reduetase (NADHcytochrome b 5 reductase) Fe~icyanide

Fig. 3

Inhibition by isocyanides and carbon monoxide

~ C y t o c h r o m e P-450~,-~O 2 (unknown factors) > Cytochrome P-450~I-~O 2

>Cytochrome b 5

-+02

Cytochrome c Neotetrazolium

Proposed scheme for microsomal electron transport systems operating at physiological concentrations of NADH and NADPH in hepatocytes. Solid a ~ o w s indicate direction of electron transport among physiological components; open arrows indicate direction of electron transport to artificial acceptors.

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ACKNOWLEDGMENTS: The authors wish to thank Prof. Howard S. Mason for his encouragement and advice. This study was supported by grants from the American Cancer Society (BC-IK) and the United States Public Health Service (AM 07180). REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Ichikawa, Y., Yamano, T., and Fujishima, H., Biochim. Biophys. Acta, 171, 32 (1969). Ichikawa, Y., and Yamano, T., J. Biochem. Tokyo, 66, 351 (1969). Glock, G. E., and Mclean, P., Biochem. J., 61, 388--(1955). Peters, T., Jr., J. Biol. Chem., 237, 1181 (1955). Singer, S. J., Advan. Prot. Chem., 22, I (1967). Ichikawa, Y., and Yamano, T., Biochim. Biophys. Acta, 200, 220 (1970). Hatefi, Y., and Rieske, J. S., in Methods in Enzymology, ed., R. W. Estabrook, and M. E. Pullman, Academic Press, New York, 1967, vol. I0, p. 225. Sottocasa, G. L., Kuylensterna, B., Ernster, L., and Bergstrand, A., J. Cell Biol., 32, 415 (1967). Dallner, G., Siekevitz, P., and Palade, G. E., J. Cell Biol., 30, 97 (1966). Horecker, B. L., and Kornberg, A., J. Biol. Chem., 175, 385 (19-~8). McCord, J. M., and Fridovich, I., J. Biol. Chem., 244, 6049 (1969). Spatz, L., and Strittmatter, P., Proc. Natl. Acad. Sci., 68, 1042 (1971). Kajiwara, T., and Hagihara, B., J. Biochem. Tokyo, 63, 453 (1968). Garfinkel, D., Arch. Biochem. Biophys., 77, 493 (19~). Omura, T., and Sato, R., J. Biol. Chem., 239, 2379 (1964). Gornall, A. G., Bardwill, C. J., and David, M. M., J. Biol. Chem., 177, 751 (1949). Takesue, S., and Omura, T., J. Biochem. Tokyo, 67, 267 (1970). Prager, B., Jacobson, P., Schmidt, P., and Stern, D., Beilstein's Handbuch der Organischen Chemie, Band XII, Springer-Verlag, Berlin, 1929, 4th ed., p. 191. Sies, H., and Kandel, M., FEBS letters, 9, 205 (1970). Estabrook, R. W., Hildebrandt, A. G., Baron, J., Netter, K. J., and Leibman, K., Biochem. Biophys. Res. Commun., 42, 132 (1971).

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