Reconstituted mammalian mixed-function oxidases: Requirements, specificities and other properties

Ph."" ae. Tber. A. Vol. 2. pp . 331-358. 1918. Pergamon Press , Printed in Greal Britain

Specialist Subject Editors: J. B. SCHENKMAN and D.

KUPFER

RECONSTITUTED MAMMALIAN MIXED-FUNCTION OXIDAsES: REQUIREMENTS, SPECIFICITIES AND OTHER PROPERTIES ANTHONY

Y. H. Lu and

SUSAN

B.

WEST

Department of Biochemistry and Drug Metabolism, Hoffmann-La Roche Inc.. Nutley. New Jersey 07110. U.S.A.

INTRODUCTION Studies of mammalian mixed-function oxidase activity have been conducted with biological systems ranging in complexity from the intact animal or perfused whole organ to isolated mitochondria and microsomes to well-defined, purified and reconstituted enzyme systems. In this article we shall limit ourselves to a discussion of reconstituted mixed-function oxidase systems and shall review their requirements, specificities and other properties. The usc of a reconstituted system rather than a more complex model to study the properties of mixed -function oxidase activity has both its advantages and its limitations. One of its many advantages is that reconstitution of multicomponent mixedfunction oxidase systems from their separated components can be used as a means of establishing the essential components for mixed-function oxidase activity. The properties and functions of each of the components can be studied separately as well as their interactions with one another. It also becomes possible to determine through its isolation and characterization whether multiple forms of cytochrome P-450 exist in mammals- Because of the absence of other interfering enzymes, the system is suitable for kinetic . ami mechanistic studies and can be used to establish and identify intermediates in the metabolism of steroids, drugs, carcinogens and mutagens. Of its limitations, one is that the reconstituted system may not be architecturally identical to the membrane-bound mixed-function oxidase, and therefore may not be .suitable for studying the spatial arrangement of the components in the membrane or the structure and function of biological membranes in general. In addition, the reconstituted system may not be suitable for establishing the total metabolic profile of all substrates due to either removal or inactivation during the isolation procedure of either one or more forms of cytochrome P-450 or another enzyme associated with the cytochrome P-450 system. In this article, we shall review the properties of reconstituted mixed-function oxidase systems from several mammalian tissues. The liver microsomal system will be described in detail, while the reconstituted mixed-function oxidase systems from other tissues will be discussed briefly, and their properties then compared with the liver microsomal system. Reconstituted mixed-function oxidase systems from bacteria or insects will not be covered. As used in this article, reconstitution refers to the restoration of catalytic activity by recombining individual components and does not imply the reconstruction of the biological membrane. RECONSTITUTED LIVER MICROSOMAL MIXED-FUNCTION OXIDASE ESSENTIAL COMPONENTS

In 1968, the liver microsomal mixed-function oxidase system was solubilized with detergent and resolved by column chromatography into three components which, when recombined, catalyzed the metabolism of fatty acids (Lu and Coon, 1968). 337

A. Y. H. Lu and S. B. WEST

338

These three components were identified as cytochrome P-450, NADPH-cytochrome c reductase (also known as NADPH-cytochrome P-450 reductase) and phosphatidylcholine (Lu et al., 1969a; Strobel et al., 1970). In subsequent studies, it was found that the same reconstituted mixed-function oxidase also catalyzed the biotransformation of steroids, drugs and a variety of other foreign compounds (Lu et al., 1969b, 1970, 1972a). The three components isolated in these early studies were not substantially purified and were cross-contaminated with one another. Nevertheless, the results clearly demonstrated that the liver microsomal mixed-function oxidase was a multi-component electron transport chain. Subsequently, several laboratories have succeeded in purifying to apparent homogeneity both cytochrome P-450 (Imai and Sato, 1974; van der Hoeven et al., 1974; Kawalek et al., 1975; Ryan et al., 1975a; Kamataki et al., 1976b; Huang et al., 1976) and NADPH-cytochrome c reductase (Vermilion and Coon, 1974; Dignam and Strobel, 1975; Yasukochi and Masters, 1976). It should be noted that there are multiple forms of cytochrome P-450 and thus a 'homogeneous' cytochrome P-450 preparation may be devoid of other proteins but may still contain more than one species of cytochrome P-450. It appears that based on catalytic and immunological studies, some of the purified cytochrome P-450 preparations do contain several different forms of cytochrome P-450 rather than a single species of cytochrome P-450 (Thomas et al., 1976a, b). Recent studies using a reconstituted system consisting of these highly purified components and synthetic phospholipid (dilauroyl phosphatidylcholine has been generally the choice) have confirmed that cytochrome P-450, NADPH-cytochrome c reductase and phospholipid are the essential components of the liver microsomal"mixed-function oxidase (Levin et al., 1974; van der Hoeven and Coon, 1974; Kamataki et al., 1976a; Philpot and Arinc, 1976; Yasukochi and Masters, 1976; Irnai, 1976). Figure 1 shows the effect of varying the concentration of cytochrome P-450, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate on the rate of benzphetamine Nsdemethylation. The detergents sodium cholate and sodium deoxycholate can stimulate this reaction (van der Hoeven and Coon, 1974; Lu et al., 1974b; Kamataki et al., 1976a, b; Imai, 1976) although the mechanism of stimulation is not clearly understood. The purification, properties and function of cytochrome P-450 and NADPH-cytochrome c reductase are briefly summarized in Table 1. Detergents, rather than the once popular proteases, have been used for the solubilization of these protein components. As shown in Table 2, when the minimal molecular weight of purified detergent-solubilized and protease-solubilized reductase as determined by SDS*-gel electrophoresis were compared, it was found that an 8000 molecular weight segment of the polypeptide was missing in the proteolytically-solubilized reductase. This alteration may be responsible TABLE

I. Components of the Liver Microsomal Hydroxylation System and their

Functions Cytochrome P-450 Prosthetic group: Iron-Protoporphyrin IX Function: I. oxygen- and substrate-binding site. 2. dete rmines substrate specificity of the system; multiple forms with overl apping substrate specificities. NADPH·cytochrome c reductase Prosthetic groups: FMN and FAD Function: transfers electrons from NADPH to cytochrome P-450. Phospholipid Active component: phosphatidylcholine and possibly other lipids: can be replaced by detergents. Function: facilitates electron transfer from the reductase to P-450 and complex formation between the two components but itself not an electron carrier. "The abbreviations used are; 50S. sodium dodecylsulfate : PS , phenobarbital; 3-MC. 3·methylcholan· threne .

339

Reconstituted mammalian mixed-function oxidases 70

70

A.

60

60

50

50

40

40

30

30

20

20

B.

10

0.05

010

0.15

0.20

I

400

I

600

800

I

1000

NAOPH - CYTOCHROME ~ REDUCTASE (unlll/ml)

CYTOCHROME P-450 (nmollml)

70

I

200

c.

70

6T1

D.

60

50

- SOOIUM CHOLAT£

10

10 DILAUROYL

20

30

40

50

PHOSPHATIDYLCHOLINE (I'g/ml)

50

100

150

200

SODIUM CHOLATE (I'g/ml)

FIG. I. Effect of concentration of rat cytochrome P-450 (A). rat NADPH-cytochrome c reductase (B). dilaurolyphosphatidylcholine (e) and sodium. cholate (D) on the rate of benzphetamine N-demethylation. The I ml reaction mixtures contained the following amounts of each component unless otherwise indicated: 0.10 nmol cytochrome P-450. 570 units NADPH-cytochrome c reductase (I unit = 1 nmol cytochrome c reduced per min assayed at 22°). lOJLg dilauroylphcsphatidylcholine (01 12:0) and 200JLg sodium chelate. In each titration. all components were present in excess except the component being varied. "C-N(methyl) benzphetamine was used as substrate and the '4C-formaldehyde formed was quantitated radiometrically (Thomas et al.. 1976a). Activity is expressed as nmol I"C-HCHO formed per 5 min.

for the inability of the proteolytically-solubilized reductase to function either in reducing cytochrome P-450 or in the overall hydroxylation reaction in the reconstituted system. SUBSTRA1'ES

Mixed-function oxidase activity has been reconstituted from the components isolated from the liver microsomes of rat, rabbit, mouse and hamster. These reconstituted systems metabolize a variety of drugs, carcinogens, insecticides, steroids, bile acids, fatty acids, and alkanes as well as many other compounds (Table 3). Thus, the reconstituted systems like intact microsomes have broad substrate specificities and catalyze a variety of different reactions such as N- and Ordealkylation. aliphatic, aromatic and N-hydroxylation, dehalogenation and desulfuration.

340

A. Y. H. Lu and S. B. WEST TABLE

2. Comparison of Purified Rat Liver Microsomal NADPH-Cytochrome c Reductase Obtained by Detergent Solubilization or Proteolytic Solubilization"

Property Molecular weight: (a) by SDS·polyacrylamide gel electrophoresis . (b) by gel filtration or other techniques in the absence of SDS Prosthetic group: Catalytic activity: (a) reduction of cytochrome c (b) reduction of Ierricyanide (c) reduction of cytochrome b,

Detergent-solubilized NADPH-cytochrome c reductase

Protease-solubilized NADPH-cytochrome c reductase

79,000

71,000

520,000 FAD and FMN (I: I)

71,000 FAD and FMN (l: l)

yes yes yes

yes yes only in the presence of high salts

yes

no

yes

no

yes

yes

(d) reduction of cytochrome P-450 in the presence of phospholipid (e) function in overall cytochrome P-450-dependent hydroxylation Interaction with antibody against protease-solubilized NADPH-cytochrorne c reductase

*Data compiled from the following references: Lu et al.; 1%9a: Masters et al., 1971: Iyanagi and Mason, 1973; Welton et al., 1973; Prough and Masters, 1974; Vermilion and Coon, 1974; Dignam and Strobel, 1975; Yasukochi and Masters, 1976. TABLE

3. Substrates of the Reconstituted Liver Microsomal Hydroxylation System

Drugs: benzphetarnine, aminopyrine, ethylmorphine, hexobarbital, pentobarbital, chlorcyclizine, norcodeine, zoxazolamine, p-nitroanisole (Lu et al., 1970, 1973b: Levin et al., 1974; van der Hoeven and Coon, 1974: Haugen et al., 1975; Tomaszewski et al., 1976) Steroids and bile acids: testosterone (Levin et al., 1974; Haugen et al., 1975), cholesterol (Biorkhern et al., 1974b), taurodeoxycholic acid, lithocholic acid, taurochenodeoxycholic acid (Bj5rkhem et al., 1974a), 5a-androstane-3a, 17J3-diol (Gustaffsson and Ingelman-Sundberg, 1976) Carcinogens: benzo(a)pyrene (Holder et al.. 1974; Ryan et al., 1975a), dimethylnitrosamine, 2-acetaminofluorene (Lotlikar et al., 1974, 1976), aflatoxin B, (Guengerich, 1977) Insecticides: parathion (Kamataki et al.• 1976a) Anesthetics: 1,1.2-trichloroethane (Gandolfi and Van Dyke, 1973) Fatty acids: octanoic acid, lauric acid (Lu et al., 1970) Alkanes: octane, hexane, heptane, nonane, decane, dodecane, tetradecane, cyclohexane (Lu et al., 1970) Other compounds: acetanilide (Selander et al., 1974), chlorobenzene (Selander et al., 1975), naphthalene (Dansette et al., 1974), biphenyl. ethoxyresorufin (Burke and Mayer, 1975), coumarin, 7-ethoxycoumarin (Thomas et al., 1976a; Philpot and Arinc, 1976; Huang et al.• 1976), aniline (Lu et al., 1972b; Fujita and Mannering. 1973; Imai and Sato, 1974: Haugen et al., 1975), ethanol (Teschke et al., 1974; Miwa et al., 1977)

DEPENDENCY ON LIPID

Phosphatidylcholine has been identified as the active component in the lipid fraction and its fatty acid composition is critical (Strobel et al., 1970). Of the long chain fatty acids, the unsaturated dioleoyl derivative is much more active than the saturated distearoyl or dipalmitoyl derivatives: If phosphatidyl derivatives containing saturated fatty acids are compared (Fig. 2), the dilauroyl derivative with 12 carbons is much more active than those containing 8, 10, 14 or 16 carbons (Lu et al., 1976). Thus, like many other lipid-requiring enzymes, the phospholipid requirement of the recon-

Reconstituted mammalian mixed -function oxidases

341

100 0112 -0

600

500 2 0 ~

ct

...J ~

4001-

::.

>= Ul

.... 2

lIJ 0

3001-

a:

LoJ

a,

200,..

01100 0180

10001140 • 01160

SATURATED CARBON CHAIN LENGTH

FIG. 2. Effect of the saturated carbon chain length of phosphatidylcholine on the metabolism of benzo(a)pyrene. Benzo(a)pyrene metabolism was assayed by the f1uorimetric assay in the presence of fixed amounts of rat cytochrome P-448 (0.13 I-lM), rat NADPH-eytochrome c reductase (100 units, I unit = I nmol cytochrome c reduced per min assayed at 22°), optimal concentrations of each phospholipid. 80 I-lM benzofalpyrene (in 0.04 ml acetone), 100 mM potassium phosphate buffer , pH 6.8 and necessary cofactors. Percent stimulation was calculated relative to the metabolism in the absence of added lipid.

stituted mixed-function oxidase system is not specific. In fact, several nonionic detergents such as Triton X-100, Triton N-lOl and Emulgen 911 at appropriate concentrations can replace the lipid and function effectively in the reconstituted system (Lu et al., 1974b). In most cases, the concentration of nonionic detergent which gives maximal stimulation falls within a very narrow range; higher 'concentrations often strongly inhibit the overall reaction .. Ionic detergents such as I cholate and deoxycholate can also replace lipid but arc much less effective. Studies have appeared in the literature reporting little or no stimulatory effect by lipid on the rate of metabolism in a reconstituted system. The lack of effect of lipid in these studies may very well be due to the presence of sufficient amounts of either lipid or detergent in these preparations of cytochrome P-450 and NADPH-cytochrome c reductase. When the levels of lipid and detergent in both enzyme preparations are sufficiently low, all reconstituted systems reported to date show a significant dependency on lipid or detergent for catalytic activity. In our own laboratory, we have observed that using the same enzyme preparations and similar assay conditions in metabolism studies with the reconstituted system. the degree of lipid dependency varied depending on the substrate (Table 4). For example, in the absence of lipid, reaction rates range from less ' than 10 per cent for benzphctamine N-demethylation to as high as 80 per cent for the deethylation of ethoxycoumarin as compared to the rate in the presence of Iipid. iAlthough the mechanism of this lipid dependency is unclear, it is tempting to speculate that the lipid, detergent or lipid-detergent micelle may influence the concentration of neutral substrate available to the enzyme for metabolism.' A systematic study encompassing the physical properties of substrate pKa and partition character has not yet been done. Alternatively, those substrates which show the least dependency on lipid ,may not only function as substrates but also behave in the reconstituted system in a manner similar to lipid and facilitate complex formation and electron transfer between reductase and cytochrome P-450.

A. Y. H. Lu and S. B.

342 TABLE

WEST

4. Lipid Requirement for the Metabolism of Various Substrates

Substrate Benzphetamine Benzo(a)pyrene Zoxazolamine Chlorobenzene Ethoxycoumarin

Percentage activity in the absence of added lipid <10 20-30

40 44 60-80

ROLE OF LIPID

Strobel et al. (1970) have shown that phospholipid is essential for efficient electron transfer from NADPH to cytochrome P-450. In the absence of lipid, the rate and the extent of cytochrome P-450 reduction by NADPH and NADPH-cytochrome c reductase are very low. Since synthetic dilauroyl phosphatidylcholine as well as certain synthetic nonionic detergents can replace the lipid, it is clear that the phospholipid does not function in the system as an electron carrier. The exact mode of action of the lipid in the reconstituted hydroxylation system is still unknown, but it appears that the phospholipid in some way facilitates electron transfer from NADPH-cytochrome c reductase to cytochrome P-450. Recently, Coon et al. (1976) have reported that the apparent binding constant for the binding of reductase to cytochrome P·450 decreases from 4.8 x 10. 7 M to 1.2 X 10-7 M in the presence of dilauroyl phosphatidylcholine. This effect of phosphatidylcholine on complex formation may partially explain the manner in which it facilitates electron transfer from NADPH to cytochrome P-450. In many earlier studies, attempts to solubilize liver microsomal cytochrome P-450 with detergents, lipase, organic solvents, or other agents often resulted in the conversion of cytochrome P-450 to P-420. Thus, it was thought for some time that the unique absorption maximum of cytochrome P-450's CO-difference spectrum was due to the close association of this pigment with the microsomal membrane. The recent successful isolation of purified cytochrome P-450 essentially free of lipid and detergent yet retaining the unique spectral properties of membrane-bound cytochrome P-450 clearly demonstrates that lipid is not responsible for the unique CO-difference spectrum of cytochrome P-450. However, it has been observed with some purified cytochrome P-450 preparations that during the measurement of their CO-difference spectra, the presence of either detergent or lipid could minimize the conversion in the cuvettes of cytochrome P-450 to P-420 presumably by stabilizing the sometimes unstable reduced form of cytochrome P-450 (Imai and Sato, 1974; Arinc and Philpot, 1976). Several studies have suggested that phospholipid may also be required for the binding of Type I substrate to cytochrome P-450 since treatment of liver microsomes with phospholipase or isooctane results in the decrease or even total loss of Type I binding (Chaplin and Mannering, 1970; Eling and DiAugustine, 1971; Leibman and Estabrook. 1971). This interpretation of the results of these experiments has, however, been questioned since the hydrolyzed products from phospholipid digestion by phospholipase may be interfering with substrate binding to cytochrome P-450. In addition, treatment of microsomes which are suspended in an aqueous medium with organic solvents such as isooctane often results in massive conversion of cytochrome P-450 to P-420. Thus, the decrease or loss in Type I binding could be very much dependent on the extent of destruction of the specific forms of cytochrome P-450 responsible for the binding of the particular substrates studied. Recently, Vore et al. (1974b) have shown that the removal of 80-90 per cent of the phospholipid by repeated extractions of lyophilized microsomes with n-butanol and acetone actually increases the magnitude of Type I binding per nanomole of cytochrome P-450 . Furthermore, purified cytochrome P-450 which is essentially free of lipid and deter-

Reconstituted mammalian mixed-function oxidases

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gent gives both Type land Type II binding spectra with a variety of substrates without addition of lipid (Levin et al., 1974). Thus, it is unlikely that phospholipid is absolutely required for substrate binding to cytochrome P-450, but phospholipid may modulate the binding. For example, Coon et al. (1976) have reported that the K. for the binding of benzphetamine to P-450 LM 2 decreases from 6.2 x 10-4 M to 1.0 X 10-4 M in the presence of dilauroyl phosphatidylcholinc. For those substrates which are totally insoluble in aqueous medium, the presence of lipid may increase their solubility and thus affect the binding of these compounds to purified cytochrome P-450. Lipid is essential not only for the' catalytic activity of the reconstituted cytochrome P-450-containing system and microsomes, but also appears to playa role in the in vivo induction of liver microsomal cytochrome P'-450 by phenobarbital. Several studies (Marshall and McLean, 1971; Norred and Wade, 1973; Cooper and Feuer, 1973; Mezey et al., 1975; Saito et al., 1975; Rowe and Wills, 1976) have shown that in rats fed a purified synthetic diet or a choline-deficient diet, the response of the liver to induction of cytochrome P-450 by phenobarbital is severely reduced in comparison to the response in rats fed a purified synthetic diet containing herring oil, linoleic acid, or 0.1 per cent oxidized sitosterol or a choline-supplemented diet. Thus, in rats, adequate synthesis of phosphatidylcholine from dietary lipid and choline is necessary for maximum phenobarbital-induced synthesis of cytochrome P-450. HYDROXYLATION BY OTHER HEMEPROTEINS

In addition to cytochrome P-450, several other hemeproteins (such as hemoglobin, myoglobin, cytochrome b~, cytochrome c and ,peroxidase) can also, catalyze the hydroxylation of aniline and several other substrates (Symms and Juchau, 1974). Thus, when hemoglobin and myoglobin are added to liver microsomes, the hydroxylation rate of aniline and acetanilide (but not' the N-demethylation of ethylmorphine and aminopyrine) is enhanced (Jonen et al., 1976). In addition, a reconstituted system containing hemoglobin and NADPH-cytochrome c.reductase can hydroxylate aniline with an efficiency similar to the reconstituted mixed-function oxidase system containing cytochrome P-450 (Mieyal et al., 1976). This system can also catalyze the N-demethylation of benzphetamine. Despite the fact that a hemoglobin-containing reconstituted system can function as a mixed-function oxidase, there are significant differences between the hemoglobincontaining system and the cytochrome P-450-containing system. (a) The former system is only partially dependent on NADPH-cytochrome c reductase while the latter system is completely dependent on the reductase for catalytic activity (if the cytochrome P-450 preparation used is not contaminated with the reductase); (b) The hemoglobin system exhibits no lipid dependency while the presence of lipid is essential in the cytochrome P-450 system. Thus, in the hemoglobin-containing system, it is not possible to study the lipid-protein interactions of the microsomal mixedfunction oxidase; and (c) Catalase and cyanide strongly inhibit the hydroxylation of aniline supported by the hemoglobin-containing system, but have no effect on the reaction catalyzed by the cytochrome P-450 system. These differences lead one to conclude that caution must be used when extrapolating the results obtained from a hemoglobin-containing system to a cytochrome P-450-containing system. ROLE OF CYTOCHROME

b,

IN HYDROXYLATION

The role of cytochrome b 5 and the mechanism of the synergistic effect of N ADH on NADPH-dependent hydroxylation has been extensively examined in liver microsomes. Based on spectral, kinetic and antibody studies, it has been suggested that cytochrome b~ plays an important role in the NADPH-dependent hydroxylation system (Hildebrandt and Estabrook, 1971; Correia and Mannering, 1973; Sasame et al., 1975). However, the direct involvement of cytochrome b s in NADPH-dependent hydroxylation has been questioned by Staudt et al. (1974) and Janssen and Schenkman (1973).

344

A. Y. H. Lu and S. B. WEST

Cytochrome b, is not an obligatory component for NADPH-supported hydroxylation in rat or rabbit liver reconstituted mixed-function oxidase system. Of .the several mixed-function oxidase systems reconstituted thus far, all can metabolize many substrates with turnover numbers equal to, or greater than, microsomal suspensions in the absence of cytochrome b s (Levin et al., 1974; van der Hoeven and Coon, 1974; Ryan et al., 1975a; Imai, 1976; Karnataki et al., 1976a). When cytochrome b, was added to the reconstituted system at a cytochrome b s ' to P-450ratio normally found in microsomes, the rate of NADPH-dependent hydroxylation was inhibited, not affected or stimulated depending on the substrate used, .the type of reaction studied. and the specific form of cytochrome P-450 employed (Lu et al., 1974a, c, 1975). Thus; while cytochrome b, is not an obligatory component of the, reconstituted mixedfunction oxidase system, it may modulate the reaction rate by interacting with the various components. Even though cytochrome b s is not directly involved in NADPH-dependent hydroxylation, it is an obligatory component of the reconstituted, NADH-dependent benzo(a)pyrene hydroxylase system (West et al., 1974) and the organic hydroperoxide reduction system (Hrycay et al., 1975). The involvement of cytochrome b s in NADHdependent hydroxylation, and in the synergistic effect of NADH has, also been established in studies with liver microsomal suspensions (Hildebrandt and Estabrook. 1971; Mannering, 1975; Sasame et al., 1975). OTHER SUGGESTED COMPONENTS

Over the years, the involvement of several other components in microsomal NADPH-dependent hydroxylation has been postulated. Suggested components include a non-heme iron protein, a soluble factor, a selenium-containing component, DT-diaphorase and protein carriers. In many of the reaction schemes depicted in the literature, a component 'X' has often been postulated as an intermediate electron carrier positioned between NADPH-cytochrome c reductase and cytochrome P-450. Mull et al. (1975) have designated one of the protein bands resulting from polyacrylamide gel electrophoresis of microsomes in the presence of SDS 'factor X'. The evidence for the identity of 'factor X' and the basis for this assignment was not given. A 'factor X' or other components have been suggested as participants in the liver microsomal mixed-function oxidase system for the foIlowing reasons: (a) since a non-heme iron protein is an obligatory component in the cytochrome P-450-containing hydroxylation system in adrenal mitochondria (Omura et al., 1966; Kimura and Suzuki, 1967) and in Pseudomonas putida (Tsai et al., 1971; Tyson et al., 1972), it is tempting to speculate that an adrenodoxin- or putidaredoxin-like protein may also be a required component in the liver microsomal mixed-function oxidase system; (b) changes in cytochrome P-450 content did not adequately account for the species, strain, age, tissue and sex differences observed in drug metabolism. At the time it was not generaIly thought that there were multiple forms of cytochrome P-450 and it was suggested that one way to account for the observed differences was to postulate specific protein carriers for the various substrates (Dallner et al., 1965). Although an iron-sulfur species has recently been found in rat liver microsomal preparations by potentiometric titration and electron paramagnetic resonance (lngledew et al., 1976), there is no direct evidence implicating any iron-sulfur protein or protein carriers in microsomal hydroxylation. Caygill et al, (1973) have suggested that a seleniumcontaining non-heme iron protein may function in microsomal hydroxylation, but have offered little convincing evidence. Other studies have shown that the induction of cytochrornes P-450 and b s by phenobarbital is impaired in selenium-deficient rats (Burk et al., 1974; Burk and Masters, 1975). However, this impairment in cytochrome P-450 induction in selenium-deficient rats has recently been shown to be due to increased degradation of hepatic heme as a result of increased heme oxygenase activity following phenobarbital treatment (Correia and Burk, 1976). The addition of the 'soluble fraction' (105,000 x g supernatant) to the liver micro-

Reconstituted mammalian mixed-function oxidases

345

somal mixed-function oxidase incubation mixture has been shown to stimulate the metabolism of several substrates, raising the question as to whether a soluble factor is a required _component in the microsomal mixed-function oxidase system. It seems unlikely, since recent studies have shown that, at least for the N-demethylation of ethylmorphine, the stimulation of activity is due mostly to the ability of the soluble fraction to reverse the inhibitory effect of lipid peroxidation and thus improve the linearity of the reaction (Karnataki et 01., 1974; Kotake et 01., 1975). It has been suggested that microsomal DT diaphorase may also play a role in the hydroxylation of benzo(a)pyrene by micro somes from 3-methylcholanthrene-treated rats (Lind and Ernster, 1974). This suggestion is .based on the parallel induction of DT diaphorase and benzo(a)pyrene hydroxylase by 3-methylcholanthrene and the inhibition of both activities by 7,8-benzoflavone. However, Huang et 01. (977) have recently shown that the purified, reconstituted mixed-function oxidase system which contains no DT diaphorase hydroxylated benzo(a)pyrene at a faster rate than micro somes and if purified DT diaphorase was added to this reconstituted system it neither stimulated nor inhibited the reaction. Since the purified, reconstituted mixed-function oxidase is able to metabolize a variety of substrates as well as, or better than, microsomes, the involvement of DT diaphorase or any of the suggested components in microsomal hydroxylation is highly unlikely. RECONSTITUTED ADRENAL MITOCHONDRIAL MIXED-FUNCTION OXIDASE The adrenal mitochondrial system was the first mammalian mixed-function oxidase to be resolved and reconstituted. This reconstituted system differs from the liver microsomal reconstituted system in several important ways. (a) In the mitochondrial system, electrons are transferred from NADPH-adrenodoxin reductase to cytochrome p-450 through adrenodoxin, an iron-sulfide protein, whereas in the reconstituted liver microsomal system, electrons are transferred directly from NADPH-cytochrome c reductase to cytochrome P-450 and no non-heme iron protein is required. (b) The mitochondrial cytochrome P-450 reducing system (i.e, NADPH-adrenodoxin reductase and adrenodoxin) can be easily solubilized without the aid of detergent by sonicating mitochondria suspended in water (Omura et 01., 1966). Once released from the membrane, both proteins remain soluble and do not aggregate. Thus, the purified adrenodoxin has a molecular weight of 19,000 (Kimura et 01., 1969) whereas the purified NADPH-adrenodoxin reductase has a molecular weight of about 54,000 (Chu and Kimura, 1973) and contains one mole of FAD per mole of enzyme. In contrast, detergent must be used to solubilize liver microsomal NADPH-cytochrome c reductase and the purified enzyme exists as an aggregate with an apparent molecular weight of about half a million. In the presence of SDS, the molecular weight of the subunit is 79,000 and each subunit contains one molecule each of FAD and FMN (Iyanagi and Mason, 1973; Vermilion and Coon, 1974; Dignam and Strobel, 1975; Yasukochi and Masters, 1976). In addition, NADPH-adrenodoxin reductase isolated from bovine adrenal mitochondria has been shown to be immunologically distinct from liver microsomal NADPH-cytochrome c reductase (Masters et 01., 1971). (c) While phospholipid is a required component of the liver microsomal reconstituted system, thus far, there is no conclusive ev idence to indicate that phospholipid is required for the mitochondrial reconstituted system. (d) The reconstituted liver microsomal system has a very broad substrate specificity (see Table 3) and is capable of metabolizing steroids, fatty acids, drugs, carcinogens and a variety of foreign compounds. In contrast, the reconstituted mitochondrial system appears to metabolize only steroids such as t t-deoxycorticosterone and cholesterol. The difference in substrate specificities of these two systems is undoubtedly due to the difference between liver microsomal and adrenal mitochondrial cytochrome P~50s. For example;Thomas et 01. (1976a) have shown by Ouchterlony double diffusion analysis that antibody to purified liver microsomal cytochrome P-450 from PB- or -3MC-treated rats does not cross-

A. Y. H. Lu dand S. B. WEST

346

react with the adrenal mitochondrial cytochrome P-450 active in 11,B-hydroxylation of deoxycorticosterone. The Pllrification of adrenal mitochondrial cytochrome P-450 has been reported by several laboratories (Schleyer et al., 1972; Mitani et al., 1973; Ramseyer and Harding, 1973; Shikita and Hall, 1973a; Horie and Watanabe, 1975; Takemori et al., 1975a, b ; Wang and Kimura, 1976). Depending on the method, the detergent used, and the particular species of cytochrome P-450 isolated, these preparations differed in their specific content (2-12 nmol P-450 per mg protein), heme content, molecular weight of the subunit determined in the presence of SDS (ranging from 46,000 to 60,000), spin state (either high or low spin or a mixture of both) and substrate specificity. Based on an earlier study of Jefcoatc et al. (1970) and the above studies, it is now evident that different forms of cytochrome P-450 are responsible for the IIf:J-hydroxylation of deoxycorticosterone and side-chain cleavage of cholesterol. The properties of these two different forms of cytochrome P-450 purified from adrenal mitochondria by Takemori et al. (1975a, b) are compared in Table 5. Shikita and Hall (l973b) have purified the cytochrome P-450 responsible for the side chain cleavage of cholesterol from bovine adrenocortical mitochondria and have found that the apparent molecular weight of the purified enzyme is 850,000. This species consists of 16 subunits but it can also exist in forms containing 8 or 4 subunits. More drastic treatment (e.g , 6 M guanidine HCl) results in the formation of a monomer with a molecular weight of 53,000. The catalytically active form of cytochrome P-450 contains 16 subunits whereas the forms containing 8 or 4 subunits are not enzymatically active (Takagi et al., 1975). More recently, the same laboratory has reported that the 16 subunit form actually contains two different subunits in equal amounts, but only one of the subunits contains heme (Tilley et al., 1976). Ideally, a cytochrome P-450 preparation should be homogeneous if its structure and the association of its . subunit are to be studied. The low specific content of P-450 (2.9 nmol/mg protein) and the high specific content of heme (9.5 nmol/mg protein) raise the Question as to TABLE

5. Properties of Two Different Forms of Adrenal Mitochondrial Cytochrome P-450· Parameters

Absorption spectrum (nrn) Oxidized Reduced Reduced-CO Endogenous Cholesterol (mol Cholesterol/mol protoheme) Minimal Molecular Weight SDS-gel electrophoresis Sedimentation in the presence of guanidine-Hel lind mercaptoethanol 11.a-Hydroxylase Activity (nmol/min/nmol P-450) Cholesterol side-chain cleavage activity (nmol/min/nOlol P-450)

P-450 116

P-450,,<

418,539,570t 394,510,645* 410,550 448,550

394, 540, 645§

insignificant

415,540 448,550 0.6-1.0 greater than

46,000

46.0001l

43,000

46,000

60

0.0

insignificant

0.7

·Cytochrome P-450 1l /l refers to the cytochrome P-4S0 catalyzing the conversion of deoxycorticosterone to corticosterone (Takemori et 01., I975b) whereas cytochrome P-450.« refers to the enzyme catalyzing the conversion of cholesterol to pregnenolone by cleaving the side chain of cholesterol (Takemori et al., 1975a). tOxidized spectrum in the absence of substrate. tOxidized spectrum in the presence of substrate. deoxycorticosterone. §Cytochrome P-450l
Reconstituted mammalian mixed-function oxidases

347

whether the preparation of Shikita and H~II (1973a) is indeed homogeneous. Also to be answered is the question whether the subunit species which appears to contain no heme is indeed cytochrome P-450. In addition, the purity of the preparation should be further examined electrophoretically in an SDS-gel system with better resolving power than the method of Weber and Osborn (1969). As an example, Levin (1977) has shown that a purified rat liver microsomal cytochrome P-450 preparation shows only one protein band using the gel system of Weber and Osborn, 1969, but the same preparation resolves into 4 bands by the method of Laemmli (1970). RECONSTITUTED ADRENAL MICROSOMAL MIXED-FUNCTION OXIIjASE The mixed-function oxidase system of bovine adrenocortical micro somes has been partially resolved without the use of detergents by Narasimhulu (1974). Lyophilized adrenal micro somes were first extracted with butanol to' remove phospholipids (80-85 per cent). The phospholipid-depleted micro somes lost 90-95 per cent of their C-21 steroid hydroxylase activity which could then be completely restored by the addition of the non-ionic detergent Triton X-114 and partially restored by the addition of Asolectin or egg lecithin plus lysolecithin micelles. The butanol-extracted micro somes were then centrifuged in a sucrose density gradient and partially resolved into a 'particulate fraction' containing cytochrome P-450 and a 'reductase fraction' containing NADPH-cytochrome c reductase. When these two fractions were recombined in the presence of Triton X-114, full activity was restored and in the presence of phospholipids, partial activity was restored. The loss of activity upon the removal of phospholipid from adrenal microsomes and the restoration of activity upon the addition of phospholipids or detergent resemble the results of studies .of Vore et al. (1974a) and Lotlikar et al. (1976) using liver microsomes. Thus, like the liver microsomal reconstituted system, the reconstituted adrenal system requires lipid or detergent for maximal catalytic activity. On the other hand, the adrenal microsomal system hydroxylates only steroids and thus does not share the broad substrate specificity of the liver microsomal system. RECONSTITUTED LUNG MICROSOMAL MIXED-FUNCTION OXIDASE The lung microsomal mixed-function oxidase system has been reconstituted from solubilized components isolated from both rats and rabbits (Philpot et al., 1975; Jernstrorn et al., 1975; Arinc and Philpot, 1976). Jernstrorn et al. (1975) have partially purified cytochrome P-450 from rat lung microsomes to a specific content of 0.66 nmol/mg protein by chromatography on an affinity column (an w-amino-n-octyl derivative of Sepharose 4B) followed by chromatography on Sephadex 0-200 and DE-52 cellulose. This partially purified lung cytochrome P-450 when recombined with NADPH-cytochrome c reductase partially purified from rat liver microsomes hydroxylated benzo(a)pyrene; partially purified NADPH-cytochrome c reductase . from lung microsomes was inactive in this reconstituted system. No requirement for phospholipid was observed, presumably due to the presence of significant amounts of lipids, detergents or both in the cytochrome P-450 and reductase fractions. Arinc and Philpot (1976) have recently made significant progress in purification of lung microsomal cytochrome P-450 and have purified it an impressive thirty-twa-fold to a specific content of 7.4 nmoljrngprotein by a combination of salt fractionation and column chromatography (DEAE-cellulose and hydroxylapatite). A unique feature .of this procedure is solubilization and ammonium sulfate fractionation in the absence of glycerol. In fact, if glycerol is included in these steps, the yield of cytochrome P-450 is greatly decreased. This observation is in contrast to studies with liver microsomal cytochrome P-450 in which the presence of glycerol is essential for purification. This partially purified lung microsomal cytochrome P-450, when recombined with partially purified, detergent-solubilized lung microsomal NADPH-cytochrome c reductase and lipid, catalyzes the O-dcethylation of 7-ethyoxycoumarin and the

348

A. Y. H. Lu and S. B. WEST

N-demethylation of :benzphetharnine. The lipid fraction is not only required for maximal activity of the reconstituted system, but is also essential for quantitation of cytochrome P-450 by its CO-difference spectrum presumably by preventing the conversion of cytochrome P-450 to P-420. Another interesting observation is that if the lung NADPH-cytochrome c reductase is solubilized with sodium cholate, it is catalytically .active in the reconstituted system, whereas if the same reductase is solubilized with sodium deoxycholate, it is not active (Philpot et al., 1975). In contrast, liver microsomal NADPH-cytochrome c reductase is active in the reconstituted liver microsomal system whether it is solubilized with cholate or deoxycholate. The inactivity of deoxycholate-solubilized lung microsomal NADPH-cytochrome c reductase observed by Philpot et al. (1975) as well as the inability of lung microsomal NADPH-cytochrome c reductase to function in the studies of Jernstrorn et al. (1975) suggest that the interaction of cytochrome P-450 and reductase in the reconstituted lung system may not be the same as their interaction in the reconstituted liver system. It is also quite 'possible that the physical, chemical, immunological and catalytic properties of purified lung microsomal cytochrome P-450 differ from those of purified liver microsomal cytochrome P-450 and a comparative study might be most interesting.

RECONSTITUTED KIDNEY MICROSOMAL MIXED-FUNCTION OXIDASE Unlike the liver microsomal system, the kidney microsomal mixed-function oxidase does not have a broad substrate specificity. It is specific for the 6J and 6J - 1 hydroxylation of fatty .acids and either does not metabolize or only poorly metabolizes drugs and other foreign compounds (EIlin and Orrenius, 1975). The kidney microsomal system has been solubilized by Ichihara et al. (971) using Triton X-tOo and resolved into two fractions by DEAE-cellulose column chromatography. Neither fraction is pure but fraction I contains cytochrome P-450 and fraction II contains NADPH-cytochrome c reductase. Neither fraction alone is catalytically active, but when the two fractions are combined they hydroxylate medium-chain fatty acids. The fraction containing NADPH-cytochrome c reductase (fraction II) can be replaced by spinach ferredoxin, a non-heme iron protein, and NADPH-ferredoxin reductase. If fraction I is extracted with ether, activity is markedly decreased but can be completely restored by addition of Triton X-lOO but not by other detergents or by phospholipids. The kidney microsomal reconstituted system has not, thus far, been shown to require lipid, however, the presence of Triton X-loo appears to be essential for maximal activity. Although kidney microsomal NADPH-cytochrome c reductase and cytochrome P-450 have now been partially purified (Ichihara et al., 1973, 1974), it is still not known if a non-heme iron protein participates in kidney microsomal hydroxylation. Hoffstrom et at. (1972) have suggested the possible involvement of an iron-sulfur protein based on electron paramagnetic resonance studies. However, at present, the evidence in support of involvement of a non-heme iron protein in kidney microsomal hydroxylation is inconclusive. The different substrate specificities of the liver and kidney microsomal hydroxylation systems are most likely due to inherent differences in their respective cytochrome P-450s. Ichihara et al., 1971, have observed comparable activities when kidney fraction I, the fraction containing cytochrome P-450, is coupled with either kidney fraction II or the same fraction isolated from liver microsomes-liver fraction II. However, if kidney fraction I is replaced by liver fraction I, hydroxylation activity is greatly decreased indicating that substrate specificities reside in fraction I and most likely in cytochrome P-450. RECONSTITUTED KIDNEY MITOCHONDRIAL MIXED-FUNCTION OXIDASE Chick kidney mitochondria catalyze the NADPH-dependent hydroxylation of 25hydroxycholecalciferol (25-hydroxyvitamin DJ ) to . t ,25-dihydroxycholecalciferol

Reconstituted mammalian mixed-function oxidases

349

(1,25-dihydroxyvitamin D J , the proposed metabolically active form of vitamin D J ) (Ghazarian and DeLuca, 1974). This mitochondrial la-hydroxylase system has been solubilized and reconstituted from the chick kidney and consists of cytochrome P-450, ferredoxin and ferredoxin reductase (Ghazarian et al., 1974; Pedersen et al., 1976). Adrenodoxin and adrenodoxin reductase isolated from adrenal mitochondria- can be substituted for ferredoxin and ferredoxin reductase in this reconstituted system. Kidney mitochondrial cytochrome P-450 appears to be distinct from kidney microsomal cytochrome P-450 based on binding studies. In contrast, ferredoxin from the kidney is very similar to adrenodoxin from ·the adrenal judging from their molecular weight and spectral, catalytic and immunological properties. RECONSTITUTED CORPUS LUTEUM MITOCHONDRIAL MIXED-FUNCTION OXIDASE The cholesterol side chain cleavage enzyme system of corpus luteum mitochondria seems to be identical to the corresponding enzyme complex in the adrenal cortex. However, unlike adrenal mitochondria, corpus luteum mitochondria possess only side chain cleavage activity and not IIf3-hydroxylase or 18-hydroxylase activity. Bovine corpus luteum mitochondrial cytochrome P-450 has been solubilized and partially purified to a specific content of I nmoljrngprotein by McIntosh et al, (1973) and was isolated in a high-spin substrate-bound form. When recombined with crude NADPHadrenodoxin reductase isolated from corpus luteum and adrenodoxin isolated from adrenal cortex, this reconstituted system catalyzed the conversion of cholesterol to pregnenolone. Ovarian adrenodoxin has not been purified because of its low content in the corpus luteum. A reconstituted system consisting of cytochrome P-450 solubilized from bovine corpus luteum, and adrenodoxin and adrenodoxin reductase purified from adrenal cortex also catalyzed the side chain cleavage of cholesterol (Caron et al., 1975). If cytochrome P-450 were made limiting and ATP, cyclic AMP, and cyclic AMPdependent protein kinase were then added to this reconstituted system, the conversion of cholesterol to pregnenolone was stimulated from 20 to 74 per cent. The stimulatory effect of the protein kinase was dependent on ATP. Protein kinase caused a phosphorylation of the cytochrome ~-450 fraction suggesting that activation of cytochrome P-450 or a component in the cytochrome P-450 fraction by protein kinase may be one of the mechanisms by which cyclic AMP mediates the effect of luteinizing hormone on steroidogenesis in the bovine corpus luteurn, RECONSTITUTED LIVER TUMOR MICROSOMAL MIXED-FUNCTION OXIDASE A variety of hepatomas are capable of metabolizing drugs and carcinogens, although at a much slower rate than liver. Recently, the microsomal mixed-function oxidase system from Hepatoma 5123 t.c, has been resolved and reconstituted (Saine and Strobel, 1976). The same three components required for liver microsomal activity, i.e. cytochrome P-450, NADPH-cytochrome c reductase and lipid, were also found to be required for liver tumor microsomal activity. The hepatoma cytochrome P-450 has been partially purified to a specific content of 1 nmol/mg protein and the reductase has· been purified to a specific activity of 8660 nmol cytochrome c reduced/min/mg protein. The K,« values of the purified hepatoma reductase for artificial electron acceptors' (such as cytochrome c and ferricyanide) were similar to those obtained with purified liver reductase; however, the K m of the hepatoma reductase for NADPH was an order of magnitude higher than that obtained for the liver enzyme as determined either by cytochrome c reduction or benzphetamine N-demethylation. These results Suggest that although the hepatoma' reductase is similar to the liver reductase, its binding site for NADPH maybe altered so that it has a reduced affinity for the electron donor. Whether the hepatoma .cytochrorne P-450 is different from liver cytochrome P-450 remains to be established.·

350

A. Y. H . Lu and S. B. WEST

SUBSTRATE SPECIFICITIES OF VARIOUS FORMS OF PURIFIED LIVER MICROSOMAL CYTOCHROME P-450 DETERMINATION OF SUBSTRATE SPECIFICITY The component responsible for determining the substrate specificity of the liver microsomal mixed-function oxidase was determined in the following way. Using the reconstituted system, two of the three components were kept constant and the source of the third component was varied to test for an effect on substrate specificity. This procedure is applicable to other mixed-function oxidase systems which have been resolved and can be reconstituted. In one of the first such studies (Lu et al., 1971). it was observed that the reconstituted system isolated from rats treated with PB exhibited high activity for benzphetamine N-demethylation but very low activity for benzo(a)pyrene hydroxylation. However, if the cytochrome P-450 fraction from PB-treated rats was replaced with the cytochrome P-448 fraction isolated from rats treated with 3-MC, the rate of bcnzphetarnine N-demethylation was greatly decreased while the rate of benzo(a)pyrene hydroxylation was greatly increased. On the other hand, the reconstituted system from 3-MC-treated rats showed good benzo(a)pyrene hydroxylase activity which was greatly decreased if the cytochrome P-448 fraction was replaced with the P-450 fraction isolated from rats treated with PB. These results indicated that the substrate specificity of the hydroxylation system resides in the cytochrome fraction rather than in the reductase or lipid fraction. Subsequent studies showed that the cytochrome fraction is primarily responsible for the difference in substrate specificities between the liver microsomes of control, PB- and 3-MC-treated rats (Lu et al., I972a, 1973a; Lotlikar et al.• 1975a). control and ethanol-treated rats (Villeneuve et al., 1976), male and female rats (Bjorkhern et al., 1974a), starved and normal rats (Bernhardsson et al., 1973), control and 3-MC-treated hamsters (Lotlikar et al., 1974) and inducible and non-inducible mouse strains (Nebert et al., 1973). More recent studies with highly purified cytochrome P-450 from a variety of sources have confirmed that the cytochrome is primarily responsible for the substrate specificity of the mixed-function oxidase (Ryan et al., 1975a; Kawalek et al., 1975; Haugen et al., 1975; Hashimoto and Irnai, 1976; Haugen and Coon. 1976). Unlike the cytochrome P-450 component, the source of the lipid has no effect on the catalytic activity of the reconstituted system (Lu et al., 1972a; Nebert et al., 1973; Joly et al., 1976). The source of the reductase also usually has no effect on the catalytic activity of the mixed-function oxidase. although with a few substrates the rates are affected but to much smaller extent than by cytochrome P-450 (Lu et al. , 1972a). Two forms of reductase have recently been purified from rats and from rabbits (Coon et al., 1977). Thus. while the substrate specificity of the mixed-function oxidase is primarily determined by the various forms of cytochrome P-450, the presence of multiple forms of reductase may also affect the rates of metabolism of some substrates. DIFFE,RENT FORMS OF CYTOCHROME P-450 FROM ANIMALS TREATED WITH DIFFERENT INDUCERS

It has been known for many years that PB and 3-MC preferentially induce the metabolism of different substrates and it was suggested that these compounds exert their effect by inducing the synthesis of different cytochrome P-450s (Conney, 1967; Kuntzman. 1969; Mannering, 1971). This hypothesis was definitively shown to be correct in 1975 when cytochrome P-450 from PB-treated rats and cytochrome P-448 from 3-MC-treated rats were purified and shown to possess different substrate specificities (Ryan et al.• 1975a). The substrate specificity of purified cytochromes P-450 and P-448 is very similar to the microsomes from which they were derived. Thus. cytochrome P·450 preferentially catalyzes the N-demethylation of benzphetamine and the metabolism of many other substrates whereas cytochrome P-448 preferentially catalyzes the hydroxylation of benzo(a)pyrene and other compounds

Reconstituted mammalian mixed-function oxidases

35t

(Ryan et al., 1975a; Levin et al., 1974). Purified cytochromes P-450 and P-448 also differ in their spectral properties (CO-difference spectra as well as ethylisocyanide difference spectra) and subunit molecular weight (48,000 vs. 53,000) as determined by gel electrophoresis in the presence of SDS (Ryan et al., 1975a). Furthermore, antibodies produced against the purified enzymes react well with their homologous antigens but cross-react poorly with their heterologous antigens (Thomas et al., 1976a). Even though more recent studies indicate that the purified cytochrome P-450 preparation from PB-treated rats contains at least 4 immunologically distinct forms and that the purified cytochrome P-448 preparation from 3-MC-treated rats contains at least 2 distinct forms (Thomas et al., 1976b), it is obvious that the cytochrome P-450 population in microsomes from PB- or 3-MC-treated rats is different. Different forms of cytochrome P-450 have also been purified from rabbits treated with PB, 3-MC or {3-naphthoflavone (Haugen et al., 1975; Haugen and Coon, 1976; Imai and Sato, 1974; Hashimoto and Imai, 1976). The 'cytochrome P-450 induced by PB (LM 2) differs from the form induced by 3-MC or {3-naphthoflavone (LM 4) in several ways. For example, LM 2 was most effective in catalyzing the metabolism of substrates such as benzphetamine, ethylmorphine and p-nitroanisole whereas LM 4 was rather poor in metabolizing these substrates. The absorption maximum of the reduced CO-difference spectrum was 451 nm for LM 2 and 447-448 nm for LM 4• In the presence of SDS, the subunit molecular weight was 48,700 for LM 2 and 55,300 for LM•. The results of amino acid analysis indicated that LM 2 and LM 4 were similar in composition but the latter protein had about 60 additional residues. The C-terminal amino acid of LM 2 was arginine whereas that of LM. was lysine. Finally, antibodies produced against these two cytochromes P-450 did not cross-react with each other (Dean and Coon, 1977). DIFFERENT FORMS OF CYTOCHROME P-450 FROM DIFFERENT ANIMALS TREATED WITH THE SAME INDUCER Although cytochrome P-450s purified from different animals treated with the same inducer have identical absorption maxima in their reduced CO-difference spectra, there is other evidence showing that these cytochrome P-450 preparations are indeed different proteins. Cytochrome P-448 purified from 3-MC-treated rats was approximately twenty-fold more effective than cytochrome P-448 purified from 3-MC-treated rabbits in catalyzing the hydroxylation of benzo(a)pyrene (Kawalek et al., 1975). The subunit molecular weight of these two enzymes differed on SDS-gel electrophoresis. In addition, antibodies produced against these two enzymes were quite specific for their homologous antigen and cross-reacted poorly with the heterologous antigen (Thomas et al., 1976a). The substrate specificity of cytochrome P-450 purified from PH-treated rats and PB-treated rabbits was very similar (Kamataki et al., 1976a, b). Despite this similarity, these two enzymes are apparently two different proteins since antibody produced against purified rat P-450 cross-reacted poorly with P-450 purified from PB-treated rabbits (Thomas et al., 1976b; Kamataki et al., 1976b). DIFFERENT FORMS OF CYTOCHROME P-450 FROM THE SAME ANIMAL (EITHER UNTREATED OR TREATED WITH AN INDUCER) Comai and Gaylor (1973) were the first to physically separate different forms of cytochrome P-450 from liver microsomes of the same animal. Three forms of the hemeprotein were separated by column chromatography as judged by their different cyanide binding affinities. These different forms of cytochrome P-450 were induced to different extents by prior treatment of rats with PB, 3-MC or ethanol. Ryan et al. (1975b) have also separated at least two forms of cytochrome P-450 from both PHand 3-MC-treated rats with different spectral and catalytic properties. More recently, multiple forms of cytochrome P-450 with different substrate specificities have been separated and purified from PH-treated rabbits (Haugen et al.,

352

A. Y. H. Lu and S. B. WEST

1975), untreated rabbits (Philpot and Arinc, 1976) and Pfl-treated mice (Huang et al ., 1976). Haugen et al. (1975) have separated four forms of cytochrome P-450 (LM::. LM l , LM h and LM7 , or LM r,7 which contains a mixture of LM. and LM 7) from PB-treated rabbits and purified some of these forms. Benzphetamine, ethylmorphine and p-nitroanisole were preferentially hydroxylated by LM 2 and benzo(a)pyrene by LM r,7 • Biphenyl was hydroxylated in both the 2 and the 4 positions by all the preparations, but the 4 position was preferentially hydroxylated by LM 2. Testosterone was hydroxylated primarily in the 16a-position·by LM 2 and the 6j3-position by LM I.7 • These {arms also differ in their subunit molecular weights and in the absorption maximum of their reduced CO-difference spectra. Two forms of cytochrome P-450 have been separated and partially purified from untreated rabbits (Philpot and Arinc, 1976). The absorption maximum of the reduced CO·difference spectrum was 450 nm {or form A and 448 nm {or form B. Even though the subunit molecular weights for both forms were very similar. form A was about twice as active as form B in a reconstituted system in supporting the hydroxylation of benzo(a)pyrene and the dealkylation of benzphetamine and 7-ethoxycoumarin. Four distinct cytochrome P-450 fractions (A h A 2, C. and C2) have been separated and purified from the liver microsomes of PB-treated hybrid mice (B6D2FdJ) by Huang et at. (1976). These forms differed in their subunit molecular weights, COdifference spectra (450 nm for AI and C2. 451 nm for A 2 and 449 nm for C.); ethylisocyanide difference spectra and catalytic properties. Benzphetamine was metabolized preferentially by Cr. benzo(a)pyrene by AI and A 2, and 7-ethoxycoumarin and coumarin by AI' Testosterone was hydroxylatcd preferentially in the 6j3-position by C. and C2 • in the 7a-position by A 2 and in the 16a-position by C •. SUBSTRATE SPECIFICITY: MICROSOMES VS. PURIFIED

CYTOCHROME P-450 It is necessary to ensure that a purified cytochrome P-450 has not been altered during solubilization and fractionation and one good criterion is a comparison of the

substrate specificity of the purified enzyme with that of the micro somes from which it was isolated. Because the mixed-function oxidase is a rather complex system it is not always possible to make a meaningful comparison of data obtained from studies with microsomal suspensions and the enzymes purified {rom them. For example. the turnover number of a particular species of cytochrome P-450 for a substrate (expressed as nmol product formed per min per nmol cytochrome P-450) should ideally be determined in the presence of limiting amounts of cytochrome P-450 and excess amounts of NADPH-cytochrome c reductase and lipid. Whereas the components in the reconstituted system can be easily adjusted to satisfy these conditions to obtain a maximal turnover number for a substrate, no such manipulation can be made with microsomal suspensions. The turnover number for a substrate determined with microsomal suspensions is only meaningful if the particular form of cytochrome P-450 in microsomes which metabolizes that substrate is the limiting component. A further complication in liver microsornes is the presence of multiple forms of cytochrome P-450 since the metabolism of a substrate by a particular form of P-450 can be inhibited by other forms of cytochrome P-450. When the turnover number of a substrate determined in the reconstituted system is equal to or greater than t~at of liver microsomes, one can conclude that the purified cytochrome P-450 "has not been altered during purification. However, when the turnover number of a substrate is decreased upon purification. . it is difficult to determine if this is due to perhaps the loss of a particular form of cytochrome P-450, or to the inherent low activity of the form isolated or to an alteration and inactivation of a form of cytochrome P-450 in the purified cytochrome P-450 preparation. For example, benzphetarnine and ethylmorphine are both rapidly Nvdernethylated by microsomes from PB-treated rats with turnover numbers of 10-20. However. with purified cytochrome P-450 from PB-treated rats. the turnover number increased to

Reconstituted mammalian mixed-function oxidases

353

40-80 for benzphetamine but decreased to 5-7 for ethylmorphine (Table 6). The reason for the low activity of purified rat P-450 toward ethylmorphine is unknown, but several possibilities can be considered: (a) The main form of cytochrome P-450 responsible for the metabolism of ethylmorphine has been removed or inactivated (either completely or partially) during the purification of cytochrome P-450; (b) The low activity for ethylmorphine is an inherent property of the purified cytochrome P-450; (c) The small amount of detergent in the cytochrome P-450 preparation could preferentially inhibit the N-demethylation of ethylmorphine but not benzphetamine; and (d) Ethylmorphine could uncouple the reconstituted system leading to the formation of H 20 2 instead of metabolic products. TABLE 6.

Turnover Numbers of Various Substrates: Microsomes vs. Purified Cytochrome P-450 nmol products formed/min/nmol P-450

Substrate Benzphetamine Benzo(a)pyrene Testosterone 6/1·0H 7a-OH 16a·OH Ethylmorphine 7-Ethoxycoumarin

Microsomes

Purified

Source of Enzymes

10-15 1.5-2.5

40-80 2.5-8.0

Rat. PB-treated Rat. 3-MC-treated

2.5 0.5 1.0 10-20 10

0.1-0.2 0.3-0.5 0.5-'1.0 5-7 15 (A,)· 4 (A z) 6 (C ,) 0.3 (C 2)

Rat. PB-treated Rat. PB-treated Rat. PB·treated Rat. PB-treated Mouse. PB-treated Mouse. PB-treated Mouse. PH-treated Mouse. PB-treated

• A" A 2• C, and C z refer to different cytochrome P-450 fractions isolated from Pll-trcatcd hybrid mice (B6D2F,/J) by the method of Huang et at. (1976).

COMMENTS ON MULTIPLICITY OF CYTOCHROME P-450 AND MICROSOMAL HYDROXYLATION The demonstration of the presence of multiple forms of cytochrome P-450 in untreated animals and in induced animals represents a significant advancement in the area of microsomal drug metabolism. The complex kinetics and patterns of inhibition seen with many substrates and inhibitors, and the complex spectral interaction seen in microsomes are best explained by', the presence of multiple forms of cytochrome P-450. These different forms of P-450 and P-448 have broad but overlapping substrate specificities. Thus, a difference in the proportion of various forms of P-450 in a microsomal preparation can perhaps explain the species, strain. age, tissue and sex differences observed in drug metabolism. Pretreatment of animals with an inducer may result in the induction of one or more of the forms of cytochrome P-450present in untreated animals; the resulting change in the proportion of the various forms would change the spectral and catalytic properties of the microsomes of the induced animals as compared with the uninduced animals. Recognition of the existence of multiple forms of cytochrome P-450 has led to a reassessment of some earlier suggested .explanations of the properties of the liver microsomal mixed-function oxidase and the ,following comments summarize some of the current thoughts on the microsomal mixed-function oxidase. (a) The catalytic activity of a, particular form of cytochrome P-450 cannot be predicted solely from the absorption maximum of its reduced CO-difference spectrum. For example, rat cytochrome P-448 is twenty times better than rabbit cytochrome P-448 in supporting benzo(a)pyrene metabolism despite the similarity of their reduced CO-difference spectra. (b) One cannot classify the different forms of cytochrome P-450 according to the types of reaction catalyzed by th~se enzymes. For example. the N-dealkylation and O-dealkylation of a compound might be catalyzed by a single cytochrome P-450 or different forms of cytochrome P-450. Likewise. theIrydroxylatiojj of a compound at

354

A. Y . H . Lu and S. B. WEST

several different positions might be catalyzed by one or several forms of cytochrome P-450. (c) It is unlikely that a particular form of cytochrome P-450 can metabolize only a limited number of substrates. The substrate specificities of various forms of cytochrome P-450 appear to be broad and overlapping. although the rate of metabolism of different forms is different.

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