Studies on the Rate-limiting Enzyme Component in the Microsomal Monooxygenase System INCORPORATION OF PURIFIED NADPH-CYTOCHROME c REDUCTASE AND CYTOCHROME P-450 INTO RAT LIVER

The identity of the rate-limiting enzyme component of the microsomal monooxygenase system has been investigated for six substrates through the incorporation of purified NADPH-cytochrome c reductase into microsomal preparations obtained from untreated, phenobarbital-and 3-meth-ylcholanthrene-treated rats. Incorporation of NADPH-cy- tochrome c reductase results in rate enhancements which depend on both the microsomal preparation and the sub- strate examined. These rate enhancements have been interpreted in terms of the variable cytochrome P-450/reductase mole ratios resulting from the multiplicity of cytochrome P-450 species in microsomal preparations. The rate-limiting component for was examined in greater detail with microsomal preparations in which either incorporated. rate

The identity of the rate-limiting enzyme component of the microsomal monooxygenase system has been investigated for six substrates through the incorporation of purified NADPH-cytochrome c reductase into microsomal preparations obtained from untreated, phenobarbital-and 3-methylcholanthrene-treated rats. Incorporation of NADPH-cytochrome c reductase results in rate enhancements which depend on both the microsomal preparation and the substrate examined. These rate enhancements have been interpreted in terms of the variable cytochrome P-450/reductase mole ratios resulting from the multiplicity of cytochrome P-450 species in microsomal preparations. The rate-limiting enzyme component for benzphetamine metabolism was examined in greater detail with microsomal preparations in which either NADPH-cytochrome c reductase or a cytochrome P-450 species, specific for benzphetamine N-demethylation, was incorporated. A rate enhancement, dependent on both incorporated reductase and cytochrome P-450 components, was observed with microsomes of untreated rats. In contrast, with microsomes derived from phenobarbital-treated rats, an increase in rate was found to depend only on the incorporated reductase component. These data indicate that the rate of benzphetamine Ndemethylation is dependent on both enzyme components in microsomes of untreated rats but becomes reductase-limited after phenobarbital induction. The absence of a kinetic isotope effect in studies with a model substrate, N&'-dimethylphentermine, also support this conclusion.
In addition, reconstitution studies with the purified enzyme components have been used to substantiate the conclusions drawn from the microsomal system.
Various purified microsomal electron transport proteins have been successfully incorporated into microsomal membranes and upon incorporation function within their respective membrane-bound electron transport systems (l-7). This technique has provided information on the hydrophobic bind-* The costs of publication of this article was defrayed in part by the payment of page charges. This article must therefore be-hereby marked 'hdvertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ing nature and the translational properties of these amphipathic proteins as well as their organization within the membrane. In addition to this information, the incorporation of one of the enzyme components of a multicomponent system could also yield information about the rate-limiting enzyme in the membrane-bound system since an increase in concentration of a rate-limiting component would be expected to increase the overall reaction rate observed for the system.
The cytochrome P-450-containing monooxygenase system which is responsible for the oxidation of many lipid-soluble xenobiotics and steroids is one of the multienzyme electron transport systems found in liver microsomes and is composed of two membrane-bound proteins: NADPH-cytochrome c reductase (NADPH-cytochrome P-450 reductase) and cytochrome P-450. The incorporation of either purified cytochrome P-450 or purified NADPH-cytochrome c reductase into microsomal membranes could, therefore, provide information about the rate-limiting enzyme component in monooxygenase reactions catalyzed by this system.
The NADPH-cytochrome c reductase isolated from the liver microsomes of uninduced, phenobarbital-or 3-methylcholanthrene-treated rats cannot be distinguished by either immunologic or catalytic properties (8,9). Although two electrophoretically distinct forms of the reductase have been recently detected in rats and rabbits, both forms catalyze the reduction of various species of cytochrome P-450 (10). Thus, the influence of incorporated NADPH-cytochrome c reductase on the hydroxylation rate of substrates metabolized by microsomal preparations, obtained from either untreated or treated rats, can be interpreted directly in terms of this electron transport component.
On the other hand, since liver microsomes contain multiple forms of cytochrome P-450 with overlapping but different substrate specificities, the incorporation of either a single species or a mixture of purified cytochrome P-450 species into microsomal membranes would not necessarily represent an increase in an enzyme component common to the microsomal preparation studied. For example, the incorporation of purified cytochrome P-448 from 3-methylcholanthrene-treated rats into microsomes of uninduced rats would not only change the quantity but also the type of major cytochrome P-450 species in the membrane (6). Therefore, the increase in benzo [alpyrene 1922  assays. We have found that the cytochrome P-450 in microsomes derived from phenobarbital-treated rats is unstable and must be used within 4 h after incorporation. NADPH-cytochrome P-450 Reductase Actiuity -NADPH-cytochrome P-450 reductase activity was determined at 22" as described by Gigon et al. (15). Samples containing 1 pmol of benzphetamine and 0.3 mg of microsomal protein/ml (total volume 2.5 ml) were bubbled with CO for 3 min before the anaerobic addition of 25 ~1 of 50 rnM NADPH via the plunger mechanism of an American Instrument Co., anaerobic cell. The reaction was monitored for at least 20 s on an Aminco DW-2a spectrophotometer set in the dual wavelength mode (AA.,,,,!,,,) before solid sodium dithionite was added to fully reduce the cytochrome P-450 in the cuvette. A 0.2 A full scale setting was used with the response control in the fast setting and a recorder scan rate of 2 s/inch. Under these conditions, the signal response is limited by the recorder which has a time constant of 240 ms for 63% of full scale response. The total cytochrome P-450 concentration was calculated from the maximum absorbance using the extinction coefficient IIa in Table I The mixtures were incubated for 10 min at 37" and the reaction terminated by the addition of 0.5 ml of 17% perchloric acid. Internal standards (25 nmol) were appropriately added and the mixtures were made alkaline with 0.5 ml of 5.5 N NaOH.
The products and internal standards were extracted into 1.5 ml of hexane and a 1.0 ml aliquot of the hexane layer transferred to a 12-ml conical test tube. Trifluoroacetic anhydride (50 ~1) was added to derivatize the secondary amine products. The samples were stored overnight at 4" to permit complete derivatization and then concentrated to a volume of 50 to 100 ~1 by evaporation of most of the solvent under a stream of dry nitrogen.
Two to five microliter volumes were injected into the gas chromatography/mass spectrometry system. Other Assays-The alkali-extractable metabolites of benzo(a)pyrene were determined by the method of Nebert and Gelboin (17)  Since NADPH-cytochrome c reductase represents less than 1% of the total microsomal protein a 5-to lo-fold increase in this component following incorporation would result in an insignificant (<6%) increase in total microsomal protein. Thus, all microsomal rate data obtained in this study are expressed per mg of protein.
The amount of NADPH-cytochrome c reductase incorporated into the microsomal membrane was quite marked (380 to 560% in Table II, 1000% in Table III, Experiment 2) as assessed by the increase in NADPH-cytochrome c reductase activity associated with the microsomes. In addition, the incorporated reductase is tightly associated with the microsomal pellet since repeated washings with buffered, 0.5 M KC1 solutions do not reduce the NADPH-cytochrome c reductase activity sedimenting with the microsomes (data not shown). The rate of reduction of cytochrome P-450 increases by about 2.5-to 4-fold after incorporation ( Fig. 1) indicating that the incorporated NADPH-cytochrome c reductase couples in the electron transport from NADPH to cytochrome P-450.
The major form of cytochrome P-450 purified from rats treated with phenobarbital can also be incorporated into microsomes and couples with the microsomal NADPH-cytochrome c reductase ( Fig. 1) in agreement with the earlier observations of Yang (6). In microsomes isolated from phenobarbital-induced rats, this results in no change in the initial rate of NADPH-dependent cytochrome P-450 reduction but does result in an increase in the total amount of cytochrome P-450 reduced after the initial linear phase. Furthermore, an increase in the total cytochrome P-450, after incorporation, is evident from the increase in the AA,,,,!,,, absorbance following reduction by sodium dithionite.

Effect of Incorporation on Microsomal Metabolism
The effect of incorporated reductase on the catalytic activity of microsomes obtained from untreated, phenobarbitaltreated, and 3-methylcholanthrene-treated rats is summarized in Table III. The degree of rate stimulation, following the incorporation of NADPH-cytochrome c reductase into microsomes, is dependent on both the substrate and the inducing agent employed. For example, when the microsomal reductase activity is increased by 4-to g-fold in untreated microsomes, little effect (~20%) is observed for testosterone hydroxylase activity or dimethylphentermine N-demethylase activity (Experiment 1). In contrast, a larger rate increase (60 to 145%) is observed for these substrates when microsomes of phenobarbital-treated rats are employed. Reductase incorporation into microsomes of 3-methylcholanthrene-treated rats results in an increase in the 7a-and 6P-hydroxylation of testosterone but has essentially no effect in 16a-hydroxytestosterone formation. Dimethylphentermine N-demethylase activity is moderately (50%) increased.
The metabolism of ethylmorphine, benzphetamine, benzo[a]pyrene, and ethoxycoumarin are all moderately increased (40 to 50%) following reductase incorporation into microsomes of untreated rats. In contrast, the metabolic activities are all markedly increased, except for benzo[a]pyrene, when reductase is incorporated into microsomes of 3-methylcholanthrene-treated rats. When the reductase activity in microsomes of untreated rats is increased from 4-to g-fold (Experiment 1) to IO-fold as in Experiment 2, a further stimulation in rate is observed for benzphetamine, benzolalpyrene, and ethoxycoumarin metab- substrates. The increase in reductase activity does not, however, increase ethylmorphine N-demethylase activity. A lo-fold increase in reductase activity markedly increases the metabolism of these four substrates (Experiment 2) when microsomes of phenobarbital-induced rats are used. The reductase-stimulated rates are particularly dramatic for benzphetamine and ethoxycoumarin and suggests that the ratelimiting enzyme in the microsomal metabolism of these substrates is NADPH-cytochrome c reductase. If this is the case, increasing the concentration of the cytochrome P-450 species responsible for the metabolism of these substrates should not affect the rate of substrate metabolism.
It is now recognized that mammalian, hepatic microsomes contain multiple forms of cytochrome P-450 with overlapping substrate specificities. The incorporation of a specific form or a number of different species of cytochrome P-450 into a microsomal system containing multiple forms of cytochrome P-450 could, therefore, result in misleading rate effects which are impossible to interpret directly in terms of a single species of cytochrome P-450. Direct deductions may be made regarding the role of cytochrome P-450 in regulating the overall hydroxylation rate by increasing the concentration of the major species of cytochrome P-450 present in a microsomal preparation and examining the effect on the catalytic activity for a substrate known to have a high turnover number with this particular species of cytochrome P-450. Microsomes derived from rats treated with phenobarbital provide such a system and the major cytochrome P-450 species from these microsomes has recently been purified to apparent homogeneity.' Under optimal conditions, this species catalyzes the Ndemethylation of benzphetamine with a turnover number equal to or greater than that observed for microsomal preparations obtained from phenobarbital-treated rats. Table IV summarizes three separate experiments in which both NADPH-cytochrome c reductase and the major form of cytochrome P-450 purified from phenobarbital-treated rats were separately incorporated into microsomes from untreated and phenobarbital-treated rats. The NADPH-cytochrome c reductase specific activity is essentially unaltered (~20%) when cytochrome P-450 is incorporated into microsomes from either source although the specific content of cytochrome P-450 is increased by 40 to 100%. In contrast, the reductase activity is markedly increased (250 to 500%) in both microsomal preparations after reductase incorporation while the cytochrome P-450 specific content is essentially unaltered (<12%).
The incorporation of either cytochrome P-450 or NADPHcytochrome c reductase moderately increases benzphetamine N-demethylase activity in microsomes derived from untreated rats. Yang and Strickhart have previously reported a moderate enhancement of benzphetamine N-demethylation following the incorporation of cytochrome P-450 into the sonicated microsomes of untreated rats(6). This pattern, however, is not observed in identical experiments using microsomes obtained from phenobarbital-treated rats. Reductase incorporation markedly increases benzphetamine N-demethylase activity while cytochrome P-450 incorporation has no effect. These data further substantiate the hypothesis that the rate-determining enzyme in benzphetamine N-demethylation is the reductase component in microsomes obtained from phenobarbital-treated rats. In contrast, microsomes derived from untreated rats demonstrate a moderate rate dependence on both the reductase and cytochrome P-450 components. The rate increase, however, following cytochrome P-450 incorporation into microsomes of untreated rats cannot be interpreted as unequivocally as after reductase incorporation since incorporation of the cytochrome P-450 species induced after phenobarbital treatment may not represent an increase in the native cytochrome P-450 species responsible for benzphetamine N-demethylation.

Studies with the Purified, Reconstituted Monooxygenase System
Three conditions may exist in microsomes containing two enzymes acting in series during the hydroxylation of a substrate.
Condition A -The reductase component is much less than saturating with respect to cytochrome P-450. By using saturating cofactor and substrate concentrations, kinetically zero order rate conditions are established for these components. This is essentially the condition for a coupled enzyme assay for the reductase enzyme and will result in the rate of hydroxylation being proportional to the amount of reductase enzyme in the system and independent of an increase in the cytochrome P-450 concentration.
Condition B -Neither component is present in excess with respect to the other. Under this condition, the substrate hydroxylation rate will be influenced by a change in concen-  will not affect the hydroxylation rate for the system. Under these conditions, the hydroxylation rate will linearly increase with increasing cytochrome P-450 concentrations, although the turnover number (expressed per mol of cytochrome P-450) will be unchanged.
The use of purified cytochrome P-450 and NADPH-cytochrome c reductase components in reconstituted drug-metabolizing systems permits the demonstration of these three conditions. Fig. 2 illustrates the rate dependence of benzphet-amineN-demethylation on reductase concentration in a reconstituted system in which the cytochrome P-450 and lipid concentrations are held constant. Under Condition A, the reductase component is much less than saturating with respect to cytochrome P-450. Under this condition, the rate of benzphetamine N-demethylation is linearly dependent on the reductase concentration but independent of an increase in the cytochrome P-450 concentration (Fig. 3  The rate of N-demethylation of the deuterium (IIb in Table I) and protium (IIa in Table I) isotopes of N,N-dimethylphentermine and the ratio of the two isotopic products were determined in separate and co-incubations of IIa and IIb by chemical ionization-gas chromatography/ mass spectrometry as described under "Experimental Procedures."