Effect of microsome-liposome fusion on the rotational mobility of cytochrome P450IIB4 in rabbit liver microsomes.

Membrane fusion of microsomes with soybean phospholipid vesicles was performed at pH 6.5 to investigate the effect of lipid-enrichment in the membrane on the rotational mobility of cytochrome P450. Rotational diffusion of cytochrome P450 in the microsomal membrane of phenobarbital-induced rabbit liver was measured by detecting the decay of absorption anisotropy after photolysis of the heme CO complex by a vertically polarized laser flash. The fusion procedures yielded three separate fractions upon sucrose density gradient centrifugation with lipid-to-protein ratio in weight (L/P) as follows: 1.5 in the bottom fraction, 2.2 in the middle fraction, and 3.9 in the top fraction. In each fraction, co-existence of mobile and immobile cytochrome P450 was observed. The percentage of rotationally mobile P450 (with the mean rotational relaxation time of phi=505-828 micros) in each of the different bands was found to be 59% in the bottom fraction, 61% in the middle fraction, and 68% in the top fraction. This increase in mobile population of P450 due to lipid-enrichment indicates that aggregated proteins in microsomal membranes dissociate with increasing L/P which is inversely proportional to the protein concentration in the membrane. With freeze-fracture electron microscopy, it was shown that the average distance increased between intramembrane particles by lipid-enrichment. Thus, the significant immobile population (32%) of P450 in microsomal membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P of 0.8. The decrease in the mobile population in the bottom fraction compared with intact microsomes was shown to be due to the pH 6.5 incubation used for fusion.

[Comparative study of the effects of essentiale and the novel Russian hepatoprotective agent "phospholiv" in a model of acute hepatitis in rats]. / Sravnitel'noe issledovanie deistviia éssentsiale i novogo otechestvennogo gepatoprotektora "fosfoliv" na modeli ostrogo gepatita u krys.

Curative effect of new preparation "Phospholiv", elaborated in Institute of Biomedical Chemistry, in acute CCl4 induced rat hepatit model was studied. The preparation consists of polyunsaturated phosphatidylcholine and glycyrrhizinic acid salt. Recovery of damaged biosynthesis of albumin and total cell liver RNA--by incorporation of C14-leucine and C14-orotic acid--were observed after 3 days Phospholiv administration, that showed on reparation of damaged protein-synthesis system. Label incorporation into liver fraction > 80S--that was decreased under CCl4 influence--was also restored after Phospholiv treatment, that may testify on its regenerative effect on wholeness of subcellular hepatocytes structures. Substantial decrease of morphologic damages of liver tissue was demonstrated as well. Other phospholipid preparation--known hepatoprotector Essentiale--gave some positive effects too, but Phospholiv influence on biochemical and morphological liver features were 1.5-2 fold as compared with that of Essentiale. Results show on efficiency of polyunsaturated phospholipids in the treatment of acute hepatit in rats--as a result of influence on hepatocytes cell membrane--and on preferential effect of new hepatoprotector Phospholiv.

[Use of a novel hepato-protective preparation "phospholiv" for inhibition of development of chronic hepatitis in rats]. / Tormozhenie s pomoshch'iu novogo gepato-protektornogo preparata "fosfoliv" razvitiia khronicheskogo gepatita u krys.

Protective influence of a new phospholipid preparation "Phospholiv" was studied using a model of chronic hepatitis. Animals were treated 45 days intraperitoneay with CCl4 with parallel intragastral administration of Phospholiv or--(for comparison)--the of other phospholipid hepatoprotector, Essential. Morphologic changes of liver, as well as protein and RNA biosynthesis were evaluated in the end of experiment--by means of measuring C14-leucine and C14-orotic acid incorporation into hepatocyte subcellular fractions. Both phospholipid preparations attenuated dystrophic liver changes, Phospholiv effect being more pronounced. They both prevented CCl4 induced inhibition of label incorporation into subcellular fraction proteins, but only Phospholiv, promoted the maintaining normal level of radioactivity incorporation into cytosol proteins and hepatocyte RNA. The results, confirming certain protective effect of Essential, show more pronounced hepatoprotective action of the new preparation Phospholiv (developed on the basis of polyunsaturated phosphatidylcholine and glycyrrhizinic acid salt). Data show also on possible fit hepatitis treatment.

Immobilization of proteins to lipid bilayers.

Phospholipid bilayers deposited on sensor surfaces are excellent substrates for immobilizing proteins via a molecular anchor. An integrated optics sensing device was used to accurately measure the binding kinetics of proteins thus anchored. By comparing the results with measurements using proteins from which the anchor had been enzymatically removed, it was shown that the anchor accounts for essentially all the irreversible binding. The insertion of the anchor into the lipid bilayer is a spontaneous process. This method of immobilization should be widely applicable to many soluble protein molecules, to which an anchor can be attached by routine methods of molecular engineering.

Dynamic interactions of rabbit liver cytochromes P450IA2 and P450IIB4 with cytochrome b5 and NADPH-cytochrome P450 reductase in proteoliposomes.

Purified liver microsomal cytochrome P450IA2 or P450IIB4 was co-reconstituted with cytochrome b5 or NADPH-cytochrome P450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P450 weight ratio of 2 by cholate dialysis procedures. The proteoliposomes catalyzed drug oxidation. Rotational diffusion of cytochrome P450 was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1 ms to a time-independent value, r3. Different rotational mobility for the two cytochrome P450s was observed. Though 20% of cytochrome P450IA2 was immobile, all cytochrome P450IIB4 molecules were rotating. The rotational relaxation time, phi, of the mobile population was 237 microseconds for cytochrome P450IA2 and 160 microseconds for cytochrome P450IIB4. The two cytochrome P450s have shown very different interactions with cytochrome b5 and NADPH-cytochrome P450 reductase. By the presence of the redox partner, the mobile population of cytochrome P450IA2 was increased significantly from 80% to 96% (plus cytochrome b5) and to 89% (plus NADPH-cytochrome P450 reductase) due to dissociation of P450 oligomers. On the other hand, the mobility of cytochrome P450IIB4 was not considerably affected by the presence of cytochrome b5 or NADPH-cytochrome P450 reductase as judged by little difference in phi and r3, keeping the mobile population of 100%. These results imply that cytochrome P450IA2 forms a transient association with cytochrome b5 and NADPH-cytochrome P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)

[Self-inactivation of cytochrome P-450 in the catalytic cycle]. / Samoinaktivatsiia tsitokhroma P-450 v kataliticheskom tsikle.

The paper deals with possible mechanisms of cytochrome p-450 self-activation during catalytic turnover. Two routes of hemoprotein inactivation are so far known. The first route studied extensively by many authors, consists in formation of active intermediates capable of modifying heme and apoenzyme. The second route revealed only lately, which results from uncoupled cytochrome P-450-catalyzed monooxygenase reactions, is yet to be clarified. Briefly, it consists in the fact that the hydrogen peroxide formed in the hemoprotein active center interacts with the enzyme-bound Fe2+, thereby generating hydroxyl radicals that bleach the heme and modify the apoenzyme. This mechanism operates with all the substrates and all cytochrome P-450 forms capable of catalyzing the partially coupled monooxygenase reactions proceeding with the formation of hydrogen peroxide as a by-product.

[The effect of covalently and noncovalently bound complexes of albumin with bilirubin on DNA and protein synthesis in liver and spleen of rats subjected to splenectomy and partial hepatectomy]. / Vliianie kovalentno i nekovalentno sviazannykh kompleksov al'bumina s bilirubinom na sintez DNK i belka v pecheni i selezenke krys so splenéktomiei i chastichnoi gepatéktomiei.

Noncovalently bound complexes of albumin and bilirubin were found to stimulate DNA and protein synthesis in partially hepatectomized regenerating rat liver tissue, while the covalently bound complex inhibited both these synthesis types in liver tissue after partial hepatectomy. Splenectomy of intact rats caused an induction of DNA and protein synthesis in liver tissue but partial hepatectomy decreased drastically the synthesis rate in spleen, thus suggesting that humoral factors stimulating the proliferation response in liver and spleen tissues were developed after spleen- and hepatectomies. The covalently bound albumin and bilirubin complex did not affect the rate of DNA and protein synthesis in liver tissue of splenectomized rats, while the complex with noncovalent bonds restored the rate of DNA and protein synthesis in the spleen of rats with partial hepatectomy. Only the noncovalently bound complex of albumin and bilirubin exhibited the properties inherent in hepatotropic growing factor whereas albumin administration was not effective. Possible structure and action of the noncovalently bound albumin and bilirubin complex are discussed.

Effects of albumin-bilirubin complexes with syngeneic or allogeneic albumin on DNA and protein synthesis in liver and spleen of partially hepatectomized rats.

The effects of non-covalently bound complexes of allogeneic or syngeneic albumin with bilirubin and of albumin alone on DNA and protein synthesis in rat liver and spleen cells after partial hepatectomy were studied. The assay procedure was based on different intravenous doses of these compounds in rats after partial hepatectomy. The allogeneic albumin-bilirubin complex (at protein doses of 0.9 and 90 micrograms/100 g body weight) stimulated DNA and protein synthesis in liver cells irrespective of the dose. At a dose of 0.9 micrograms the syngeneic albumin-bilirubin complex enhanced DNA synthesis insignificantly and produced no effect on protein synthesis, while at a dose of 90 micrograms, both DNA and protein synthesis were considerably increased. Allogeneic or syngeneic albumin at the above doses stimulated only protein, not DNA, synthesis in the liver, while the highest stimulation was at 90 micrograms allogeneic albumin. It was found also that partial hepatectomy decreased DNA and protein synthesis in spleen cells. Albumin-bilirubin complex with allogeneic or syngeneic albumin and albumin alone either significantly enhanced DNA and protein synthesis in the spleen, compared to controls, or only restored synthesis to control levels. Thus DNA and protein synthesis in the regenerating liver and spleen was significantly enhanced after the injection of small doses of the albumin-bilirubin complex, indicating the existence of small amounts of a similar endogenous complex in the blood stream.

The electron transfer reactions of NADPH: cytochrome P450 reductase with nonphysiological oxidants.

The steady-state kinetics of oxidation of rat liver NADPH: cytochrome P450 reductase (EC 1.6.2.4) by quinones, aromatic nitrocompounds, ferricyanide, Fe(EDTA)-, and cytochrome c has been studied. The logarithms of bimolecular rate constants of reduction (kcat/Km) of quinones and nitrocompounds increase with the increase in their single-electronreduction potential (E1(7)), reaching a maximum value at E1(7) > -0.15 V. The reactivities of nitroaromatics are about by an order of magnitude lower than the reactivities of quinones. For a series of nitroaromatics including the compounds with previously undetermined E1(7) values, an orthogonality was found between their reactivities toward cytochrome P450 reductase, flavocytochrome b2 (EC 1.1.2.3), and the NADPH: adrenodoxin reductase (EC 1.18.1.2)-adrenodoxin system. This indicates the absence of significant specific interactions during these reactions. The effects of ionic strength on reaction kinetics and the character of inhibition by a product of reaction, NADP+, are in accordance with the reduction of oxidants at the negatively charged site in the surroundings of FMN of P450 reductase. Quinones inactivate oxidized reductase modifying the NADP(H) binding site. The redox cycling of quinones markedly slows the inactivation. The kinetic data presented are consistent with an outer-sphere electron transfer mechanism. The analysis of kinetics of reduction of cytochrome c, ferricyanide, and Fe(EDTA)- using the model of Mauk et al. (A. G. Mauk, R. A. Scott, and H. B. Gray (1980) J. Am. Chem. Soc. 102, 4360-4363) gives calculated distances of FMN from the surface of protein globule, 0.33-0.63 nm. The data from nitroreductase reactions of cytochrome P450 reductase, flavocytochrome b2, and adrenodoxin were used for approximate evaluation of previously unknown E1(7) of nitrocompounds.

Catalytic activity of cytochrome P4501A2 in reconstituted system with Emulgen 913.

Physicochemical properties and catalytic activity of cytochrome P4501A2 in the reconstituted system with Emulgen 913 were studied. The formation of cytochrome P4501A2 monomers was shown by gel filtration at an Emulgen concentration of 8 g/liter. The catalytic activity of the monomeric monooxygenase system was low. The maximum rate of the 7-ethoxyresorufin O-deethylation reaction, 150 pmol resorufin min-1 nmol-1 cytochrome P4501A2, was observed at an Emulgen concentration of 0.1 g/liter when cytochrome P4501A2 pentamers predominated. The effects of Emulgen on cytochrome P4501A2 cumene hydroperoxide-dependent peroxidase activity, on its affinity to substrate and to cytochrome b5, and on the rate constants of dithionite-dependent reduction were insignificant. Study of the NADPH-dependent reduction of cytochrome P4501A2 in the reconstituted system showed that the rate constant and reduction level of cytochrome P4501A2 were always higher when the reaction was initiated by NADPH than when it was initiated by NADPH-cytochrome P450 reductase. This indicated that the reduction reaction initiated by the reductase was limited by a step in the cytochrome P4501A2 and reductase interaction. Correlation between 7-ethoxyresorufin O-deethylase activity and reduction level of cytochrome P4501A2 was found.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.

Cytochrome C (Fe2+) as a competitive inhibitor of NADPH-dependent reduction of cytochrome P450 LM2: locating protein-protein interaction sites in microsomal electron carriers.

The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed. Under these conditions ferrous cytochrome c was found to be a competitive inhibitor of the slow phase of the reduction process, which accounted for 80% of the total reduction amplitude. Inhibition experiments yield a dissociation constant for the LM2-reductase complex of 3.0 +/- 1.5 microM. This constant was the same both in the presence and in the absence of benzphetamine. Based on these data we conclude that cytochromes P450 and c bind to the same center on the NADPH-cytochrome P450 reductase molecule. Comparative analysis of the amino acid sequences reveals a detectable similarity between cytochrome c and cytochrome P450 LM2 at positions 68-87 and 121-145, respectively. In addition, a substantial similarity was shown for sequence fragments 204-224 of NADPH-cytochrome P450 reductase and 40-60 of cytochrome b5. Based on these findings a hypothesis for the location of the centers of intermolecular interactions on the molecules of cytochrome P450 LM2 and NADPH-cytochrome P450 reductase is proposed.

Substrate access channel geometry of soluble and membrane-bound cytochromes P450 as studied by interactions with type II substrate analogues.

The problems of alterations in the tertiary structure at the cytochrome P450 active site after isolation from the microsomal membrane and comparative analysis of the structures of the active sites of membrane-bound P450 and soluble P450cam have been studied in terms of using bifunctional compounds (I-IV). These amphiphilic compounds contain a pyridine radical, an aliphatic chain of variable length (n), and diphosphonic acid at the end of the molecule. There exists an optimal length (n) at which the interaction between I-IV and P450 is rather efficient. Comparison of the data on such interactions with microsomal P450, as well as P450 isolated from the membrane in oligomeric and monomeric states, and P450cam allows the estimation of the distance between the Fe3+ ion in the active site and the charged residues (Lys/Arg) on the enzyme surface (approximately 17 A for all P450s).

[Antioxidative properties of glycyrrhyzic acid salts and their effect on the liver monooxygenase system]. / Issledovanie antiokislitel'nykh svoistv solei glitsirrizinovoi kisloty i ikh vliianiia na mikrosomal'nuiu monooksigenaznuiu sistemu pecheni.

Antioxidative activity of potassium- and sodium glycyrrhizins was detected by means of chemoluminescence procedure. As shown by the spectral studies the glycyrrhizin salts formed complex of the I type with cytochrome P-450 from rat liver microsomes, inhibited inactivation of isolated cytochrome P-450LM2 and induced hemoprotein formation after administration into rats. The preparations of glycyrrhizic acid appear to be useful for treatment of toxic impairment of liver tissue.

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding.

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations. One group lies in the plane of the heme and remains unchanged in the two states of cytochrome P-450 LM2, the other is tilted out of the plane. The tilting angle in oligomers was smaller than in monomers. These vinyl stretching modes together with some porphyrin modes, were found to be sensitive indicators of the quaternary structure and of substrate binding. In both the oligomer and the monomer, substrate binding causes changes of the relative intensities of some porphyrin modes and the vinyl stretching vibrations which may reflect modifications of the electronic transitions due to hydrophobic interactions between the bound substrate and the heme. In contrast to the monomeric cytochrome P-450 LM2, benzphetamine binding to the oligomers of this isozyme additionally produces a shift of the spin-state equilibrium. This indicates that in the oligomer the substrate-binding pocket is converted by protein-protein interaction to a structure that forces substrates to interfere with the sixth ligands, inducing an increase of the five-coordinated high-spin configuration. In the monomer the substrate-binding pocket can accommodate benzphetamine without affecting the spin state. Binding of imidazole to the monomeric and oligomeric cytochrome P-450 LM2 produces essentially the same resonance Raman spectra. Apparently the replacement of the native sixth ligand by imidazole disturbs the structure of the active site in such a way that it becomes insensitive to protein-protein interactions. H2O2-dependent N-demethylation of benzphetamine and aniline p-hydroxylation by cytochrome P-450 LM2 did not depend on its state of aggregation.

[The role of rat liver microsomes in the metabolism of methylmethacrylate to formaldehyde]. / Rol' mikrosom pecheni krys v metabolizme metilmetakrilata do formal'degida.

Rat liver microsomal carboxylesterase catalyzed hydrolysis of butyl methacrylate (BMA), butyl acrylate (BA), methyl acrylate (MA) and methyl methacrylate (MMA). The rate of butanol formation from BA and BMA was 4-fold higher as compared with methanol production from MA and MMA. Methanol was oxidized to formaldehyde at the next step of microsomal metabolism. The rate of methanol oxidation was increased 2.2-fold after addition of the NADPH-generating system into the incubation medium. In the mixture containing MMA, NADPH-generating system and liver microsomes, preinduced with phenobarbital or methyl cholanthrene, the rate of formaldehyde generation was increased 7- and 2-fold, respectively, as compared with the analogous experiments involving liver microsomes of control rats. Production of methanol and generation of formaldehyde were not detected after incubation of MMA with inactivated microsomes. The microsomal MMA-methanol oxidizing system was inhibited by CO or after addition of microsomes from rats pretreated with CoCL2. Sodium azide (0.05 mM) decreased 8-fold the rate of formaldehyde generation.

[Inactivation of sodium dithionite reduced cytochromes P-450 of different origins]. / Issledovanie inaktivatsii vosstanovlennykh ditionitom natriia tsitokhromov P-450 razlichnogo proiskhozhdeniia.

The inactivation of five dithionite reduced soluble cytochrome P-450 isoforms has been studied. The inactivation of microsomal rabbit liver isoform LM2 and bacterial linalool cytochrome P-450 is followed by its conversion into cytochrome P-420. Microsomal rabbit liver isoform LM4, bacterial camphor and p-cymene cytochromes P-450 were not inactivated under these conditions. The inactivation of linalool cytochrome P-450 and LM2 isoform is a first order reaction; the rate constants for linalool cytochrome P-450 and LM2 are 0.3 and 0.1 min-1, respectively. In the case of linalool cytochrome P-450 its carboxycomplex (Fe2+-CO) is inactivated, while in the case of LM2 the inactivation affects its oxycomplex (Fe2+-O2). The amino acid residues of linalool cytochrome P-450 are probably modified due to a direct electron transfer in its carboxycomplex. The amino acid residues of LM2 isoform are modified, presumably due to oxidation by oxygen active species which are released during the oxycomplex decay.

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers.

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only. The addition of benzphetamine and/or flavoprotein to cytochrome P-450 monomers did not cause the spin equilibrium shift and the low-spin form content was higher than 85% in all cases. Investigation of the dependence of the initial rates of NADPH-dependent cytochrome P-450 reduction and benzphetamine oxidation on the stoichiometry of the flavoprotein and cytochrome P-450 at their constant total concentration showed that the molar ratio of 1:1 was required for maximal activity. Thus this system works in full accordance with the mass action law.