Effect of purified cytochrome P450 incorporation into liposomal membranes on its properties]

The physico-chemical properties and hydroxylase activity of three forms of cytochrome P450, i. e. purified soluble hemoprotein, purified hemoprotein incorporated into the liposomal membrane and microsomal cytochrome P450, were studied. Soluble cytochrome P450 binds type I substrates in a lesser degree than does its microsomal form. The incorporation of hemoprotein into phosphatidyl choline liposomes restores the ability of purified cytochrome P450 to interact with these substrates. The soluble and lipid-bound forms of cytochrome P450 do not differ in their thermal stabilities and protease digestion. The liposome-bound cytochrome P450 has higher dimethylaniline, aniline and p-nitroanisol hydroxylase activities as compared to its soluble form. The aniline hydroxylase activity of microsomal, proteoliposomal and soluble forms of cytochrome P450 is inhibited by the tyrosinecopper complex with NADPH or cumole hydroperoxide as cosubstrates. The inhibiting effect of the complex on other hydroxylase activities depends on the type of cytochrome P450 and the cosubstrates and substrates used.     

Benzonal--a phenobarbital-type of inducer of the mono-oxygenase system]

It has been found that the Soviet anticonvulsant drug Benzonal is an inducer of the liver cytochrome P-450 of the phenobarbital type. The drug causes formation of the cytochrome P-450 form which is immunologically identical to the phenobarbital inducible form of the hemoprotein with identical molecular mass determined with the SDS-gel electrophoresis method in PAAG. The microsomes, obtained from the rats treated with Benzonal display increased rates of metabolism of 7-pentoxiresorufin and 16 beta-hydroxylation of androstenedione which are specific substrates for the cytochrome P-450b.     

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b₅, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.     

Reduction kinetics of purified rat liver cytochrome P-450. Evidence for a sequential reaction mechanism dependent on the hemoprotein spin state

The anaerobic reduction kinetics of purified rat liver ferric cytochrome P-450 from phenobarbital-treated rat liver microsomes, reconstituted with saturating NADPH-cytochrome P-450 reductase, have been investigated and were shown not to be monophasic. From experiments correlating changes in the rate of fast-phase reduction with the spin state of the heme iron existing at preequilibrium, data were obtained consistent with a model for spin-state control of cytochrome P-450 reduction wherein the high-spin form of the hemoprotein is more rapidly reduced than the low-spin form. In addition, the temperature dependence of the reduction process in the presence of the substrate benzphetamine was studied. From the results obtained it is suggested that the endothermic nature of the low- to high-spin transition largely accounts for the apparent activation energy observed for the reduction of high-spin cytochrome P-450 being relatively temperature insensitive when compared to the rate constant for reduction of the membrane-bound form of the hemoprotein.     

Kinetics of cytochrome P-450 reduction: evidence for faster reduction of the high-spin ferric state

Results are presented that support our hypothesis [Backes, W. L., Sligar, S. G., & Schenkman, J. B. (1980) Biochem. Biophys. Res. Commun. 97, 860-867] that the multiphasic reduction kinetics of cytochrome P-450 are, in part, due to the spin equilibrium of the ferric hemoprotein. The disappearance of the high-spin charge-transfer band at 650 nm during reduction of the hemoprotein by NADPH was fast, exhibiting a rate constant greater than that of the fast phase of reduction measured by formation of the carbon monoxide adduct. In contrast, the disappearance of the ferric low-spin form of the cytochrome was at a considerably slower rate. A mathematical expression of the fractional content of high-spin cytochrome P-450 was obtained by comparing the ratio of the initial rate of change in the fraction of total oxidized cytochrome remaining to the initial rate of change in the fraction of high-spin ferric P-450 remaining. Results supporting the model were obtained by using both microsomes and purified cytochrome P-450 RLM5. The calculation from experimental data yielded results that were similar to those obtained by different extrapolation methods used for estimation of the amount of high-spin cytochrome P-450, supporting further the proposed relationship between the spin equilibrium and the reduction kinetics of this hemoprotein.     

Purification and characterization of a new form of cytochrome P-450 (LM2b) from untreated male rabbit liver microsomes

A new form of cytochrome P-450 has been purified from untreated male rabbit liver microsomes. This form was designated P-450 LM2b on the basis of its electrophoretic mobility on SDS polyacrylamide gel, where it migrates as a polypeptide of apparent molecular weight of 50,250. This hemoprotein exhibits a maximum at 448.5 nm in the Soret band of the CO-Ferrous state spectrum. On the basis of its molecular, spectral, enzymologic and immunologic data, P-450 LM2b was shown to be distinct from the other P-450 forms, already characterized in rabbit liver microsomes. However P-450 LM2b and P-450 LM3b appear to be immunologically related proteins.     

Purification and characterization of cholesterol 7 alpha-hydroxylase cytochrome P-450 of untreated rabbit liver microsomes

Cytochrome P-450 which catalyzes the 7 alpha-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome per mg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5. The P-450 catalyzed the 7 alpha-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome b5 the activity was maximum at 0.4 M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome b5 is an essential component of the cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes.     

Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo[a]pyrene hydroxylation from liver microsomes of untreated rats

A form of cytochrome P-450 generally catalyzing benzo[a]pyrene (B[a]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/B[a]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/B[a]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/B[a]P indicated that P-450/B[a]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of B[a]P). B[a]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/B[a]P antibody. These results demonstrate that P-450/B[a]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes B[a]P hydroxylation in liver microsomes of untreated rats.     

Cytochrome P-448 induction in liver microsomes of mice of inbred strains after administration of xenobiotics of MC-type

Using immunochemical methods, the identity of cytochrome P-448 from liver microsomes of mice of "inducible" and "non-inducible" lines during induction by xenobiotics of MX-type (3-methylcholanthrene, 3,4-benzpyrene, 2,3,7,8-tetrachlorodibenzodioxin) was established. This hemoprotein form was shown to play a role in 3,4-benzpyrene metabolism. Monospecific antibodies to purified cytochromes P-448 and P-450 were obtained; the cytochrome P-448 content in microsomes was measured by rocket immunoelectrophoresis. The content of cytochrome P-448 in control and phenobarbital-induced microsomes makes up to 10-15% of the total hemoprotein content determinable from the CO-spectra. 3-Methylcholanthrene and 3,4-benzpyrene injected into "non-inducible" mice cause no increase in the content of this hemprotein form, whereas in mice induced with 2,3,7,8-tetrachlorodibenzodioxin it rises to 50%. Under these conditions, an almost 100% inhibition of 3,4-benzpyrene metabolism by antibodies to cytochrome P-448 is observed. Antibodies against cytochrome P-448 obtained from liver microsomes of 3-methylcholanthrene-induced mice cause a 90% inhibition of 3,4-benzpyrene in microsomes induced with 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzodioxin.     

Isolation and partial characterization of a rifampicin induced rabbit liver microsomal cytochrome P-450

Rifampicin administration to New Zealand male rabbits increased the concentration of an LM3 form of cytochrome P-450 to up to 30% of the microsomal P-450 concentration. This enzyme was purified to electrophoretic homogeneity with a yield of 8% of the original total microsomal P-450 concentration. Isolated as a low spin hemoprotein in its substrate free oxidized form, it displays in its reduced CO-complexed form an absorption maximum at 449 nm. Immunological assays, as well as activity measurements, in particular its stereospecific progesterone hydroxylation in the 6 beta-position, show a relationship between LM3,Rif and LM3c (from untreated rabbits).     

Soluble cytochrome P-450 from housefly microsomes. Partial purification and characterization of two hemoprotein forms.

Housefly microsomes contain two spectrally different forms of cytochrome P-450 which we have termed P-450 and P-450I. Methods have been developed for the fractionation and chromatographic purification of these two hemoprotein forms. Microsomes are solubilized first with Triton X-100 in the presence of glycerol, dithiothreitol, ethylenediaminetetra-acetic acid, and phenobarbital. Cytochrome P-450 is recovered in a floating pellet after the addition of 25% ammonium sulfate followed by centrifugation, whereas cytochrome P-450I remains in the 25% ammonium sulfate supernatant fluid. Cytochrome P-450 is purified further by Sephadez G-200 and DEAE-Sephadex A-50 column chromatography, which also allows the isolation of cytochrome b5 and NADPH-dependent cytochrome P-450 reductase in good yields and with little cross-contamination. Cytochrome P-450 apparently is free of cytochromes b5 and P-420 as well as of reductase and is obtained in a final yield of approximately 16% with a 6.9-fold purification. Its maximum absorbance is at 45 mn in the CO-difference spectrum and its average extinction coefficient is 103 cm-1 nm-1. Cytochrome P-450I is purified by Sephadex G-25 column chromatography but still contains some cytochromes b5 and P-420 as well as reductase. Its maximum absorbance is at 448.5 nm in the CO-difference spectrum and its extinction coefficient is 83 to 86 cm-1 mM-1. Both cytochromes hydroxylate type I substrates such as aminopyrine. Sufficient amounts of reductase are present in the cytochrome P-450I preparation to sustain activity, but the reductase has to be added to cytochrome P-450 in a reconstituted system for activity. Cytochrome P-450 is fairly stable, whereas cytochrome P-450I can be isolated only when protected by a substrate (phenobarbital). Detergent-solubilized housefly cytochromes P-450 and P-450I seem to correspond to either aggregates or oligomeric proteins. Cytochrome P-450 appears to correspond to a tetramer, each subunit having a molecular weight of 45,000, whereas cytochrome P-450I may correspond to an aggregate of at least 10 subunits. The cytochrome P-450 aggregate is dissociated by 6 M urea, but cytochrome P-450I remains as such.     

Spectrophotometric study of cytochrome P-450 (11 beta) interaction with physiological effectors

Using the optical absorbance spectroscopy method, the interaction of a number of biospecific ligands (steroids, adrenodoxin) with homogeneous cytochrome P-450 (11 beta) from bovine adrenal mitochondria was investigated. The parameters of the steroid-protein interaction in a number of substrates and products of the 11 beta- and 18 (19)-hydroxylation with the active site of cytochrome P-450 (11 beta) were determined. A sharp decrease in the cytochrome affinity for steroids upon the insertion of the first hydroxy group was observed, which provides for a predominant formation of monohydroxylated products from the substrate and minimum amounts of dihydroxylated ones, despite the presence of more than one position for the substrate hydroxylation by cytochrome P-450 (11 beta). Some structural elements of the steroid molecule were determined as any alterations in these strongly affect the enzyme affinity for the steroid. These structures are: 1) delta 4-3-oxo structure; 2) either 21-hydroxy group of pregnen steroids or the one fulfilling its functions, 17 beta-hydroxy or 17-oxo group of androsten steroids, and 3) the 11th position of all the substrates under study. It was shown that the binding of various substrates into stoichiometric (1:1) steroid-protein complexes provides a transition to high spin state from 30-40% (cortisol, corticosterone) to 90-95% (11-deoxycorticosterone) of hemoprotein iron. Using the experimental system containing individual cytochrome P-450 (11 beta) and adrenodoxin, as well as the steroid and nonionic detergent Tween 20, it was shown that the parameters of substrate binding and hemoprotein spin equilibrium did not differ from the corresponding parameters of the cytochrome-adrenodoxin dienzyme complex. The peculiarities of the multiligand interactions in the 11 beta-hydroxylase system, involving cytochrome, substrates and ferredoxin demonstrate some analogy with a bacterial camphor hydroxylase system and some differences from the mitochondrial system for the side chain cleavage of cholesterol.     

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.     

Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein

The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.     

Studies on the substrate-binding sites of liver microsomal cytochrome P-448.

The interaction of substrates of the microsomal mixed-function oxidases with cytochromes P-450 and P-448 was investigated by using liver microsomes from rats pretreated with phenobarbital or 3-methylcholanthrene, and with purified forms of the cytochromes isolated from rabbit liver. The two forms of the cytochrome have different substrate specificities; cytochrome P-450 has one type 1 substrate-binding site that can accommodate a large variety of substrates, but in contrast cytochrome P-448 may possess two type 1 substrate-binding sites, one of which is different to that of cytochrome P-450 in that it shows a specificity for substrates such as safrole and 9-hydroxy-ellipticine. These findings explain why the two forms of the cytochrome have different substrate specificities and play contrasting roles in the activation and deactivation of xenobiotics.     

Nitric oxide synthase is a cytochrome P-450 type hemoprotein.

Nitric oxide has emerged as an important mammalian metabolic intermediate involved in critical physiological functions such as vasodilation, neuronal transmission, and cytostasis. Nitric oxide synthase (NOS) catalyzes the five-electron oxidation of L-arginine to citrulline and nitric oxide. Cosubstrates for the reaction include molecular oxygen and NADPH. In addition, there is a requirement for tetrahydrobiopterin. NOS also contains the coenzymes FAD and FMN and demonstrates significant amino acid sequence homology to NADPH-cytochrome P-450 reductase. Herein we report the identification of the inducible macrophage NOS as a cytochrome P-450 type hemoprotein. The pyridine hemochrome assay showed that the NOS contained a bound protoporphyrin IX heme. The reduced carbon monoxide binding spectrum shows an absorption maximum at 447 nm indicative of a cytochrome P-450 hemoprotein. A mixture of carbon monoxide and oxygen (80%/20%) potently inhibited the reaction (73-79%), showing that the heme functions directly in the oxidative conversion of L-arginine to nitric oxide and citrulline. Additionally, partially purified NOS from rat cerebellum was inhibited by CO, suggesting that this isoform may also contain a P-450-type heme. NOS is the first example of a soluble cytochrome P-450 in eukaryotes. In addition, the presence of FAD and FMN indicates that this is the first catalytically self-sufficient mammalian P-450 enzyme, containing both a reductase and a heme domain on the same polypeptide.     

Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.     

Multiple forms of cytochrome P-450. Characteristics of isolated aminopyrine-N-demethylase (cytochrome P-450AP)

A previously unidentified cytochrome P-450AP possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on hydroxyl apatite. Using radioisotope techniques, it was found that 4-isopropylaminoantipyrine induces cytochrome P-450AP synthesis de novo. The isolated cytochrome P-450AP has the following characteristics: Mr = 49,000 Da. CO-peak maximum at 450.5 mm, rate of aminopyrine demethylation in a reconstituted system-20 nmol HCHO/min/nmol of cytochrome P-450, benzphetamine-15. The hemoprotein synthesis is paralleled with the synthesis of a protein with Mr of 51,000 Da. Immunochemical analysis permitted to identify the latter protein as cytochrome P-450b. It was demonstrated that cytochrome P-450AP does not interact with the antibodies to the major phenobarbital-induced form, i.e., with cytochrome P-450b.     

Spectral and catalytic properties of cytochrome P-450 from a diazinon-resistant housefly strain

Microsomes from the diazinon-resistant Rutgers strain of housefly contain amounts of cytochrome P-450 that are larger than those reported for rat liver, but the specific activity expressed as nmole of cytochrome P-450 per mg protein is much lower. The hemoprotein shows that spectral changes type I, II and IV are essentially in the low-spin form as judged by the n-octylamine and ethyl isocyanide difference spectra, and is unstable at pH below 6.5 and above 8.0. Cytochrome P-420 is also produced with time when CO-difference spectra are recorded. This is accelerated at pH above 8.0. The presence of contaminating amounts of cytochrome P-420, due to denaturation during spectral analysis or to the method used to isolate the microsomes, makes questionable the practice of characterizing the hemoprotein on the basis of the 455 nm peak in the ethyl isocyanide spectra, since a 434 nm peak is produced with concomitant decrease of the 455 nm peak. Microsomes hydroxylate naphthalene, aminopyrine and aniline, but the activity when expressed as nmole of product per nmole of cytochrome P-450 is the same or lower than that reported for other resistant housefly strains.     

Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450. Monospecific antibodies against domains F1 and F2

Highly specific antibodies to adrenocortical cytochrome P-450scc as well as fragments F1 and F2 representing the N- and C-terminal sequences of the hemoprotein obtained by limited trypsinolysis were raised in rabbits. Antibodies to cytochrome P-450scc as demonstrated by the Ouchterlony diffusion analysis, immunoelectrophoresis and immunoblotting techniques interact with the hemoprotein and both fragments. Antibodies to cytochrome P-450scc fragments interact with the hemoprotein and corresponding antigens, but do not cross-react. To determine the localization of antigenic determinants in the polypeptide chain of cytochrome P-450scc, the interaction of antibodies to the hemoprotein and to its fragments F1 and F2 with limited trypsinolysis products was studied. All antibodies were found to effectively inhibit cholesterol transformation into pregnenolone in a reconstituted system. Using SDS electrophoresis followed by immunoblotting, the cross-reactivity of antibodies to cytochrome P-450scc and to its fragments with microsomal cytochromes P-450scc LM2 and LM4 as well as with mitochondrial cytochrome P-45027 was revealed. This finding testifies to the presence of common antigenic determinants in the hemoproteins.