Effects of gamma-hexachlorocyclohexane and L-3,3',5-triiodothyronine on rat liver cytochrome P4502E1-dependent activity and content in relation to microsomal superoxide radical generation

Liver microsomal cytochrome P4502E1-dependent p-nitrophenol (PNP) hydroxylation and expression of cytochrome P4502E1 were studied in rats subjected to gamma-hexachlorocyclohexane (HCCH) or L-3,3,5-triiodothyronine (T3) administration as a possible mechanism contributing to superoxide radical (O2.-) generation. HCCH treatment (a single dose of 40 mg/kg body wt) produced a 43% increase in the content of total cytochrome P450, whereas T3 (daily doses of 0.1 mg/kg body wt for two consecutive days) led to a 37% decrease. NADPH-dependent O2.- generation was elevated by HCCH and T3, expressed as either per mg of protein or per nmol of cytochrome P450, with a 135% enhancement in the O2.- production/superoxide dismutase (SOD) activity ratios being observed in both conditions. This was partly due to depression of SOD activity. Concomitantly, the molecular activity of NADPH-cytochrome p450 reductase was enhanced by 90 and 69% by HCCH and T3, respectively. In these conditions, microsomal PNP hydroxylation showed increases of 58 and 45% in HCCH- and T3-treated rats over control values, respectively, with a parallel 31% (HCCH) and 41% (T3) enhancement in the content of cytochrome P4502E1 assessed by western immunoblotting. We conclude that HCCH and T3 enhance the expression and activity of cytochrome P4502E1 and that of NADPH-cytochrome P450 reductase in rat liver, regardless of the changes in total cytochrome P450 content, representing major contributory mechanisms to microsomal NADPH-dependent O2.- generation.     

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 micrograms/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 micrograms/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6 beta-hydroxytestosterone (51%), 2 beta-hydroxytestosterone (18%), 15 beta-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1 beta-hydroxytestosterone (3%), 16 beta-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16 beta-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited greater than 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin- or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.     

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.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Nitric oxide metabolism in mammalian cells: substrate and inhibitor profiles of a NADPH-cytochrome P450 oxidoreductase-coupled microsomal nitric oxide dioxygenase

Human intestinal Caco-2 cells metabolize and detoxify NO via a dioxygen- and NADPH-dependent, cyanide- and CO-sensitive pathway that yields nitrate. Enzymes catalyzing NO dioxygenation fractionate with membranes and are enriched in microsomes. Microsomal NO metabolism shows apparent KM values for NO, O2, and NADPH of 0.3, 9, and 2 microM, respectively, values similar to those determined for intact or digitonin-permeabilized cells. Similar to cellular NO metabolism, microsomal NO metabolism is superoxide-independent and sensitive to heme-enzyme inhibitors including CO, cyanide, imidazoles, quercetin, and allicin-enriched garlic extract. Selective inhibitors of several cytochrome P450s and heme oxygenase fail to inhibit the activity, indicating limited roles for a subset of microsomal heme enzymes in NO metabolism. Diphenyleneiodonium and cytochrome c(III) inhibit NO metabolism, suggesting a role for the NADPH-cytochrome P450 oxidoreductase (CYPOR). Involvement of CYPOR is demonstrated by the specific inhibition of the NO metabolic activity by inhibitory anti-CYPOR IgG. In toto, the results suggest roles for a microsomal CYPOR-coupled and heme-dependent NO dioxygenase in NO metabolism, detoxification, and signal attenuation in mammalian cells.     

Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.     

Changes in the ratio of microsomal electron carriers in reconstituted microsomal membranes

The reconstitution of microsomal membrane monooxygenase system with variable contents of the hydroxylating chain enzymatic components was carried out. It was found that during self-assembly of microsomal membranes solubilized with 4% sodium cholate and gel filtration through Sephadex LH-20 in the presence of isolated microsomal enzymes, two forms of cytochrome P-450, i. e. phenobarbital- and 3-methylcholantrene-induced ones, and NADPH-cytochrome P-450 reductase, the exogenous enzymes are incorporated into the microsomal membrane matrices of control and methyl-cholantrene-treated animals. In the membranes reconstituted from the microsomes of the methylcholantrene-induced animals the catalytic activity of cytochrome P-448 in the metabolism of benz(a)pyrene at varying cytochrome P-448 and NADPH-cytochrome P-450 reductase contents were investigated.     

Rotation of cytochrome P-450. Complex formation of cytochrome P-450 with NADPH-cytochrome P-450 reductase in liposomes demonstrated by combining protein rotation with antibody-induced cross-linking

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles by a cholate dialysis technique. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme X CO complex by a vertically polarized laser flash. All cytochrome P-450 was found to be rotationally mobile when co-reconstituted with equimolar amounts of NADPH-cytochrome P-450 reductase in lipid to cytochrome P-450 ((L/P450)) = 1 (w/w] vesicles. Antibodies against NADPH-cytochrome P-450 reductase were raised. Their specificity was demonstrated by Ouchterlony double diffusion analysis. Antireductase Fab fragments were prepared from antireductase IgG by papain digestion. The N-demethylation of benzphetamine, catalyzed by the proteoliposomes, was significantly inhibited by antireductase IgG and by antireductase Fab fragments. Cross-linking of NADPH-cytochrome P-450 reductase by antireductase IgG resulted in complete immobilization of cytochrome P-450 in L/P450 = 1 vesicles. Antireductase IgG also immobilized cytochrome P-450 in L/P450 = 5 vesicles, although the degree of immobilization was slightly smaller. No immobilization of cytochrome P-450 in L/P450 = 1 vesicles was detected in the presence of antireductase Fab fragments or preimmune IgG. These results further support the proposal of the formation of monomolecular complexes between cytochrome P-450 and NADPH-cytochrome P-450 reductase in liposomal membranes (Gut, J., Richter, C., Cherry, R.J., Winterhalter, K.H., and Kawato, S. (1982) J. Biol. Chem. 257, 7030-7036).     

Target inactivation analysis applied to determination of molecular weights of rat liver proteins in the purified state and in microsomal membranes

In principle, target inactivation analysis provides a means of determining the molecular weights (Mr) and states of aggregation of proteins in native environments where they are functionally active. We applied this irradiation technique to the rat liver microsomal membrane proteins: cytochrome b5, epoxide hydrolase, flavin-containing monooxygenase, NADH-ferricyanide reductase, NADPH-cytochrome P-450 reductase, and seven different forms of cytochrome P-450. Catalytic activities, spectral analysis of prosthetic groups, and sodium dodecyl sulfate-polyacrylamide electrophoresis/peroxidase-coupled immunoblotting were used to estimate apparent Mr values in rat liver microsomal membranes. Except in one case (cytochrome P-450PCN-E), the estimated Mr corresponded most closely to that of a monomer. Purified cytochrome P-450PB-B, NADPH-cytochrome P-450 reductase and epoxide hydrolase were also subjected to target inactivation analysis, and the results also suggested monomeric structures for all three proteins under these conditions. However, previous hydrodynamic and gel-exclusion results clearly indicate that all three of these proteins are oligomeric under these conditions. The discrepancy between target inactivation Mr estimates and hydrodynamic results is attributed to a lack of energy transfer between monomeric units. Thus, while P-450PCN-E may be oligomeric in microsomal membranes, target inactivation analysis does not appear to give conclusive results regarding the states of aggregation of these microsomal proteins.     

Cytochrome P450 IIIA1 (P450p) requires cytochrome b5 and phospholipid with unsaturated fatty acids.

In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1)2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b5. A polyclonal antibody against purified cytochrome b5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b5 caused a approximately 60% inhibition of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by approximately 70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b5 and phospholipid containing unsaturated fatty acids.     

Reaction of monosubstituted hydrazines and diazenes with rat-liver cytochrome P450. Formation of ferrous-diazene and ferric sigma-alkyl complexes

The alkyldiazenes RN = NH (R = CH3 or C2H5) react with reduced microsomal cytochrome P450 leading to complexes exhibiting a Soret peak at 446 nm. Upon oxidation of the [cytochrome P450-Fe(II)(CH3N = NH)] complex with limited amounts of dioxygen, a new complex characterized by a Soret peak at 486 nm is formed. The latter complex was also formed upon slow reaction of methyldiazene with microsomal cytochrome P450-Fe(III) or in situ oxidation of methylhydrazine by limited amounts of O2 or ferricyanide. This complex is rapidly destroyed by O2 or ferricyanide in excess and more slowly by excess dithionite in the presence of CO. Reactions of ethyldiazene or benzyldiazene with cytochrome P450-Fe(III) afforded similar complexes characterized by Soret peaks around 480 nm. These results, when compared to those recently described on reactions of monosubstituted hydrazines RNHNH2 and diazenes RN = NH with hemoglobin and iron-porphyrins, are consistent with a [cytochrome P450-Fe(II)(RN = NH)] structure for the 446-nm-absorbing complexes and a sigma-alkyl cytochrome P450-Fe(III)-R structure for the complexes characterized by a Soret peak around 480 nm. They also suggest a sigma-cytochrome P450-Fe(III)-Ph structure for the complex derived from phenylhydrazine oxidation, recently described in the literature. Finally, they provide the first evidence that cytochrome P450-Fe(III)-R complexes are formed upon microsomal oxidation of alkyl or phenylhydrazines.     

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.     

Loss of cytochrome P-450 from hepatic nuclear membranes of rats fed 2-acetylaminofluorene

The cytochrome P-450 content of nuclear membranes isolated from the livers of male Sprague-Dawley rats fed a semipurified diet containing 0.05% w/w 2-acetylaminofluorene (AAF) for 3 weeks, was only about 20% of the values in control rats fed the same diet devoid of AAF. This effect was apparent after only 1 week of AAF treatment and persisted in nuclear membranes from isolated hyperplastic nodules (HPN) generated by 4 cycles of interrupted AAF-feeding. The microsomal cytochrome P-450 content, on the other hand, remained at control levels after 1 week of AAF treatment, and it was only slightly decreased after 3 weeks. In contrast, microsomes from HPN generated by prolonged AAF treatment had markedly decreased amounts of cytochrome P-450. The AAF treatment also caused changes in cholesterol epoxide hydrolase activity, which paralleled those observed for cytochrome P-450 content. Nuclear membranes from livers of rats fed AAF for 3 weeks, and from isolated HPN, had only 30-50% of the cholesterol epoxide hydrolase activity present in controls, whereas the microsomal enzyme activity remained at control levels after 3 weeks of AAF feeding but was 50% depressed in microsomes from HPN. The selective loss of cytochrome P-450 and of cholesterol epoxide hydrolase in hepatic nuclear membrane, but not in microsomes, of rats fed AAF for 3 weeks suggests independent control for these enzymes in these two membrane fractions. Cytochrome P-450 plays a role both in the activation of AAF (N-hydroxylation) as well as in its detoxification (ring hydroxylation) whereas cholesterol epoxide hydrolase initiates the detoxification of cholesterol epoxide. Therefore, our findings suggest the hypothesis that AAF treatment causes an early loss, at the surface of the nucleus, of the last line of defense for detoxification of transforming or promoting metabolites generated by microsomal activation of natural substances such as cholesterol and of xenobiotics such as AAF.     

Phosphorylation of rabbit liver cytochrome P-450 LM2 and its effect on monooxygenase activity

The phosphorylation of rabbit liver microsomal cytochrome P-450 LM2 by catalytic subunit of cyclic AMP-dependent protein kinase (W. Pyerin et al. (1983) Carcinogenesis 4, 573) has now been studied in detail with purified soluble form of cytochrome P-450 as well as with the purified protein incorporated into model membranes. The apparent Km values for P-450 of the phosphorylation reaction in all experimental systems were in a range of 2-8 microM, while the Vmax values were dependent on the state of P-450. Upon phosphorylation, the reconstituted enzyme activities with benzphetamine (N-demethylation) and 7-ethoxycoumarin (O-deethylation) as substrates were reduced to 30-40% of control.     

Modulation of rat liver cytochrome P450 activity by prolonged red wine consumption

Cytochrome P450-dependent oxidation of lauric acid, p-nitrophenol and ethanol by liver microsomal fractions were studied in control rats and in animals given either ethanol, red wine, or alcohol-free red wine for 10 weeks. Ethanol increased the total cytochrome P450 and the isoenzyme 2E1 content, as well as the p-nitrophenol hydroxylation and ethanol oxidation. These effects of ethanol treatment were attenuated by red wine administration. Red wine increased the total antioxidant capacity of plasma, whereas the alcohol-free red wine decreased the cytochrome P450 content and decreased the oxidation of lauric acid, p-nitrophenol and ethanol to values lower than control. It is concluded that red wine administration attenuates the ethanol-induced enhancement in liver microsomal parameters dependent on cytochrome P450 2E1 activity, an affect that seems to be accomplished by the non-alcoholic constituents of red wine known to have antioxidant properties.     

B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

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.     

An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein.

Immunoreactive cytochrome b5 and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from rat liver microsomal fractions were measured by using an enzyme-linked immunoadsorbent assay (e.l.i.s.a.) as a function of age, sex and type of inducer (phenobarbital or 3-methylcholanthrene), and the values were compared with those obtained by spectral measurement (for cytochrome b5) or enzymic assay (for reductase). In untreated animals, there was more cytochrome b5 and NADPH-cytochrome P-450 reductase when measured by an e.l.i.s.a. than was seen spectrally or enzymically. However, for microsomal preparations from phenobarbital-pretreated animals, spectrally obtained values for cytochrome b5 and immunoreactive-cytochrome b5 values were similar. Values from control animals suggest that there is about 20-30% more immunoreactive cytochrome b5 than that which is spectrally detectable.