Drug-induction decreases the mobility of cytochrome P-450 in rat liver microsomes: protein rotation study

Effect of drug-induction on the rotation of cytochrome P-450 and on lipid fluidity in rat liver microsomes was examined. Rotational diffusion of cytochrome P-450 was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. Microsomal lipid fluidity was measured by observing fluorescence anisotropy of DPH incorporated in the lipid bilayer. The absorption anisotropy decayed within 2 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the drug-induction with PB, MC, and PCB when compared with non-induced CON-microsomes. The observed values for the normalized time-independent anisotropy r(infinity)/r(0) are r(infinity)/r(0) = 0.41 (CON-microsomes), 0.54 (PB-microsomes), 0.52 (MC-microsomes), and 0.57 (PCB-microsomes). The average rotational relaxation time phi = 580-690 microseconds was almost unchanged over all microsomes presently examined. A significantly high value of r(infinity)/r(0) = 0.41-0.57 implies the co-existence of mobile and immobile populations of cytochrome P-450. Based on the assumption that the heme tilts about 55 degrees from the membrane plane for all species of P-450s besides P-450PB, 59% (CON-microsomes), 46% (PB-microsomes), 48% (MC-microsomes), and 43% (PCB-microsomes), respectively, of the cytochrome P-450 in microsomes is calculated to be mobile. Upon drug-induction the microsomal membrane was fluidized to some extent as judged by the steady-state fluorescence anisotropy of 0.156 for CON-microsomes and 0.139-0.148 for drug-induced microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of cytochrome P-450 reduction: studies in bovine adrenocortical microsomes

The results of the present study indicate first that in the microsomal preparation, the components of the P-450 reduction system are heterogeneously distributed, comprising dissociable and nondissociable parts. Second, the P-450 reduction curve can be adequately described by a sum of two exponential functions, indicating two concurrent first-order reactions. Third, the two phases can be altered independently. The addition of the substrate increased the extent of the fast phase while it had little or no effect on that of the slow phase. Changes in the interaction of the dissociable and nondissociable components affected the extent of the slow phase while they were without effect on that of the fast phase. Experiments with different steroids indicated that the independence of the two phases is not due to functionally different P-450's and that the cytochrome reduced in both phases is essentially P-450C-21. The results are interpreted as follows: Transformation of P-450 from the low- to the high-spin state controls the total P-450 reduced. The rate and the biphasicity of the reduction are functions of the interaction of P-450 and the reductase.     

Conversion of cholesterol to pregnenolone mobilizes cytochrome P-450 in the inner membrane of adrenocortical mitochondria: protein rotation study

Rotation of cytochrome P-450 was examined in bovine adrenocortical mitochondria before and after an enzymatic transformation of cholesterol into pregnenolone by cytochrome P-450scc in the presence of malate. Rotational diffusion was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate substrate-dependent intermolecular interactions of cytochrome P-450 with other redox components. Rotational mobility of cytochrome P-450 was significantly dependent on the decrease in cholesterol content by side chain cleavage reaction catalyzed by cytochrome P-450scc. In a typical experiment, the observed value for the normalized time-independent anisotropy r(infinity)/r(0) was decreased from 0.78 in control mitochondria to 0.60 after conversion of 21% of cholesterol to pregnenolone, while no significant change was observed for the average rotational relaxation time phi of about 700 microseconds. Significantly high values of r(infinity)/r(0) = 0.78 and 0.60 imply co-existence of mobile and immobile populations of cytochrome P-450. Since we observed that the heme angle tilted 55 degrees from membrane plane, 22% (control mitochondria) and 40% (after conversion of cholesterol to pregnenolone) of cytochrome P-450 in mitochondria are calculated to be mobile in the preparation. The significant mobilization of cytochrome P-450scc molecules caused by the conversion of cholesterol to pregnenolone is likely due to changes in protein-protein interactions with its redox partners, since the lipid fluidity was kept unchanged by the cholesterol depletion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system

The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.     

Effect of spin state on reduction of cytochrome P-450 (P-450C21) from bovine adrenocortical microsomes

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450C21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17 alpha-hydroxyprogesterone-bound P-450C21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25 degrees C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450C21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25 degrees C. The steady-state results showed that the rate of P-450C21 reduction was not affected by the high spin content of substrate-bound P-450C21 but was very slow without a steroid substrate. Biphasic reduction of P450C21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17 alpha-hydroxyprogesterone-bound and progesterone-bound P-450C21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450C21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450C21.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Studies of cytochrome P-450 in testis microsomes

Studies on the role of cytochrome P-450 in mouse, rat, and chick testis microsomes showed that this CO-binding hemoprotein is involved in the activity of the 17α-hydroxylase. A 70–80% inhibition by CO of the 17α-hydroxylase activity was detected in rat and chick testis microsomes. In the mouse testis, the level of the enzyme activity is ten times greater than that of the rat. This partly explains why an acceleration of NADPH oxidation by progesterone can be observed in mouse but not in rat testis microsomes. In rat testis microsomes, type I binding spectra of cytochrome P-450 was observed with pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. The apparent K_s values for progesterone and 17-hydroxyprogesterone were 0.50 and 1.00 μm, respectively.When NADPH is used to measure cytochrome P-450 levels in rat testis microsomes, CO formation resulting from a stimulation in lipid peroxidation by phosphate or Fe²⁺ was sufficient to bind with 50% of the total amount of cytochrome P-450. Substitution of phosphate by Tris reduced the amount of lipid peroxidation to minimal levels. On a comparable basis, no CO formation was observed in avian testis microsomes.An increase in the testicular levels of cytochrome P-450 resulted upon the administration of HCG and cyclic-AMP to 1-day-old chicks. The lack of stimulation of the cytochrome P-450 levels by progesterone and pregnenolone suggest that the hormonal stimulation of the P-450 levels is not due to substrate induction.     

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).     

Constraint on the substrate cytochrome P-450 binding reaction in bovine adrenocortical microsomes at physiological temperature.

The addition of cholate to the microsomes at 37.5 degrees C resulted in a striking decrease in the apparent substrate dissociation constant (K's) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the Ks and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome P-450 is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substrate-cytochrome -450 binding reaction and in the temperature dependency of the Ks.     

Rotation and interaction with epoxide hydrase of cytochrome P-450 in proteoliposomes

Purified rat liver cytochrome P-450MC or P-450PB was co-reconstituted with epoxide hydrase in liposomal vesicles made of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a lipid to protein weight ratio of 5 by the cholate dialysis procedure. Rotational diffusion of the cytochromes was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane-normal" model. The measurements were used to investigate interactions of cytochrome P-450MC or P-450PB with epoxide hydrase. Different rotational mobilities of the two cytochromes were observed. The amount of mobile molecules was 78% for cytochrome P-450MC and 91% for P-450PB, and the rest was immobile within the experimental time range of 1 ms. In the presence of epoxide hydrase 85% of cytochrome P-450MC and 96% of P-450PB were mobile. Cross-linking of epoxide hydrase by anti-epoxide hydrase antibodies resulted in a drastic immobilization of the cytochromes, reducing the mobile population to 49% for P-450MC and to 60% for P-450PB. The rotational relaxation times phi of the mobile populations ranged from 210 to 283 microseconds. These results imply that both cytochromes P-450MC and P-450PB transiently associate with epoxide hydrase in liposomal membranes. Further analysis of the data showed that the angle between the heme plane of P-450MC and the membrane is 48 degrees or 62 degrees, different from the value of 55 degrees reported previously for P-450PB (Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S. (1983) J. Biol. Chem. 258, 8588-8594).     

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.     

Purification of cytochrome P-450 from bovine adrenocortical mitochondria

One soluble cytochrome P.450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The purified enzyme catalyses side-chain cleavage of cholesterol and to a much lesser extent 11β-hydroxylation (<13% side-chain cleavage) but shows no 18-hydroxylase activity. The molecular weight of this P.450 is approximately 800,000.     

Binding of nitrosamines to cytochrome P-450 of liver microsomes.

The interactions of 5 carcinogenic and 1 non-carcinogenic nitrosamines with hepatic microsomal cytochrome (cyt.) P-450 were investigated, using both optical difference and electron paramagnetic resonance (EPR) spectroscopic methods. Liver microsomes from phenobarbital (PB)-pretreated mice and 3-methylcholanthrene (3-MC)-pretreated rats were used, in order to have an increased specific content of cyt. P-450 and cyt. P-448 respectively. The optical and EPR spectral data obtained in the oxidised state suggest that nitrosamines are able to bind both as substrates and as ligands to the hemoprotein cyt. P-450, depending on the concentration of nitrosamine, its chemical identity and the cytochrome species present. After reduction with dithionite or NADPH in the optical difference spectrum a Soret band developed between 444 and 453 nm to an extent, which is dependent on the particular nitrosamine present. This initial nitrosamine-induced spectrum might represent a ferrous nitric oxide (NO)-cyt. P-450 complex. It appears unstable and is converted kinetically into a spectrum lacking a Soret band, but with a predominant absorbance minimum at about 425 nm. A visible band is located at 585 nm. In the EPR spectrum a sharp 3-line signal around g = 2.01 appears concomitantly. Both spectral parameters are typical of a NO-cyt. P-420 complex. These results, in conjunction with metabolic studies, indicate that nitrosamines are denitrosated by a reductive process in which cyt. P-450 appears to be involved. The resulting NO-cyt. P-450 complex denatures to a NO-cyt. P-420 complex when the dioxygen level is not sufficiently high to complete successfully.     

Electron paramagnetic resonance studies of cytochrome P-450 in plant microsomes.

The technique of electron paramagnetic resonnance spectrometry has been applied to the study of plant microsomal electron-transport components. Only tulip-bulb microsomes were found to give strong enough signals to allow detailed study. At 77 K in the oxidised state, signals were observed at g values of 2.40, 2.25 and 1.93, characteristic of cytochrome P-450 in the low-spin state, and also at g = 4.27, attributable to ferric iron in a rhombic environment. The signals at g = 2.40, 2.25 and 1.93 disappeared upon reduction with sodium dithionite. At 10 K in the oxidised state, signals at g = 8.3 and 3.3 appeared, and these were attributed to high-spin cytochrome P-450. At this temperature a further signal at g = 6, due to cytochrome P-420, was seen in aged tulip-bulb microsomes. Redox titration of both high-spin and low-spin cytochrome P-450 gave the same apparent midpoint potential of -315 +/- mV at pH 6.8 and 25 degrees C. The significance of this value is discussed. Addition of "type I" or "type II" ligands to oxidized cytochrome P-450 caused an increase and a decrease, respectively, in the ratio of the high-spin to the low-spin form. A second effect of aniline, a type II ligand of cytochrome P-450, was to remove the g = 6 signal, suggesting that it also interacts with cytochrome P-420. No iron-sulphur proteins similar to those found in some other cytochrome P-450 electron-transport chains could be detected in any of the microsomes analysed.     

Multiple forms of cytochrome P-450 in rabbit colon microsomes

Three cytochrome P-450 preparations, designated as cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction, were separated and purified about 23-, 50-, and 29-fold, respectively, from the cholate extracts of rabbit colon mucosa microsomes. Their specific contents were 1.2, 2.6, and 1.5 nmol of cytochrome P-450 per mg of protein, respectively. Cytochrome P-450ca and cytochrome P-450cb migrated as heme-containing polypeptide bands with molecular weights of about 53,000 and 57,000, respectively, on SDS-polyacrylamide gel electrophoresis. The CO-reduced difference spectra of cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction showed maxima at 451, 450, and 449 nm, respectively. Cytochrome P-450ca efficiently catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1) and the omega- and (omega-1)-hydroxylation of caprate, laurate, and myristate in the reconstituted system containing cytochrome P-450ca, NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. In contrast, cytochrome P-450cb and cytochrome P-448c fraction had no detectable activity toward PGA1 and fatty acids. Both catalyzed aminopyrine and benzphetamine N-demethylation. Cytochrome P-448c fraction also hydroxylated benzo(a)pyrene, and phosphatidylinositol or phosphatidylserine exhibited a stimulatory effect on this activity. The results show that rabbit colon microsomes contain catalytically different cytochrome P-450, one of which is specialized for the omega-oxidation prostaglandins, the others being involved in the metabolism of exogenous compounds such as drugs and polycyclic hydrocarbons.     

Spectroscopic quantitation of cytochrome P-450 in human lung microsomes

The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.     

Steroid interactions with cytochrome P-450 from testis microsomes

The cytochrome P-450 of gonadal microsomes is an integral component of the steroid converting enzymes, 17 alpha-hydroxylase and 17,20-lyase. Interaction of the steroid substrates with this cytochrome results in a shift in the Soret band as measured by difference spectroscopy. In these studies it is shown that in contrast to placental microsomal cytochrome P-450 which binds C19 steroids, testis microsomal cytochrome P-450 primarily binds C21 steroids. However, addition of a 17 alpha- methyl, 17 beta-acetate or a 17 beta-benzoate group to testosterone permits interaction. The addition of hydroxyl or methyl groups to other positions does not affect binding. The presence of multiple oxygen functions on C21 steroids, as in cortisol and corticosterone, precludes interaction. At least one oxygen function seems necessary for binding as 5 alpha- and 5 beta-pregnane do not bind whereas 20-deoxypregnenolone (5-pregnen-3 beta-ol) does bind. These findings indicate that factors in addition to hydrophobic interactions dictate the binding of steroid substrates to testis microsomal cytochrome P-450.     

The active form of cytochrome P-450 from bovine adrenocortical mitochondria.

Cytochrome P-450 from bovine adrenocortical mitochondria exists in three forms of molecular weight: 850,000 (protein 16), of one-half (protein 8), and of one-quarter of this value (protein 4). The forms of the enzyme are named according to the number of subunits and all appear to be active in converting cholesterol to 3beta-hydroxy-5-pregnen-20-one (side chain cleavage) (Shikita, M., and Hall, P.F. (1973) J. Biol. Chem. 248, 5606). To determine whether all three forms are active at their characteristic molecular weights, the three cytochromes were each layered onto separate sucrose density gradients and centrifuged at 49,000 rpm for 60 min; the gradients contained all the factors necessary for side chain cleavage including one of the following substrates: cholesterol, 20S-hydroxycholesterol, and 20S,22R-dihydroxycholesterol. Regardless of the form of P-450 layered onto the gradient and regardless of the substrate, enzyme activity (side chain cleavage) was observed only in fractions corresponding to a sedimentation coefficient of 20 to 22 S which is that for protein 16. No activity was observed at S values corresponding to either protein 8 or protein 4. These findings indicate that the active form of cytochrome P-450 from adrenocortical mitochondria is that containing 16 subunits, i.e. the form in which the cytochrome is normally isolated from adrenal mitochondria. Forms consisting of eight and four subunits which can be prepared from protein 16 become active only by forming protein 16, at least in an aqueous medium in vitro.