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1.
Z Sípal P Anzenbacher Z Putz J Chlumsky O Krivanová 《Acta biologica et medica Germanica》1979,38(2-3):483-494
Hydrocarbons of different structures interact with microsomal and solubilized cytochrome P-450 from liver of phenobarbital-pretreated rats forming a high spin enzyme-substrate type complex. The affinity of cytochrome P-450 for hydrocarbons increases with increasing lipophilicity independently of the chemical structure. The binding capacity of microsomal P-450 for aliphatic hydrocarbons is generally higher than for aromates. Mutual influence in binding of two different hydrocarbons by microsomal P-450 is stronger among aromatic than among aliphatic hydrocarbons; in both cases it appears to be effected rather by specific interaction of both substances with the binding site than by a nonspecific influence on the microsomal membrane. Only one fraction of low spin form of solubilized cytochrome P-450 from rat liver interacts with hydrocarbons. The binding capacity for aromatic and aliphatic substances corresponds to that found in microsomes. The affinity for the most lipiphilic substrate, perhydrophenanthrene, is equal in microsomal and solubilized preparation; with decreasing lipophilicity the affinity of solubilized P-450 decreases faster than in microsomes. The LM2 fraction of cytochrome P-450 from phenobarbital-pretreated rabbits interacts only with aliphatic hydrocarbons with wide variation of the binding capacity. The affinity is generally one order of magnitude lower than in microsomes. Active fractions of solubilized P-450 from both species are rapidly converted to P-420 by dithionite. The extent of this conversion is strongly reduced by saturation with substrate. 相似文献
2.
The effects of modified cyclodextrins (CDs) hydroxypropyl-beta-CD and methyl-beta-CD were studied in vitro on cDNA-expressed human cytochrome P-450 (CYP) activities (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). The modified CDs inhibited the activities of CYP2C19 and CYP3A4 while enhancing CYP2C9 activity by 140 to 176% relative to the control values at lower concentrations. In addition, methyl-beta-CD inhibited CYP1A2 and CYP2D6 at higher concentrations. 相似文献
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Spectrophotometric, affinity chromatography and cross-linking experiments provided evidence that cytochrome P-450scc from bovine adrenocortical mitochondria forms a tight complex with cytochrome b5 from rabbit liver microsomes. In the reconstituted system cholesterol side chain activity of cytochrome P-450scc was enhanced by the addition of cytochrome b5. 相似文献
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The effect of linear monoamines on dimyristoylphosphatidylglycerol and dimyristoylphosphatidylcholine multilamellar liposomes was studied as a function of their length and compared with the behavior of linear carboxylic acids. The role of the hydrophobic interactions was demonstrated and the free energy of the binding for each interacting carbon atom was determined. The thermotropic behavior of the liposomes was characterized by differential scanning calorimetry and it was shown that these molecules affect the temperature and the cooperativity of the gel to fluid state transition of the membrane differently. In particular, it appeared that membrane perturbation was maximum when the chain length of the amphipathic molecules ranged between 7 and 9 carbon atoms, with more pronounced effects in the case of monoamines. Molecules shorter than 3-4 carbon atoms did not produce any observable change in the transition temperature. The study was extended to linear alpha,omega-diamines to investigate the amphipathic character of long diamines and to investigate the role of bridging bonds established with neighboring phospholipids. 相似文献
6.
Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed. 相似文献
7.
John Y.L. Chiang 《Archives of biochemistry and biophysics》1981,211(2):662-673
The interactions between purified microsomal cytochrome P-450 and cytochrome b5 has been demonstrated by aqueous two-phase partition technique. Major forms of cytochrome P-450 induced by phenobarbital (P-450LM2) and β-naphthoflavone (P-450LM4) are almost exclusively distributed in the dextran-rich bottom phase (partition coefficient, K = 0.06), whereas NADPH-cytochrome P-450 reductase and cytochrome b5 are mainly distributed in the polyethylene glycol-rich top phase (K = 3.5 and 2.5, respectively), when these enzymes were partitioned separately in the dextran-polyethylene glycol two-phase system. The mixing of P-450LM with cytochrome b5 changes the partition coefficients of both P-450LM and cytochrome b5 indicating that molecular interaction between P-450LM and cytochrome b5 occurred. Complex formation was also confirmed by optical absorbance difference spectral titration, and the stimulation of the P-450LM-dependent 7-ethoxycoumarin and p-nitrophenetole O-deethylase activities by equal molar quantity of detergent-solubilized cytochrome b5, but not trypsin-solubilized enzyme, in the reconstituted system. Cytochrome b5 decreases the Km's of both substrates for P-450LM2-dependent O-deethylations and increases the V's of both reactions by two- to three-fold. This stimulatory effect requires the presence of phospholipid in the reconstituted enzyme system. These results suggest that cytochrome b5 plays a role in some reconstituted drug oxidation enzyme systems and that molecular interactions among cytochrome P-450, reductase, and cytochrome b5 are catalytically competent in the electron transport reactions. 相似文献
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J F Crivello 《Archives of biochemistry and biophysics》1986,248(2):551-561
The antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), nordihydroguiaretic acid (NDGA), benzyl sulfoxide (BS), ferulic acid (FA), caffeic acid (CA), dimethyl sulfoxide (Me2SO), protocatechuic acid (PCA), and P-450 inhibitor metyrapone all acted to slow the previously noted loss of vitamin D3 1 alpha-and 24-hydroxylase activities in cultured bovine proximal tubule cells. The slowing of the loss of hydroxylase activities by antioxidants was increased by culturing cells in 5% O2 vs 19% O2. These same antioxidants also directly inhibited 1 alpha- and 24-hydroxylase activities. For a single antioxidant, or metyrapone, Ki's for inhibition of both hydroxylases were equal, ED50's for stabilization of both hydroxylase activities were equal, and Ki's and ED50's were not significantly different. These antioxidants prevented tert-butylhydroperoxide (tert-BOOH)-mediated proximal tubule cell death at concentrations, i.e., 0.1 mM, which were effective in stabilizing hydroxylase activities. When added together, the antioxidants H2SeO3, uric acid, and trolox c gave slight stabilization of hydroxylase activities without inhibiting hydroxylase activities. Singly, these antioxidants did not stabilize or directly inhibit hydroxylase activities. This antioxidant combination augmented BHA- or BHT-mediated stabilization of both hydroxylase activities independent of any effects on inhibition. But the most potent antioxidants which acted to stabilize hydroxylase activities in culture also directly acted to inhibit hydroxylase activities. Antioxidant effects were additive for both inhibition and stabilization of hydroxylase activities. Stabilization of hydroxylase activities was dissociated from inhibition in the presence of maximal FA, CA, and BHA or FA, CA, and BHT combinations. Bovine renal mitochondrial cytochrome P-450 levels decreased in cultured bovine proximal tubule cells to nondetectable levels by 8 days in culture. When cultures were treated with BHA and BS, mitochondrial P-450 levels were almost twofold greater than in untreated controls. Percentage changes in mitochondrial P-450 levels closely paralleled percentage changes in hydroxylase activities elicited by antioxidant treatment regimes. Antioxidants which were effective inhibitors of hydroxylase activities in cultured bovine proximal tubule cells were also effective in inhibiting hydroxylase activities in isolated proximal tubule mitochondria, supplemented with a NADPH-generating source. Ki's for inhibition of hydroxylase activities were very similar in cultured cells and in isolated mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
10.
Using the electron paramagnetic resonance (EPR) method with spin-labeled fatty acids and gel-penetrating chromatography, the effect of phosphatidylglycerol on cytochrome P-450 incorporation into liposomes from dimyristoylphosphatidylcholine was investigated. An addition of phosphatidylglycerol caused an increase in the protein content of the proteoliposomes as well as their accelerated formation at temperatures below and above the liposome phase transition temperature (Ts). The dependence of the proteoliposome formation rate on the phosphatidylglycerol content in the liposome mixture is described by complex kinetics with a maximum in the presence of 10 mol.% of the negatively charged phospholipid. The mechanism of proteoliposome formation is discussed in terms of asymmetric distribution and phase state of the phospholipids in the original vesicles. 相似文献
11.
Cytochrome P-450 and NADPH-cytochrome P-450 REDUctase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphoaditylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80--90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the sysTEM IN A SIMILAR WAY TO THE FAST-PHASE REDUCTION OF CYTOCHROME P-450, though the latter is not the rate-limiting step of the overall reaction. 相似文献
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R Misselwitz G R J?nig H Rein E Buder D Zirwer K Ruckpaul 《Acta biologica et medica Germanica》1975,34(11-12):1755-1766
In order to characterize the substrate binding sites, difference spectroscopic titrations in microsomal and solubilized cytochrome P-450 from induced and non-induced rat liver microsomes were performed. The binding constants determined show differences depending on the physicochemical nature of the substrate and the degree of integration of the enzyme system. In hydrophilic substrates the differences of the binding to the microsomal or solubilized form are less pronounced than in lipophilic ones. From the comparison of the parameters obtained at various levels of integration it is concluded that the micromilieu of the binding site is of great importance for the binding of the substrate of cytochrome P-450. 相似文献
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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. 相似文献
14.
D Müller-Enoch P Churchill S Fleischer F P Guengerich 《The Journal of biological chemistry》1984,259(13):8174-8182
Phospholipid has been reported to be necessary for optimal catalytic activity of a number of mammalian cytochrome P-450 (P-450) systems. We also confirm that a number of individual phospholipids and mixtures, used as soluble monomers or phospholipid vesicles, show activation of 7-ethoxycoumarin O-deethylase activity by an enzyme system composed of rat liver microsomal P-450PB-B and NADPH-P-450 reductase. However, by preincubating a mixture of P-450 and NADPH-P-450 reductase at high concentrations, optimal activity can be obtained in the absence of phospholipid. The catalytic activity of the complex formed is concentration dependent in the absence of lipid or in the presence of soluble lipid. The activity in phospholipid vesicles is optimal and concentration independent. The apparent Km for NADPH-P-450 reductase in P-450-dependent oxidation systems is lowered severalfold in the presence of phospholipid. The apparent Km for the P-450 substrate, 7-ethoxycoumarin, and the temperature dependence of 7-ethoxycoumarin O-deethylase activity were unaffected by the addition of phospholipid to a preformed complex of P-450PB-B and NADPH-P-450 reductase. The effect of lipid on a number of other P-450 isozymes was also examined and in no case did lipid enhance the catalytic activity of the preformed complex. These results lead to the conclusion that the major effect of phospholipids in P-450-based enzyme systems is the facilitation of an active P-450:NADPH-P-450 reductase complex. This is the first report that maximum P-450 supported monooxygenase activity can be obtained in the absence of phospholipid. 相似文献
15.
John W. Greiner Robert E. Kramer Howard D. Colby 《Prostaglandins & other lipid mediators》1979,17(4):587-597
Studies were carried out to investigate the effects of prostaglandins (PG) on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE1, PGE2, PGA1, PGF1α or PGF2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism. 相似文献
16.
Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles 总被引:2,自引:0,他引:2
J Gut C Richter R J Cherry K H Winterhalter S Kawato 《The Journal of biological chemistry》1982,257(12):7030-7036
Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes. 相似文献
17.
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. 相似文献
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A protein-protein association of cytochrome P-450 LM2 with NADPH-cytochrome P-450 reductase, with cytochrome b5, and with both proteins was demonstrated in reconstituted phospholipid vesicles by magnetic circular dichroism difference spectra. A 23% decrease in the absolute intensity of the Soret band of the magnetic CD spectrum of cytochrome P-450 was observed when it was reconstituted with reductase. A difference spectrum corresponding to a 7% decrease in absolute intensity was obtained when cytochrome b5 was incorporated into vesicles that already contained cytochrome P-450 and cytochrome P-450 reductase compared to a decrease of 13% in absolute intensity when cytochrome b5 was incorporated into vesicles that contained only cytochrome P-450. The use of the magnetic circular dichroism confirmed that protein-protein associations that have been detected by absorption spectroscopy between purified and detergent-solubilized proteins also exist in membranes. High ionic strength was shown to interrupt direct electron flow from cytochrome P-450 reductase to cytochrome P-450 but not the electron flow from reductase through cytochrome b5 to cytochrome P-450. Upon incorporation of cytochrome b5 into cytochrome P-450- and cytochrome P-450 reductase-containing vesicles, an increase of benzphetamine N-demethylation activity was observed. The magnitude of this increase was numerically identical to the residual activity of the reconstituted vesicles measured in the presence of 0.3 M KCl. It is concluded that there is a requirement for at least one charge pairing for electron transfer from reductase to cytochrome P-450. These observations are combined in a proposed mechanism of coupled reversible association reactions in the membrane. 相似文献
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