Reconstitution of liver endoplasmic reticulum membranes from solubilized proteins and lipids by removal of detergent]

A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown. A removal of the detergent results in a formation of membranes, which are chemically close to the original ones but ultrastructurally very different from the latter. On the other hand, absorption or dialysis of cholate-solubilized proteins and lipids results in reconstituted membranes with asymmetrically arranged intramembrane particles located on the hydrophobic surfaces of the membrane halves. The number and size of these particles are similar to those of the original microsomal membranes.     

Incorporation of the cytochrome P-450 monooxygenase system into large unilamellar liposomes using octylglucoside,especially for measurements of protein diffusion in membranes

Cytochrome P-450 and NADPH cytochrome P-450 reductase were incorporated into large unilamellar lipid vesicles (200–300 nm in diameter) removing octylglucoside from mixed micelles by dialysis. The large size of the protein-containing liposomes guarantees a negligibly small vesicle tumbling. Such large vesicles are better suited for studies of protein rotation in reconstituted membranes than vesicles prepared by use of bile salts. At present the octylglucoside reconstituted monooxygenase system seems to be the most appropriate model for studying protein-protein and protein-lipid interactions in liver microsomes due to the similarity with respect to the main structural and functional properties, including size.     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

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.     

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.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

Association of cytochrome b5 and cytochrome P-450 reductase with cytochrome P-450 in the membrane of reconstituted vesicles

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.     

Localization of the active center of microsomal cytochrome P-450

To solve the problem of localization of the active center of cytochrome P-450 in microsomal membranes, new bifunctional compounds (I-IV), which contain pyridine radical, aliphatic chain of variable length and diphosphonic acid ("floating" molecules) have been applied. These compounds inhibit oxidation and binding of the substrates of cytochrome P-450 (aminopyrine and aniline), inhibition being of a competitive character. Measurements of distribution coefficients between water and membranes of microsomes and liposomes from egg phosphatidylcholine evidence that the microsomal proteins are necessary for providing effective interaction of I-IV with microsomal membrane. The 1H-NMR method has demonstrated compounds to be incorporated into lipid bilayer so that the non-polar part is in the inner membrane volume. The results obtained confirm our previous conclusion (Krainev A.G., Weiner L.M., Alferyev I.S., Slynko N.M. (1985) Biochim. Biophys. Acta, 818, 96-104) about localization of the active center of microsomal cytochrome P-450 at the depth of approximately 18 A from the hydrophilic surface of a membrane.     

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.     

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.     

Incorporation of bacterial membrane proteins into liposomes: factors influencing protein reconstitution

Meningococcal and gonococcal outer membrane proteins were reconstituted into liposomes using detergent-mediated dialysis. The detergents octyl glucopyranoside (OGP), sodium cholate and Empigen BB were compared with respect to efficiency of detergent removal and protein incorporation. The rate of OGP removal was greater than for cholate during dialysis. Isopycnic density gradient centrifugation studies showed that liposomes were not formed and hence no protein incorporation occurred during dialysis from an Empigen BB containing reconstitution mixture. Cholate-mediated reconstitution yielded proteoliposomes with only 75% of the protein associated with the vesicles whereas all of the protein was reconstituted into the lipid bilayer during OGP-mediated reconstitution. Essentially complete protein incorporation was achieved with an initial protein-to-lipid ratio of 0.01:1 (w/w) in the reconstitution mixture; however, at higher initial protein-to-lipid ratios (0.02:1) only 75% protein incorporation was achieved. Reconstituted proteoliposomes were observed as large (>300 nm), multilamellar structures using cryo-electron microscopy. Size reduction of these proteoliposomes by extrusion did not result in significant loss of protein or lipid. Extruded proteoliposomes were unilamellar vesicles with mean diameter of about 100 nm.     

Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes

Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.     

Substrate binding site of microsomal cytochrome P-450 directly faces membrane lipids

Cytochrome P-450, purified from liver microsomes of phenobarbital-treated rabbits, was incorporated into dimyristoylphosphatidylcholine liposomes. The binding of benzphetamine to the liposome-bound cytochrome P-450 was examined by measuring the benzphetamine-induced spectral change at various temperatures. The van't Hoff plot of the apparent spectral dissociation constant showed a distinct break at the temperature of phase transition of the synthetic lipid. On the other hand, no such break was observed for benzphetamine binding to microsomal bound cytochrome P-450. These results suggest that the substrate binding site of cytochrome P-450 is embedded in the apolar interior of phospholipid bilayer membranes.     

Conformational state and functional activity of NADPH-dependent cytochrome P-450 reductase isolated and incorporated into phospholipid bilayers

It was shown that the alpha-helix content in both isolated and incorporated into phospholipid bilayer NADPH-dependent cytochrome P-450 reductase is 20%. NADPH or dithionite reduction of the flavoprotein is not followed by conformational changes. The incorporation of the NADPH-dependent cytochrome P-450 reductase molecule into the phospholipid bilayer does not affect its catalytic properties. It was found that the protein does not interact with the phospholipid bilayer of phosphatidyl choline liposomes but incorporates readily into the liposomal membrane from a microsomal phospholipid mixture with a binding constant of 17.4 microM.     

Phosphatidylcholine-induced repair of damaged hepatocyte membranes in heliotrine poisoning

Hepatocyte membranes destruction in experimental toxic hepatitis caused by heliotrine administration was accompanied by a 10-fold increase in blood serum activity of aldolase fructose-I-monophosphate, a decrease in cytochrome P-450 content, an increase in the rate of cytochrome P-450 inactivation, as well as a decrease in microsomal glucose-6-phosphatase activity. Administration of phosphatidylcholine liposomes decreased the activity of aldolase twofold, which indirectly shows partial reconstitution of liver cell membranes. Phosphatidylcholine protective action is also manifested in an increase in the activity of glucose-6-phosphatase, a microsomal marker enzyme, up to its control level and in a 20% reduced rate of cytochrome P-450 inactivation. It has been shown that destroyed liver cell membranes may be repaired by the introduction of phosphatidylcholine in the form of multilayer liposomes.     

Temperature quenching of cytochrome P-450 fluorescence in proteoliposomes with different physical states of the lipid bilayer

Studies were carried out of temperature quenching of self-fluorescence of cytochrome P-450 in solution and liposomes from natural phosphatidylcholine, dimiristoylphosphatidylcholine, dipalmitoylphosphatidylcholine. The fluorescence spectrum of cytochrome P-450 is a superposition of triptophane and tyrosine components. During protein incorporation into liposomes a significant short-wave shift of the emission spectrum takes place. Temperature dependence of the intensity of cytochrome P-450 self-fluorescence in solution has bends at 30, 45 and 50 degrees C. When protein is incorporated into liposomes the location of bends depends on individual properties of lipids forming the bilayer. Effect of lipid surrounding on temperature conformational rearrangements of cytochrome P-450 molecule is discussed.     

Reconstitution studies on the involvement of radiation-induced lipid peroxidation in damage to membrane enzymes

The effect of radiation on the drug-metabolizing enzyme system of microsomes, reconstituted with liposomes of microsomal phospholipids, NADPH-cytochrome P-450 reductase and cytochrome P-450, was examined to elucidate the role of lipid peroxidation of membranes in radiation-induced damage to membrane-bound enzymes. The reconstituted system of non-irradiated enzymes with irradiated liposomes showed a low activity of hexobarbital hydroxylation, whereas irradiated enzymes combined with non-irradiated liposomes exhibited an activity equal to that of unirradiated controls. Irradiation of liposomes caused a decrease in cytochrome P-450 content by destruction of the haem of cytochrome P-450 and also inhibited the binding capacity of cytochrome P-450 for hexobarbital. The relationship between radiation-induced lipid peroxidation and membrane-bound enzymes is discussed.     

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).     

Isolation and catalytic activity of cytochrome P-450 from ventral prostate of control rats

5 alpha-Androstane-3 beta, 17 beta-diol hydroxylase (3 beta-diol hydroxylase), a form of cytochrome P-450, was purified from rat ventral prostate, and its regulation as a function of age and 5 alpha-dihydrotestosterone (DHT) treatment was examined. Cytochrome P-450 could be quantitated by its CO difference spectrum only after partial purification from the microsomal membrane, and this was achieved by chromatography on p-chloroamphetamine-coupled Sepharose. Further purification of prostate microsomal P-450 by anion exchange chromatography yielded a preparation with a P-450 content of 8-10 nmol/mg of protein, which upon sodium dodecyl sulfate electrophoresis showed, in the molecular weight region between 50,000 and 60,000 where P-450 is expected to migrate, a single protein band of Mr 54,000. This preparation upon reconstitution with cytochrome P-450 reductase and microsomal lipid catalyzed the formation of three triols, 5 alpha-androstane-3 beta, 7 beta, 17 beta-triol, 5 alpha-androstane-3 beta, 6 alpha, 17 beta-triol, and 5 alpha-androstane-3 beta, 7 alpha, 17 beta-triol from 3 beta-diol in the ratio 1:7:3. Both turnover number and the ratio of the three products in the reconstituted system were similar to that found in prostate microsomes. These data indicate that a single form of P-450 catalyzes the formation of all three triols and that 3 beta-diol hydroxylase is the major, if not the only, form of P-450 in the prostate microsomes of untreated rats. The yield of P-450 from prostate microsomes varied as a function of age from a high level of 0.05 nmol/mg of microsomal protein in 6-week-old rats to 0.002 nmol/mg of microsomal protein in rats 11 weeks or older. 3 beta-Diol hydroxylase activity followed a similar age-related pattern varying between 2,000 and 4,000 nmol of triols formed/g of tissue/h in 6-week-old rats to 100 nmol of triols formed/g of tissue/h in 11-week-old rats. Treatment of 6-week-old rats with DHT did not prevent the age-related decrease in 3 beta-diol hydroxylase activity. However, DHT does play a role in the regulation of this enzyme since castration resulted in a loss of catalytic activity from the prostate and treatment of castrated rats with DHT caused an induction of the enzyme.     

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.     

Membrane topology of microsomal cytochrome P-450: saturation transfer EPR and freeze-fracture electron microscopy studies

The rotation of cytochrome P-450 LM2 (CYPIIB4) incorporated into large microsomal-like lipid vesicles was investigated by saturation transfer EPR using 15N- and 2H-substituted spin labels. In combination with rotational diffusion, the distribution and size of protein particles in the bilayer were studied by freeze-fracture electron microscopy. The data from both methods suggest an oligomeric and membrane-spanning aggregate for the topology of microsomal cytochrome P-450.