Microsomes and Drug Oxidations: Proceedings of the Third International Symposium, Berlin, July 1976

ROSENTHAL

OTTO ROSENTHAL, THE MAN AND HIS WORK

Herbert Remmer,     Institute of Toxicology, University of Tübingen, Wilhelmstraße 56, D-7400 Tübingen, W. Germany

Lieber Herr Rosenthal: But now I should use the American manner of addressing you: Dear Otto, dear colleagues and friends, members of this Faculty of the Free University of Berlin: I am deeply moved by having the privilege to honour Professor Rosenthal!

A Berliner addresses you as a Berliner! You lived in this beloved, marvellous and great city for 35 years, the best years of your life, before you were banished and went over to the United States.

As a student of the Berlin University and a former member of this Faculty I address you, who was also a student of this University in the 20th and a research assistant and professor until you had to leave.

It speaks now a friend to his fatherly friend, a colleague to his admired colleague, who remembers so many hours talking with you about the new situation in our country and discussing the latest news about cytochrome P-450, when I visited Philadelphia, and we were sitting in your small and modest office room which no German professor would accept! You have never been pretentious. Your reserved but warm appearance and, sometimes, shy behaviour might be the reason why many scientists, even friends, overlooked your unique contributions in biochemistry, particularly, in the field which is the topic of this symposium.

Thus, a scientist, having worked about several aspects of cytochrome P-450, addresses a senior scientist whom he admires because of his imagination to use procedures which he transfered from Berlin to the Harris Department of Biochemistry, belonging to the Clinic of Surgery in Philadelphia.

I refer to the Warburg Apparatus and the reaction vessels which came from Berlin, reminding all the students and younger people here in the audience, who are probably not so familiar with these fundamental work of Otto Rosenthal and his associates, Ron Estabrook and David Cooper, that it was his idea to use the method he learnt here in Dahlem and in the Charité, the old University Clinic, today in the eastern part, but formerly in the center of our now divided city. With this procedure Otto Rosenthal has been able to obtain such great scientific achievements. Figure 1 presents the vessel in which microsomes from bovine adrenal cortex were incubated with NADPH and 17-hydroxyprogesterone. The 21-hydroxylation of this steroid could be stopped by adding CO. Irradiation with light released the inhibiting effect of CO. The so called chemical action spectrum delivered a maximal energy for reversing the diminished enzyme activity produced by CO at a wavelength of 450 nm. The idea of cytochrome P-450 was born (1).

Fig. 1 The arrangement for light irradiation employed for the determination of the light reversibility of CO–inhibition of cortexolone formation.

This ingenious concept, dear Otto, would have never been created if scientific work has not been originated in this city!

I am too young to tell from own experience something about the stimulating scientific life in Berlin during your early years. It is no exaggeration if I say that here in Berlin, 50 years ago, in the 20th, lived and worked world’s scientific elite! Let me quote Werner Heisenberg, one of the leading atomphysicists, Nobel-Price-Winner, who deceased just recently, several months ago, who wrote in his memoirs that he, as a young man of 26 years of age and as a Bavarian - my German friends know what a Berliner thinks about people from Bavaria -, was invited to give a seminar about his new atomic concept before the most distinguished society here in Berlin. He describes his feeling and says: The University of Berlin was at that time viewed as a most prominent strong-hold of physics in Germany and in the world. Here worked at the same time Max Planck, Albert Einstein, von Laue and Nernst. Here Planck discovered the quanten-theory, and Rubens confirmed it with measurements of thermic radiation, and here Einstein in the year 1916 formulated the concept of relativity and the theory of gravitation. (2)

But I should continue now and mention that just in the neighbourhood, here nearby in Dahlem, stand even today well preserved the buildings of the former Kaiser-Wilhelm-Gesellschaft. Most of them belong now to the Free University and some to the Max-Planck-Society. In one, Herr Rosenthal, you should remember, was located the Institute of Biology. The director was the famous geneticist Goldschmidt. In this building worked during the 20th an enthusiastic crew of young scientists, all becoming Nobel-Price-Winners in later days. I should remind you of such names, as Otto Warburg, Hans Krebs, Fritz Lippmann, Meyerhoff, Ochoa. Excuse me, if I miss one or the other name!

Also famous pharmacologists, as Nachmansohn and Blaschko, worked in this outstanding department for awhile.

In the neighbouring building beyond the road was situated the Department of Chemistry; even today it is used as Institute of Chemistry of the Free University. In the basement Otto Hahn possessed a very small laboratory where he performed his outstanding experiments proving the scission of uranium, which initiated a new era.

And, Herr Rosenthal, if you walked from the Department of Chemistry just over the street, you would arrive at a building, formerly the Department of Biochemistry of the Kaiser-Wilhelm-Gesellschaft, and now the Department of Pharmacology of the Free University. The Director in those days was the famous Neuberg. In this department, you remember, you tried your first steps on the road to science. Even before you ended your medical studies, you worked in this institute. Not satisfied, I suppose, with the informations the students received, and wanting to know more, you probably went every day from the Charité to Dahlem and worked as a student in the laboratory of Neuberg. I should mention your first paper which appeared in 1923 in the Biochemische Zeitschrift; Neuberg and Rosenthal are the authors (3). This road on which you started passed the Charité where you stayed for nearly ten years, and ended in Philadelphia at the Harris Foundation.

In one dark year the road you went was blocked. The flourishing scientific life in Dahlem was almost deadly hurted, and science in Germany, even after nearly 50 years, never recovered again to this old and highly successful standard. This embarrassing situation in 1933 was masterly described by Hans Selye in his book From Dream to Discovery (4). Let me quote him: The great Galileo was exiled from his home and had to wander from city to city, until, thrown into prison, he was asked to disclaim his discoveries as despicable errors. Descartes, after also being exiled, was forced to lead a hazardous existence as soldier, physician, philosopher, and physicist until he died in a foreign land. Vesalius had to live a vagabond’s life, being severely penalized for imaginary crimes and eventually dying of hunger. Copernicus did not dare publish his great discoveries, and it was only on the day of his death that he finally saw them in print. Kepler, though pensioned by his emperor, lived in misery, because he never received his pension. Let no one think that this kind of persecution could happen only in the dark Middle Ages; it can happen in a highly cultured, modern state right now. It happened a short time ago in Hitler’s Germany. The science of Albert Einstein, Otto Loewi, Otto Warburg, and many others among the nations’ and the world’s greatest minds was labeled Jewish and, hence, unacceptable. All these men had to flee their country in shame … Can this be ever forgotten?

Honouring you, Otto Rosenthal, we honour with you all the nameless scientists of Jewish origin who were exiled, and found, as you did, a new home and a place of peace for scientific work in the United States!

It is an old law in history that the ‘genius loci’, the spirit of a place and of an epoch, if it vanishes, can never be revived. Let us wish that all the young people here in this Free University, in the Klinikum, students, assistants and professors, which have the privilege to teach and to work as scientists in a university with an old tradition, be it here in Steglitz or nearby in Dahlem, in the old buildings of the Kaiser-Wilhelm-Gesellschaft, might sometimes breath and feel the air of this spirit, enabling them to experience the imagination and creativity of a generation who lived and worked here 50 years ago.

Celebrating Otto Rosenthal and his contribution, we all might feel some of the ‘genius loci’ of science which governed in the 20th here over Dahlem and in Berlin!

REFERENCES

1. Estabrook, R.W., Cooper, D.Y., Rosenthal, O. The light reversible carbon monoxide inhibition of the steroid C21–hydroxylase system of the adrenal cortex. Biochem. Z. 1963; 338:741.

2. Heisenberg, W. Der Teil und das Ganze. München: Piper Verlag, 1969; 90.

3. Neuberg, C., Rosenthal, O. Über die Cellase der Taka-Diastase. Biochem. Z. 1923; 143:399.

4. Selye, H. From Dream to Discovery. New York: McGraw–Hill, 1964; 122.

I

BIOCHEMISTRY

LIPID STRUCTURE OF LIVER MICROSOMAL MEMBRANES, LIMITATIONS OF A MODEL

A. Stier, W. Kühnle and R. Rösen,     Max Planck Institute for Biophysical Chemistry, 34 Göttingen, Germany

SUMMARY

Rigid and fluid lipid areas are present in rabbit liver microsomes depending on temperature.Rigid lipid areas (boundary lipid) form haloes around the cytochrome P–450 system.The boundary lipid undergoes a transition from rigid to fluid state around 35°C.The results suggest that the lateral organization of lipids in microsomal membranes exhibits the dynamics of a multiphase system. The lipid phase diagram is modulated by conformation of cytochrome P–450 and of other integral membrane proteins and by many physiological and experimental factors perturbing lipid structure. But the lipid states may determine assemblage of the cytochrome P–450 system as well as conformation of its enzyme components and therefore biotransformation of drugs qualitatively as well as quantitatively. This may impose limitations for microsomes as an in vitro model.

RIGID AND FLUID AREAS IN LIVER MICROSOMES

Part of the lipids of microsomal membranes form lamellae existing in a state of high fluidity as shown previously by spinlabel experiments (Ref 1). An order parameter (S = 0.71 at 23°C) which is a measure for the angular deviation of the amplitude of anisotropic rotation of a spin-labeled fatty acid chain from the normal to the membrane (Ref 2) and therefore a measure of lipid fluidity can be evaluated (see Fig. 4). It lies in the lower range of the order parameters measured so far by this method in biological membranes. The fluidity of this part of the membrane allows for a high rate of lateral diffusion (several μm/sec) of a stearic acid within it.

Fig. 4 Temperature dependence of the order parameter S of ESR spectra of a fatty acid spinlabel (N-oxyl-4,4′-dimethyloxazolidine derivative of stearic acid, the doxyl group positioned on C-5, Syva Inc. Palo Alto) incorporated in rabbit liver microsomes, in a cytochrome P-450 preparation described in Fig. 3 and in resuspended lipid extracts of the two preparations recorded on a Varian E9 spectrometer. The small step seen in the curve of the cytochrome P-450 preparation around 36°C was consistently observed in 5 experiments using preparations from different animals. (from Ref 3)

Other lipids are segregated into areas which are not accessible for the fatty acid spin probe due to their low fluidity. Proton magnetic resonance spectra show (see Fig. 1), underlying the sharp resonances of the cholinomethyl, methylene and methyl groups of lipids in a highly fluid state, broad bands representing immobilized lipids that are nearly absent in microsomal lipid extracts, redispersed by ultrasonication, or are strongly reduced in microsomal preparations to which cholate has been added. The sharp band of the cholinomethyl group is remarkable, it is absent in microsomes (Ref 5). This indicates a quite different protein lipid interaction of these two membrane types: The lipid layer in liver microsomes may extend over large areas devoid of surface proteins, allowing rapid lateral phase separation processes, rapid lipid exchange with other cell membranes, and with regard to biotransformation of drugs unhindered substrate access to and product release from the membrane. The strongest immobilization is suffered by the methylene groups, an effect which can also be seen in carbon-13 natural abundance NMR spectra (Ref 19). The amount of immobilized lipid is distinctly higher in microsomes from untreated animals compared to phenobarbital pretreated ones, and much higher for both microsomal preparations at 40°C than at 20°C. It should be noted that the values in Table 1 may not be taken as absolute, there being unresolved doubts about interpretation of spectra from differently sized vesicles (Ref 7). The higher amount of lipid immobilization in microsomal membranes enriched in cytochrome P-450 might give a clue to the topography of the rigid lipid areas (see below).

TABLE 1

Proportion of Fluid Lipid Appearing in Proton Resonances of Rabbit Liver Microsomes¹)

¹)Areas below sharp resonances of H¹ NMR spectra taken in the presence of an external standard from microsomal suspensions and from lipid extracted from these suspensions and resuspended by ultrasonication in the same volume of buffer have been compared (from Ref 4).

²)determined by delayed Fourier transform technique (Ref 7).

Fig. 1 Proton NMR spectra of liver microsomes (15 mg/ ml protein) from phenobarbital stimulated rabbits, of cholate (final cone. 10−3 M) treated microsomes and of resuspended microsomal lipid extracts (0.6% W/W) in 0.01 M potassium phosphate D2O buffer pD 7.4 recorded by Fourier transform technique on a Bruker WH 270 spectrometer. Vertical scaling factors are different. Microsomes have been washed 3 times in D2O and once in the buffer used for final suspension. Lipid extracts (Ref 4) have been resuspended by low power ultrasonication at 40°C under N2 after exhaustive evaporation of solvents under high vacuum. (from Ref 3)

The sharp methylene and methyl resonances in proton NMR spectra increase with temperature in a nonlinear way as shown by the upbend curves in Fig. 2a in contrast to microsomal preparations to which cholate or hexobarbital had been added and also in contrast to the resuspended lipid extract of these microsomes (see Fig. 2b). This upbend shape of the curves which is less noticeable in microsomes from untreated animals might originate from a superposition of two different temperature gradients of fluidity belonging to two different lipid areas within the membrane, one being fairly fluid over the whole temperature range from 20-40°C, the other existing in a rigid crystalline state below about 35°C and undergoing a kind of lipid phase transition at that point. A similar temperature dependence of the signal height of bulk methylene resonances in the carbon-13 NMR spectra can be seen (Ref 19).

Fig. 2a Temperature dependence of signal intensities of sharp resonances of methylene (triangles) and methyl resonances (circles) of proton NMR spectra of liver microsomes (15 mg/ml protein) from phenobarbital stimulated rabbits, in presence (closed signs, dashed curves, right scale) and absence (open signs, continuous curves, left scale) of cholate (final cone. 10−3 M) as determined by application of delayed Fourier transform technique (Ref 7) in presence of an external standard. For other experimental conditions see Fig. 1. (from Ref 3)

Fig. 2b Temperature dependence of signal intensities of methylene resonances (triangles, right scale) and methyl resonances (discs, left scale) of proton NMR spectra of sonicated lipids (0.45% W/W) of rabbit liver microsomes (same preparation as in Fig. 2a).

BOUNDARY LIPID AND THE CYTOCHROME P-4 50 COMPLEX

Is the rigid lipid associated with the cytochrome P-450 system? Is its thermotropic polymorphism correlated to a conformational change of cytochrome P-450? A cytochrome P-450 preparation (Ref 8) in which cytochrome P-450 accounts for about 50% of proteins can occur in 2 states: A metastable state shows a NMR-spectrum composed of sharp and broad methylene and methyl resonance bands (similar to microsomes). It can be irreversibly converted to a state associated with strong immobilization of lipids. The conversion sets in abruptly when the temperature reaches 36°C (Fig. 3). The strong lipid immobilization can also be seen from the high order parameter of a stearic acid spinlabel incorporated into this preparation (see Fig. 4), it is much higher than in resuspended lipid extracts of the same preparation and compared to the corresponding preparations of microsomes. The temperature curve of the cytochrome P-450 preparation shows a distinct inflection at 36°C. These experiments do not simulate the original situation in microsomes but indicate different thermotropic polymorphic conformational states of cytochrome P-450 which are associated with different states of the lipid. Other examples of functions of endoplasmic reticulum membrane of liver depending critically on temperature between 30 – 40°C are aniline hydroxylation (Ref 10), UDP-glucuronyltransfer (Ref 11), CCl4 stimulated lipid peroxidation (Ref 12), NADPH dependent reduction of a stearic acid nitroxide (Ref 1), NADPH oxidation (Ref 13), formation of a stable nitroxide from 2-aminonaphthalene (Ref 14) and biliary excretion of lead (Ref 15).

Fig. 3 Temperature dependence of the methylene (triangles) and methyl resonances (circles) of Proton NMR spectra of a cytochrome P-450 enriched membrane fraction (8nM cytochrome P-450/mg protein) of liver microsomes from phenobarbital stimulated rabbits. The fraction was separated from bulk membrane constituents by column chromatography on a AH Sepharose 4B of cholate treated microsomes (Ref 8) and dialyzed against 0.01 M potassium phosphate D2O buffer pD 7.4. (from Ref 3)

From the P-NMR spectra we see a preference of cytochrome P-450 for lecithin and sphingomyelin over phosphatidylethanolamine (see Fig. 5). Perhaps it reflects also an asymmetry of its transversal distribution with a preference to face the luminal site in view of the known asymmetric distribution of lipids (Ref 9).

Fig. 5 Phosphorous NMR spectra of lipid extracts (Ref 4) of liver microsomes from phenobarbital stimulated rabbits (upper spectrum) and of a cytochrome P-450 enriched microsomal fraction prepared as described in Fig. 3 (lower spectrum) recorded on a Bruker WH 270 Fourier transform NMR spectrometer. Note the reduced proportion of phosphatidylethanolamine (PE) relative to sphingomyelin (SPM) and phosphatidylcholine (PC) appearing in the P-resonances of the lower spectrum. (from Ref 3)

PROSPECTS: MICROSOMAL MEMBRANES – A MULTIPHASE SYSTEM, LIMITATIONS OF A MODEL

In conclusion these results may be interpreted in the following way: The lateral organization of lipids in microsomal membranes exhibits the dynamics of a multiphase system. The lipid phase diagram is modulated by the conformation of cytochrome P-450 and of other integral membrane proteins and by many physiological and experimental factors perturbing lipid structure. The whole system is in metastable state around 37°C, to put it in another way, on a phase border in a phase diagram. But the lipid states may determine conformation and assemblage of the cytochrome P-450 system (as shown schematically in Fig. 6) and therefore influence biotransformation reactions quantitatively and qualitatively: cytochrome P-450 may act in the assembled complex as mixed function oxygenase, in the disassembled state as oxidase or peroxidase.

Fig. 6 Two interconvertible states of lateral organization of cytochrome P-450 (large light circles), of cytochrome P-450 reductase (large heavy circles) and of 2 types of membrane lipids (small open and closed circles) in a multiphase model of liver microsomes.

Moreover the lipid phase separation dynamics – due to the cooperativity of the supramolecular structure of lipid bilamellae (Ref 16) – may link the functions of the cytochrome P-450 system to other functions localized in areas of the endoplasmic reticulum membrane distant from the latter (e.g. biotransformation phase II reactions), an aspect pertinent to entanglement of cytochrome P-4 50 in the physiology as well as pathology of the liver (Ref 17 and 18).

This concept of lipid dynamics imposes limitations for microsomes as an in vitro model for biotransformation of drugs which have been surveyed more comprehensively (Ref 19). Loss of control of some ER functions in isolated microsomes may perturb lipid structure and therefore influence the biotransformation system, especially in the cases where these functions are lipid mediated linked to the functions of the cytochrome P-450 system. Some of the experimental conditions may shift the membrane to a stable but nontheless irrelevant state with regard to the in vivo situation. Thereby the results may become even more reproducible but erroneous as is often found in biological assays involving membranes if these are aged. The better model might be hepatocytes in which the nearly complete product pattern can be detected in situ by carbon-13 NMR spectroscopy as pilot studies using carbon-13 labeled fatty acids show.

REFERENCES

1. Stier, A., Sackmann, E. Spin label as enzyme substrates heterogeneous lipid distribution in liver microsomal membranes. Biochim. Biophys. Acta. 1973; 311:400–408.

2. Hubbel, W.L., McConnell, H.M. Molecular motion in spin-labeled phospholipids and membranes. J. Amer. Chem. Soc. 1971; 93:314–326.

3. A. Stier and R. Rösen, in preparation.

4. Bligh, E.G., Dyer, W.J. Rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 1957; 37:911–917.

5. Robinson, J.D., Birdsall, N.J.M., Lee, A.G., Metcalfe, J.C. ¹³C and ¹H nuclear magnetic resonance relaxation measurements of the lipids of sarcoplasmic reticulum membranes. Biochemistry. 1972; 11:2903–2909.

6. Kroon, P.A., Kainosho, M., Chan, S.I. Proton magnetic resonance studies of lipid bilayer membranes Experimental determination of inter- and intramolecular nuclear relaxation rates in sonicated phosphatidylcholine bilayer vesicles. Biochim. Biophys. Acta. 1976; 433:282–293.

7. Seiter, C.H.A., Feigenson, G.W., Chan, S.I., Hsu, M. Delayed Fourier transform proton magnetic resonance spectroscopy. J. Amer. Soc. 1972; 94:2535–2537.

8. Imai, Y., Sato, R. An affinity column method of partial purification of cytochrome P-450 from phenobarbital-induced rabbit liver microsomes. J. Biochem. 1974; 75:689–697.

9. Nilson, O., Dallner, G. Distribution of constitutive enzymes and phospholipids in microsomal membranes of rat liver. FEBS Lett. 1975; 58:190–193.

10. Schenkman, J.B. The effects of temperature and substrates on component reactions of hepatic microsomal mixed-function oxidase. Mol. Pharmacol. 1972; 8:178–188.

11. Eletr, S., Zakim, D., Vessey, D.A. A spin-label study of the role of phospholipids in the regulation of membrane-bound microsomal enzymes. J. Mol. Biol. 1973; 78:351–362.

12. T.F. Slater, personal communication.

13. J. Chayen, Histochemistry of drug metabolism systems, Fourth European workshop on drug metabolism, Mainz, 1974.

14. A. Stier and R. Clauss, in preparation.

15. Klaasen, C.D., Shoeman, D.W. Biliary excretion of lead in rats, rabbits, and dogs. Toxicol. appl. Pharmacol. 1974; 29:434–446.

16. Shimshick, E.J., McConnell, H.M. Lateral phase separation in phospholipid membranes. Biochemistry. 1973; 12:2351–2360.

17. Schmitt, F.O., Schneider, D.M., Crothers, D.M., eds. Functional linkage in biomolecular systems. Raven Press, New York, 1975.

18. A. Stier, Membrane fluidity, in T.F. Slater (ed.), Biochemical mechanisms of liver injury, Academic Press, London, in press.

19. Stier, A. Lipid structure and drug metabolizing enzymes. Biochem. Pharmacol. 1976; 25:109–113.

20. A. Stier and E. Sube, unpublished results.

INTERACTIONS BETWEEN CYTOCHROME P-450 AND NADPH-CYTOCHROME P-450 REDUCTASE IN THE MICROSOMAL MEMBRANE

Chung S. Yang,     Department of Biochemistry, New Jersey Medical School, CMDNJ, Newark, New Jersey 07103 U.S.A.

Publisher Summary

The endoplasmic reticulum of liver cells contains a monooxygenase system that catalyzes the biotransformation of steroids, fatty acids, drugs, carcinogens, and various other xenobiotics. In this enzyme system, the reducing equivalents from NADPH are transferred through NADPH-cytochrome P-450 reductase to cytochrome P-450, which in turn catalyzes the oxygenation of various substrates. Cytochrome P-450 can account for as much as fifteen percent of the microsomal protein, and the number of this hemoprotein can be 10–25 times greater than that of the reductase molecule in the membrane. The existence of a lipophilic mobile electron carrier between these two enzymes has never been demonstrated. It appears that one reductase molecule has to interact with a great number of cytochrome P-450 molecules for efficient catalysis. The NADPH-cytochrome P-450 reductase is an amphipathic protein with a molecular weight of about 80,000 Da, and cytochrome P-450 is an integral membrane protein with a molecular weight of about 50,000 Da. The chapter presents an experiment that studies the interactions of these two enzymes in the membrane and attempts to elucidate the nature of the organization of the microsomal monooxygenase enzymes.

The endoplasmic reticulum of liver cells contains a monooxygenase system which catalyzes the biotransformation of steroids, fatty acids, drugs, carcinogens, and various other xenobiotics (1-3). In this enzyme system, the reducing equivalents from NADPH are transferred through NADPH-cytochrome P-450 reductase to cytochrome P-450 which in turn catalyzes the oxygenation of various substrates. Cytochrome P-450 can account for as much as fifteen percent of the microsomal protein, and the number of this hemoprotein can be 10-25 times greater than that of the reductase molecule in the membrane (4). The existence of a lipophilic mobile electron carrier between these two enzymes has never been demonstrated. It appears that one reductase molecule has to interact with a great number of cytochrome P-450 molecules for efficient catalysis. The NADPH-cytochrome P-450 reductase is an amphipathic protein with a molecular weight of about 80,000 daltons and cytochrome P-450 is an integral membrane protein with a molecular weight around 50,000 daltons (5). The organization and mobility of these enzymes in the membrane are not very well understood (6-9). This paper reports the interactions of these two enzymes in the membrane and attempts to elucidate the nature of the organization of the microsomal monooxygenase enzymes.

Organizational Models of the Monooxygenase Enzymes

Two different concepts of organization of cytochrome P-450 and the reductase are illustrated by the rigid and nonrigid models in Fig. 1. With the rigid model, the reductase molecule is surrounded by many cytochrome P-450 molecules to form individual electron transfer complexes and translational diffusion of these proteins is not required for catalysis. With the nonrigid model, both the reductase and cytochrome P-450 are mainly surrounded by phospholipids; lateral mobility, at least short ranged diffusion, of these enzymes is required for their catalytic functions. The nonrigid model does not imply that the monooxygenase enzymes are randomly distributed in the membrane; rather certain domains of the membrane may have high local concentrations of these enzymes.

Fig. 1 .

Chemical Modification Studies

As a first test of these two models, the chemical modification approach of Franklin and Estabrook (6) was used. The study is based on the rationale that, for example, when 50% of the reductase is inactivated, only 50% of the cytochrome P-450 can be reduced enzymically according to the rigid model. Based on the nonrigid model, however, it can be predicted that more than 50% or almost all of the existing cytochrome P-450 should be reducible by NADPH, although it may proceed at a slow rate. Mersalyl (sodium 0-[(3-hydroxymercuri-2-methoxypropyl)carbamyl]phenoxyacetate) was used to inactivate the NADPH-dependent reductase activity and its effect on the enzymic reduction of cytochrome P-450 was measured. The results of this study (7) favor the nonrigid over the rigid model. For example, when approximately 75% of the reductase was inactivated, about 70% of the cytochrome P-450 could still be reduced in 5 min. A series of experiments also showed that when the reductase activity was inhibited to different extents (up to 85% inhibition) by different amounts of mersalyl, almost all the cytochrome P-450 molecules in the system could still be reduced by NADPH within a period of 20-25 min. These results suggest that cytochrome P-450 and the reductase do have translational mobility, although the rate and extent of the movement may not be as great as have been suggested for the cytochrome b5 reduction system (10-11).

Incorporation of Purified Cytochrome P-450 into the Microsomal Membrane

According to the nonrigid model, purified cytochrome P-450, when added to microsomes, should be able to incorporate into the membrane, to receive electrons from the reductase, and to catalyze monooxygenase reactions. This has been demonstrated experimentally (8). The addition of partially purified cytochrome P-448 (isolated from 3-methylcholanthrene-pretreated rats) to control microsomes greatly enhanced the microsomal benzo[a]pyrene hydroxylase activity and a maximum of 5-fold enhancement was observed (8). This is due to the binding or incorporation of the cytochrome P-450 into the membrane. As shown in Fig. 2, the enriched microsomes can be separated from the unbound cytochrome P-450 by gel filtration with a Sepharose 4B column. Similar results were also obtained with binding studies using cytochrome P-448. The relationship between the bound or incorporated cytochrome P-448 and the enhanced benzo[a]pyrene hydroxylase activity was also studied. It was observed that the enhanced hydroxylase activity was proportional to the amount of cytochrome P-448 incorporated and the cytochrome P-448 was about 2.5 times as active as the endogenous cytochrome P-450 (8). The results suggest that the added cytochrome P-448 can incorporate into the membrane and become a functional part of the monooxygenase system.

Fig. 2 Elution profiles of microsomes, cytochrome P-450, and enriched microsomes. Sonicated control microsomes were incubated with solubilized cytochrome P-450 (from phenobarbital-pretreated rats) at 37° for 30 min. The sample was applied to a Sepharose 4B column (1.5 × 40 cm) and eluted with Buffer A (0.1 M potassium buffer, pH 7.4, containing 5 mM MgCl2 and 0.1 mM EDTA). One ml fractions were collected. The traces are: —, 0.7 ml of microsomes (4.2 mg protein) incubated with 0.4 ml of cytochrome P-450 (55 nmol); —-, 0.7 ml of microsomes incubated with 0.4 ml of a buffer containing 0.05 M potassium phosphate, pH 7.4, 0.1 mM EDTA, 0.1 mM DTT and 20% glycerol, in which the cytochrome P-450 sample had been dialyzed; …., 0.2 ml of cytochrome P-450 incubated with 0.4 ml of Buffer A.

Previously, we have reported that upon binding to microsomes, the added cytochrome P-448 can be reduced by NADPH, but were unable to determine whether the exogenous cytochrome P-448 can obtain electrons directly from the reductase molecules or indirectly from the endogenous cytochrome P-450 molecules (8). To examine these possibilities, the effects of added cytochrome P-448 on linoleic acid hydroperoxide pretreated microsomes were studied (Table 1). The hydroperoxide treatment inactivated more than 95% of the cytochrome P-450 in control microsomes but retained about 70% of the reductase activity. As a consequence, less than 2% of the benzo[a]pyrene hydroxylase activity was retained. The microsomes, however, can regain hydroxylase activity after incubating with cytochrome P-448. Similar results were also observed with microsomes from phenobarbital or 3-methylcholanthrene pretreated animals. It is also noted that after incubation with cytochrome Microsomes, with quantities indicated, were preincubated with 3.6 nmol of cytochrome P-448 in 0.25 ml of Buffer A at 37° for 30 min. The benzo[a]-pyrene hydroxylase activity was assayed (8) at 37° with an incubation period of 5 min, and is expressed as nmol of product formed per min per unit of reductase activity. Control, PB, and MC microsomes were obtained from control, phenobarbital-pretreated, and 3-methylcholanthrene-pretreated rats, respectively. After treatment with linoleic acid hydroperoxide, they are referred to as Treated Microsomes.

TABLE 1

Effect of Cytochrome P-448 on Benzo[a]pyrene Hydroxylase Activities

P-448, the hydroperoxide treated control and PB microsomes had higher activities than the corresponding untreated ones. This is probably because that in the treated microsomes the incorporated cytochrome P-448 does not have to compete with the less active endogenous cytochrome P-450 for the reductase molecules. These results suggest that the bound cytochrome P-448 molecules have a direct access to the NADPH-cytochrome P-450 reductase molecule and a heme to heme electron transfer from the endogenous cytochrome P-450 to the added cytochrome P-448 is not required for the enhanced catalysis.

Cytochrome P-450 can also bind to microsomes and enhance oxidative demethylase activity (Fig. 3). Upon the addition of cytochrome P-450 to freshly thawed microsomes, no enhancement in demethylase activity was observed.

Fig. 3 Effects of solubilized cytochrome P-450 on microsomal benzphetamine demethylase activity. Microsomes were incubated with different amounts of cytochrome P-450 in 0.3 ml of Buffer A at 37° for 30 min; then more buffer and substrates were added for the assay of benzphetamine demethylase activity. The activity was assayed by the method of Thomas et al. () corresponding to 0.32, 0.32, or 1.26 mg of protein, respectively.

However, when the microsomes were converted into smaller vesicles by a brief sonication, significant enhancement in benzphetamine N-demethylase activity was seen. A more dramatic increase in the demethylase activity was demonstrated when the microsomes had been pretreated with linoleic acid hydroperoxide. Such treatment inactivated about 90% of the cytochrome P-450 molecules but retained most of the reductase activity.

The microsomal ethoxycoumarin dealkylase activity was also enhanced by the addition of partially purified cytochrome P-448. The effect of preincubation on the extent of enhancement is shown in Fig. 4. The enhancement is not instantaneous and the results suggest that a temperature-dependent incorporation of cytochrome P-448 into the microsomes is required. At 37°, it took 10 min to attain most of the enhancement and 30-40 min to reach a plateau. Such a preincubation requirement was also observed for the binding of cytochrome P-448 or P-450 to microsomes and for the enhancement of benzo[a]pyrene hydroxylase and benzphetamine demethylase activities.

Fig. 4 ) for the periods indicated. Buffer A (at 20°) and substrates were added, and the cuvette was equilibrated at 20° for 2-3 min. The reaction was initiated with 80 nmol of NADPH and the rate was measured at 20°. One (fluorescence) unit of dealkylase activity corresponds to the production of 16 pmol of 7-hydroxycoumarin per min.

All the results described so far are consistent with a nonrigid model based on structural considerations. The data, however, do not indicate whether translational diffusion is involved in the catalysis of the monooxygenase reactions. The rate and extent of the lateral movement of these enzymes in the membrane remain to be determined. If we assume that these enzymes have the diffusion rate of rhodopsin in outer rod segments (13), then a collision rate of 16 per msec is predicted if these two enzymes are separated by 100 A (14). Such a rate is too fast to be rate-limiting in the enzymic reduction of cytochrome P-450 (9) and in monooxygenase reactions.

Effect of Temperature on Monooxygenase Reactions

The effect of temperature on the rate of the ethyl morphine demethylase reaction of PB microsomes is shown in Arrhenius plots (Fig. 5). A linear plot was not observed. Rather, the data can best be fitted by two straight lines of different slopes. This was observed when either NADPH or NADP was used to initiate the reaction. Similar breaks were also observed with the NADPH-dependent demethylation of benzphetamine, aminopyrine, and p-nitroanisole. The results of these experiments are summarized in Table 2. These reactions have activation energies of 10-12 and 19-21 Kcal per mol at temperature ranges above and below the break temperature (at about 24°), respectively. A similar break was also observed in the ethylmorphine demethylation reaction catalyzed by control microsomes, except that the activation energy at temperatures below the break is 16-18 Kcal per mol. The results are consistent with the results of Duppel and Ullrich (15), obtained with the dealkylation of 7-ethoxycoumarin and p-nitroanisole; but differ from those of Schenkman (16) and Holtzman and Carr (17). The effect of temperature on the NADPH-dependent reduction of cytochrome c was also studied and a break in the Arrhenius plot was not observed (data not shown), consistent with previous results (16, 17). The results suggest that the NADPH-dependent reductase alone is not responsible for the break in the Arrhenius plots of monooxygenase reactions. The observed break is probably due to a membrane effect, since treating microsomes with 30% glycerol abolished the break of the Arrhenius plot (data not shown). The crystal-liquid crystal phase transition of phospholipid has been used to explain the discontinuity of the Arrhenius plots of the reactions catalyzed by many membrane enzymes (18-20). However, it is not known whether the present results can be attributed to the phase transition of membrane lipids. Thus, although the presently observed break in Arrhenius plots is possibly due to the interactions of cytochrome P-450 and the reductase in the membrane, alternative interpretations are equally possible. For example, different rate-limiting steps may be involved under different experimental conditions and the change of temperature may cause a shift in the rate-limiting steps of the mono-oxygenase reactions.

TABLE 2

Energy of Activation of Microsomal Demethylase Reactions

Fig. 5 Arrhenius plots of the ethylmorphine demethylase reaction. The reaction mixture contained 1.5 mg microsomal protein, a NADPH-generating system, and 0.65, 1.00, 2.00, or 5.00 mM of ethylmorphine. In set A, the reactions were initiated by NADPH. The Vmax values (in nmol HCHO/min/mg) were obtained from double-reciprocal plots. In set B, the assay mixture contained 2.5 mg of microsomal protein and the reaction was initiated by NADP.

With ethylmorphine as a substrate, Vmax values were used for the Arrhenius plot. With other substrates, the observed velocity at selected substrate concentrations, i.e., benzphetamine at 0.2 or 0.5 mM, aminopyrine at 1.0 or 2.0 mM, and p-nitroanisole at 0.5 or 1.0 mM, were used. Since the substrate concentration did not affect the activation energy of the reactions, results were summarized and expressed as mean ± standard deviation.

Requirements of a Nonrigid Model

Considering the approximate dimensions of cytochrome P-450, the reductase, and the microsomal membrane, it is difficult to envision how a reductase molecule can be rigidly associated and react efficiently with 20-25 cytochrome P-450 molecules. In a rigid model, are some of the cytochrome P-450 molecules located in the inner circle surrounding the reductase and others in the outer circle? In such an arrangement, where are the cytochrome b5-and cytochrome b5 reductase molecules which are known to interact with tne cytochrome P-450 system? Do the outer circle cytochrome P-450 molecules obtain electrons from the reductase through the inner circle cytochrome P-450 molecules during active catalysis? Since the reductase probably has only one site that can transfer electrons to cytochrome P-450, even the electron transfer from the reductase to all the inner circle cytochrome P-450 molecules would not be favorable if these molecules were rigidly associated in a molecular complex in which rotational mobility is hindered.

Although translational diffusion of monooxygenase enzymes in the membrane is a feature of the nonrigid model, it is not known whether such a process is rate-limiting in the enzymic reduction of cytochrome P-450. Even if the diffusion is rate-limiting for cytochrome P-450 reduction, the Arrhenius plot of the cytochrome P-450 reduction rate may or may not show a break above 4°, depending on the properties of the phospholipids surrounding the system. The properties of the lipids may be altered due to the binding to proteins and a mixture of different phospholipid molecules may not show a clear thermally-induced phase transition. This point has been clearly demonstrated in the NADH-dependent reduction of cytochrome c, a reaction in which translational diffusion of cytochrome b5 and cytochrome br reductase is involved (10, 11). In this case, a break in the Arrhenius plot of the reduction of cytochrome c cannot be observed with microsomes, but can be demonstrated when the two enzymes were incorporated into dimyristoyl lecithin liposomes (14). Thus, the lack of a break in the Arrhenius plot of the fast phase of the reduction of cytochrome P-450 in microsomes (9, 21), may not be in conflict with the nonrigid model. In addition, the interesting observation that 48% of the cytochrome P-450 could still be reduced in the fast phase when 95% of the NADPH-cytochrome P-450 reductase was removed from the microsomes by trypsin digestion (21), is consistent with the nonrigid model. According to the nonrigid model, it can be predicted that the addition of purified NADPH-cytochrome P-450 reductase to microsomes should enhance some monooxygenase activities. This has recently been demonstrated (22). Thus, the above results and discussions are in favor of a nonrigid organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the membrane. The nonrigid model also requires the translational diffusion of the monooxygenase enzymes in catalysis, a property which remains to be