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Monooxygenase activity of rat liver microsomes immobilized by entrapment in a crosslinked prepolymerized polyacrylamide hydrazide
204
Biochimica et Biophysica A eta, 798 (1984) 204-209 Elsevier
BBA 21683
MONOOXYGENASE ACTIVITY OF RAT LIVER MICROSOMES IMMOBILIZED BY ENTRAPMENT IN A CROSSLINKED PREPOLYMERIZED POLYACRYLAMIDE HYDRAZIDE A M I N A D A V Y A W E T Z a, A L B E R T S. PERRY ~, AM1HAY F R E E M A N b and E P H R A I M K A T C H A L S K I - K A T Z 1 R
b,*
Institute for Nature Conservation Research and h Center for Biotechnology, The George S. Wise Faculty of Life Sciences, TeI-Aoiv University, Tel- Aviv (Israel) (Received August 4th, 1983)
Key words: Monooxygenase," Microsome entrapment; Polyacrylamide hydrazide; (Rat liver)
Rat liver mierosomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37"C of I/max --2.3 nmol p-nitrophenoi/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The K m value for the p-nitroanisole-immobilized microsomai cytochrome /-450 system (1.2.10 -s M) was rather close to that of microsomes in suspension (0.8.10- 5 M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25"C was considerably lower than that of the control (microsomes in suspension, 78 nmoi/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37°C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37°C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.
Introduction
Liver microsomal cytochrome P-450, the terminal oxidase of an NADPH-dependent electron transport pathway, is known to oxidize a variety of structurally unrelated compounds ranging from endogenous substrates such as steroids and fatty acids to exogenous compounds, including drugs, insecticides, hydrocarbons and chemical carcinogens [1-3]. The diversity in substrate specificity has been attributed to the presence of multiple forms of cytochrome P-450 in the endoplasmic * To whom correspondence should be addressed. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V,
reticulum of liver and other tissues [4,5]. The hepatic microsomal cytochrome P-450 system [4], consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and phosphatidylcholine, is rather unstable, and quite rapidly loses its catalytic activity in vitro. Immobilization of the system by covalent binding of its components to bromocyanogen-activated Sepharose has been found to lead to a considerable loss in catalytic activity [6]. We thus assumed that if covalent binding could be avoided and the whole intact hepatic microsomal cytochrome P-450 system could be entrapped in a suitable polymer network it might be possible to obtain microsomal
205 gels of high monooxygenase activity which do not lose enzyme activity rapidly. A new mild method for whole cell immobilization, based on gel entrapment of the cells in chemically crosslinked prepolymerized polyacrylamidehydrazide, was recently developed in our laboratories [7]. The procedure, which also allows enzyme entrapment yields gels of remarkably high biological activity. It permits crosslinking of the preformed linear acrylamide polymer chains under extremely mild conditions, yielding a polymer network in which the cells or enzymes are immobilized [7-9]. The newly developed immobilization technique readily enabled the entrapment of rat hepatic microsomes containing the cytochrome P-450 system. The enzymic activity of the immobilized microsomes thus obtained was investigated and compared to that of microsomes in suspension. Materials and Methods
Chemicals. NADP, NADPH, glucose 6-phosphate, glucose-6-phosphate dehydrogenase and cytochrome c were purchased from Sigma Chemical Co., St. Louis, MO. p-Nitroanisole was purchased from Aldrich Chemical Co., Milwaukee, WI. All other chemicals were of analytical grade. Gel components. Prepolymerized linear polyacrylamide, partially substituted with acylhydrazide groups (M r = 180000; 0.75 mmol acylhydrazide groups per g) was prepared essentially as previously described by Freeman and Aharonowitz [7]. Glyoxal hydrate (trimer, Art. No. 804192) was purchased from Merck-Schuchardt, Darmstadt, F.R.G. Preparation of microsomes. Male Wistar rats, about 1 month old (120 g), were killed after light anaesthesia with ether. The livers were excised, rinsed in 0.15 M KCI until free of blood, blotted on filter paper and cut into small slices. Using a Potter-Elvehjem homogenizer with a Teflon pestle, the slices were homogenized in 7 vol. of ice-cold 0.1 M potassium phosphate buffer, pH 7.4, containing EDTA and dithiothreitol at a concentration of 0.1 mM and glycerol (20%). The homogenate was centrifuged at 12000 × g for 30 min and the precipitate was discarded. The super-
natant fraction was centrifuged at 105 000 × g for 60 min in a Beckman Model L5-50 refrigerated ultracentrifuge. The microsomal pellet obtained was rinsed with buffer, resuspended in the same medium and recentrifuged at 105000 × g for 60 min. The washed microsomal pellet was stored at - 9 0 ° C until use. All operations were carried out at 0-4°C. p-Nitroanisole O-demethylase activity, pNitroanisole O-demethylase activity was assayed by measuring the formation of p-nitrophenol. The reaction mixture consisted of NADP (2 pmol), glucose 6-phosphate (20 pmol), p-nitroanisole (2 pmol), glucose-6-phosphate dehydrogenase (3 units) and immobilized microsomes (containing 1.5-2.7 nmol P-450) all in 2 ml of 0.1 M phosphate buffer, pH 7.4. Incubation was carried out aerobically in 5 ml flasks using a constant temperature shaker at 37°C. The 3 units of glucose-6phosphate dehydrogenase were added only after preincubation of the mixture for 5 min at 37°C. A control containing all the above ingredients, except for the NADPH-generating system, was run simultaneously with each experiment. After 10 min of incubation the reaction was terminated by filtering the reaction mixture through moist filter paper. The absorbance of the gel-free clear solution was recorded at 400 nm, using the control as a blank, p-Nitrophenol concentration was determined using an extinction coefficient of 14.5 mM -1. cm -1 [13]. Determination of the demethylase activity in microsomal suspensions was carried out essentially as described above, but using microsomal suspensions instead of immobilized microsomes. The reaction was terminated by the addition of 0.1 rnl of 20% trichloroacetic acid. p-Nitrophenol was extracted from the reaction mixture into 2 ml of chloroform/ether (5: 1). The layers were separated, and the organic phase was transferred to another test tube and shaken with 2 ml of 0.005 M Na2CO 3 in 25% ethanol. Absorbance of the basic ethanol layer was determined at 400 nm, and p-nitrophenoxide ion concentration was obtained assuming a value of 18.9 m M - l - c m -1 for the molar extinction coefficient of the aromatic anion [14]. Sustained demethylase activity in immobilized microsomes. The ability of the immobilized mi-
206 crosomes to O-demethylate p-nitroanisole for several hours was investigated by placing 5 ml of the immobilized microsomal gel containing 3.6 nmol cytochrome P-450 in a column (1.5 × 20 cm) equipped with a water jacket and flow adaptor. The flow adaptor was adjusted above the gel and the column was maintained at 37°C by passing water through the jacket. Phosphate buffer (0.1 M, pH 7.4) was pumped from an open reservoir through the gel and then back to the reservoir at a rate of 15 ml/h. The total volume of the fluid in the system was 15 ml. The temperature of the reservoir, like that of the column, was maintained at 37°C. To the reservoir were added 150 ~1 of a solution of 0.1 M pnitroanisole in acetonitrile and 0.375 ml of a NADPH-generating system containing NADP (14 /xmol), glucose-6-phosphate (140 ~mol), and 100 units of glucose-6-phosphate dehydrogenase. At zero time, after 0.5 h, 1 h and thereafter at 1 h intervals, 1 ml samples were withdrawn from the reservoir, their absorbance determined at 400 nm, and the samples then returned to the reservoir. In order to maintain a relatively high concentration of NADPH, 0.375 ml of fresh NADPH-generating system was added at 1.5 h intervals. The increase in fluid volume caused by the addition of the NADPH-generating system approximately compensated for the loss of fluid by evaporation. Entrapment of microsomes. The combined microsomal pellet obtained from the livers of six rats was resuspended in 8 ml of 0.1 M phosphate buffer, pH 7.4. Aliquots (0.1 ml) of the suspension were each diluted 20-fold in the same buffer and the microsomal suspensions thus obtained were used for determination of cytochrome P-450 and protein content. Protein determination was carried out according to the method of Bradford [10]. Water-soluble linear polyacrylamide was prepared according to the procedure described previously [7]. Microsomal suspension (8 ml) was added to 40 ml of this prepolymer aqueous solution (2%, w/v), and the mixture was homogenized by gentle magnetic stirring over ice for 1 min. Aqueous glyoxal solution (2.5 ml, 5% w / v , adjusted to pH 7.0) was then added with stirring. The gel thus obtained was allowed to harden overnight at 4°C. It was then pressed through a 20 ml syringe (outlet diameter 2 mm), after which it was ground in an
Omni mixer at 7000 rpm for 0.5 min in 20 ml of ice-cold 0.1 M phosphate buffer, pH 7.5. The gel was then placed in a 1.5 × 20 cm column and washed with several volumes of the same buffer until the eluted fluid had no absorbance at 430 nm. Cytochrome P-450 and protein content that were not entrapped in the gel were determined in the eluted solution. Determination of cytochrome P-450. The content of cytochrome P-450 in the various preparations used was derived from the carbon monoxide difference spectrum according to Omura and Sato [11], using an extinction coefficient of 91 mM - t . cm-1.
NADPH-dependent cytochrome c reductase activity. The NADPH-dependent cytochrome c reductase activity was derived from the increase in absorbance at 550 nm following the reduction of cytochrome c. The reaction mixture consisted of immobilized microsomes containing 0.315 mg of protein, oxidized cytochrome c as an electron acceptor ( 5 . 1 0 5 M), KCN (1-10 3 M), and N A D P H (1 - 10 -4 M) as an electron donor in 0.1 M phosphate buffer, pH 7.4 (total volume 2 ml). Controls containing the above ingredients, except for N A D P H , were run simultaneously. Incubation was carried out aerobically with shaking in 5 ml flasks using a constant temperature shaker at 25°C. After 5 min the reaction was terminated by filtering the reaction mixture through moist Whatman No. 1 filter paper. The absorbance of the gel-free clear solution was recorded at 550 nm in a double-beam spectrophotometer, using the control as a blank. The amount of reduced cytochrome c formed was determined by assuming an extinction coefficient of 19.6mM 1.cm 1112]. Determination of the reductase activity of the microsomal suspensions was carried out by a similar procedure using approximately the same amount of microsomal protein as that in the reaction mixture. The increase in absorbance at 550 nm after 5 min of incubation was recorded directly by placing the reaction mixture in the cuvettes. A microsomal suspension was run simultaneously and used as control. It contained approximately the same amount of cytochrome P-450 and all the ingredients mentioned above, in a total volume of 15 ml. The amounts of p-nitrophenol
207
formed under conditions similar to those specified for the system using immobilized microsomes were determined according to the procedure described ahove. No degradation of p-nitroanisole in 0.1 M phosphate buffer, pH 7.4, was detected after its incubation at 37°C for 10 h.
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Results and Discussion
The cytochrome P-450 monooxygenase system is rather unstable in vitro [11]. An attempt was therefore made in the present study to stabilize the system by immobilizing it in a crosslinked gel. Rat hepatic microsomes were chosen to represent the monooxygenase system, and entrapment was effected in prepolymerized polyacrylamide-hydrazide crosslinked with glyoxal. This immobilization technique was chosen since it has been shown that it does not affect the biological activity of various microbiological and plant cells [7-9]. Under the experimental conditions employed we succeeded in immobilizing most of the cytochrome P-450 content of the microsmal suspensions used. The cytochrome P-450 which was not entrapped within the gel showed the characteristic CO-difference spectrum, thus making it possible to estimate its proportion (approx. 12% of the total) within the immobilization reaction mixture. The immobilization of approx. 88% of microsomal cytochrome P-450 in the three-dimensional gel could thus be predicted. All of the immobilized cytochrome P-450 showed full demethylase activity in the presence of a large excess of p-nitroanisole. The entrapped microsomal cytochrome P-450 preparation used in the present study contained 3.6 nmol of cytochrome P-450 and 6.3 mg protein per ml of packed gel. A constant rate of demethylation of p-nitroanisole occurred for at least 1 h in the presence of immobilized microsomal cytochrome P-450 under the conditions specified in Materials and Methods. The Irmax value for the O-demethylation of pnitroanisole was essentially the same for the immobilized and for the freely suspended microsomal cytochrome P-450 systems (2.3 nmol pnitrophenol formed/min per nmol cytochrome P450). The Michaelis constant for the immobilized system ( K m = 1.2-10 -5 M), however, was some-
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what higher than that recorded for the freely suspended microsomal system (Km = 0.8.10 -5 M). The K m values recorded for both systems seem to suggest that because of the hydrophilic character of the three-dimensional polymer network in which the microsomal system is entrapped, and the hydrophobic nature of the substrate p-nitroanisole, the concentration of substrate within the gel network at equilibrium is somewhat lower than in the surrounding medium. The pH activity curves for a microsomal cytochrome P-450 system suspended in 0.1 M phosphate buffers of different pH values, and for an immobilized microsomal system maintained under similar conditions, are given in Fig. 1. The data presented show that the pH activity curve of the immobilized system is shifted by approximately half of a pH unit towards more alkaline values. This is most probably due to the presence of some negatively charged groups on the crosslinked polyacrylamide network. Rat hepatic microsomes are known to contain cytochrome P-450 reductase in addition to cyto-
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Fig. 2. Effect of temperature on the initial rate of p-nitroanisole demethylation. Demethylation by a microsomal suspension (O). Demethylation by immobilized microsomes (O). v, rate of p-nitrophenol formed in n m o l / m i n per nmol cytochrome P-450. T, absolute temperature. A. Arrhenius plot of o vs. 1/T in the temperature range of 10°C to 37°C. B. Original data from which the Arrhenius plot was derived.
chrome P-450. The activity of the reductase was assayed by using oxidixed cytochrome c as an electron acceptor. The immobilized microsomal system reduced oxidized cytochrome c at a rate of 11.7 nmol cytochrome c per min per mg microsomal protein. Freely suspended microsomes reduced the oxidized cytochrome c at a rate of 78.0 nmol cytochrome c per min per mg microsomal protein. The reduced rate of electron transfer to cytochrome c recorded for the immobilized system undoubtedly results from the appearance of a relatively high permeability barrier to the high molecular weight cytochrome c (12 000). In this connection it is worth mentioning that the temperature variation of the demethylation of p-nitroanisole by the immobilized microsomal system was similar to that of microsomes in suspension (see Fig. 2B). Linear, almost parallel plots were obtained within the temperature range recorded in Fig. 2A, yielding apparent activation energies for p-nitroanisole O-demethylation of 5.7
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J Incubation time (hrs) Fig. 3. O-Demethylation of p-nitroanisole by immobilized microsomes (©), and by freely suspended microsomes (e). A continuous flow system (shown in the insert) was used to assay the activity of the immobilized microsomes, a, water jacket maintained at 37°C; b, immobilized microsomal gel; c, 15 ml of 0.1 M K2HPO 4 buffer, pH 7.4, containing substrate and NADPH-generating system, maintained at 37°C; d, peristaltic pump. The NADPH-generating system was added at 1.5 h intervals, as indicated by the arrows, both to the immobilized microsomal system and to the microsomes in suspension.
kcal/mol and 6.0 kcal/mol for the entrapped and freely suspended systems, respectively. The demethylase activity of immobilized microsomal cytochrome P-450 was retained considerably longer than that of microsomes in suspension. It is well known that cytochrome P-450 is rather unstable in vitro and rapidly loses its activity upon standing due to its transformation to the inactive form cytochrome P-420. The polyacrylamide gel apparently protects cytochrome P-450 from inactivation but the mechanism of this protection is not known. The addition of adequate amounts of the NADPH-generating system to the immobilized microsomes, as described in Materials and Methods, resulted in continuous p-nitroanisole demethylase
209
activity for up to 5 h, yielding 5.6% conversion of the substrate into the correspondingp-nitrophenol. This value is 3.5-fold higher than that recorded for the freely suspended microsomal cytochrome P-450 containing approx, the same amount of protein (Fig. 3). Addition of the NADPH-generating system to the microsomes in suspension at 1.5 h intervals did not increase p-nitrophenol formation beyond the initial 1 h interval (Fig. 3). This suggests that the sustained demethylase activity of the immobilized microsomes might be due to the protection afforded by the polyacrylamide gel in keeping the integrity of the microsomal structure. Orienting tests not shown in the text have shown that the immobilized microsomes in 15 ml suspension exhibited a somewhat higher activity than microsomes in suspension. However, as shown in Fig. 3, immobilized microsomes in the packed column and the circulating nature of the substrate proved to have a much higher enzyme activity. In conclusion, our results demonstrate that the immobilization of a highly active rat hepatic microsomal cytochrome P-450 system under the experimental conditions employed prevented rapid loss in activity. This immobilization technique thus enables continuous and repeated operation within a time limit of several hours. Stable immobilized microsomes might in due course be expected to serve as catalyst derivatives of choice for the oxidation of drugs, environmental pollutants and a variety of endogenous metabolites.
Acknowledgments
We acknowledge with gratitude the technical help rendered by Mr. Jacob Mozel and Mrs. Tova Blank-Koblenc. References
1 Conney, A.H., Levin, W., Ikeda, M., Kuntzman, R., Cooper, D.Y. and Rosenthal, O. (1968) J. Biol. Chem. 243, 3912-3915 2 Conney, A.H. (1967) Pharmacol. Rev. 19, 317-366 3 Gillette, J.R., Davis, D.C. and Sasame, H.A. (1972) Annu. Rev. Pharmacol. 12, 57-84 4 Conney, A.H., Levin, W., Jacobson, M. and Kuntzman, R. (1969) in Microsomes and Drug Oxidation (Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., Fouts, J.R. and Mannering, F.J., eds.), pp. 279-295, Academic Press, New York 5 Lu, A.Y.H. and Levin, W. (1974) Biochim. Biophys. Acta 344, 205-240 6 Baess, D., Janig, G.R. and Ruckpaul, K. (1975) Acta Biol. Med. Ger. 34, 1745-1754 7 Freeman, A. and Aharonowitz, Y. (1981) Biotech. Bioeng. 23, 2747-2759 8 Pines, G. and Freeman, A. (1982) Eur. J. Appl. Microbiol. Biotechnol. 16, 75-80 9 Galun, E., Aviv, D., Dantes, A. and Freeman, A. (1983) Planta Medica, in press 10 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 11 Omura, T. and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378 12 Yonetani, T. (1965) J. Biol. Chem. 240, 4509-4514 13 Netter, K.J. and Seidel, G. (1964) J. Pharmacol. Exp. Therap. 146, 61-65 14 Shigematsu, H., Yamano, S. and Yoshimura, H. (1976) Arch. Biochem. Biophys. 173, 178-186
Biochimica et Biophysica A eta, 798 (1984) 204-209 Elsevier
BBA 21683
MONOOXYGENASE ACTIVITY OF RAT LIVER MICROSOMES IMMOBILIZED BY ENTRAPMENT IN A CROSSLINKED PREPOLYMERIZED POLYACRYLAMIDE HYDRAZIDE A M I N A D A V Y A W E T Z a, A L B E R T S. PERRY ~, AM1HAY F R E E M A N b and E P H R A I M K A T C H A L S K I - K A T Z 1 R
b,*
Institute for Nature Conservation Research and h Center for Biotechnology, The George S. Wise Faculty of Life Sciences, TeI-Aoiv University, Tel- Aviv (Israel) (Received August 4th, 1983)
Key words: Monooxygenase," Microsome entrapment; Polyacrylamide hydrazide; (Rat liver)
Rat liver mierosomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37"C of I/max --2.3 nmol p-nitrophenoi/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The K m value for the p-nitroanisole-immobilized microsomai cytochrome /-450 system (1.2.10 -s M) was rather close to that of microsomes in suspension (0.8.10- 5 M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25"C was considerably lower than that of the control (microsomes in suspension, 78 nmoi/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37°C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37°C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min.
Introduction
Liver microsomal cytochrome P-450, the terminal oxidase of an NADPH-dependent electron transport pathway, is known to oxidize a variety of structurally unrelated compounds ranging from endogenous substrates such as steroids and fatty acids to exogenous compounds, including drugs, insecticides, hydrocarbons and chemical carcinogens [1-3]. The diversity in substrate specificity has been attributed to the presence of multiple forms of cytochrome P-450 in the endoplasmic * To whom correspondence should be addressed. 0304-4165/84/$03.00 © 1984 Elsevier Science Publishers B.V,
reticulum of liver and other tissues [4,5]. The hepatic microsomal cytochrome P-450 system [4], consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and phosphatidylcholine, is rather unstable, and quite rapidly loses its catalytic activity in vitro. Immobilization of the system by covalent binding of its components to bromocyanogen-activated Sepharose has been found to lead to a considerable loss in catalytic activity [6]. We thus assumed that if covalent binding could be avoided and the whole intact hepatic microsomal cytochrome P-450 system could be entrapped in a suitable polymer network it might be possible to obtain microsomal
205 gels of high monooxygenase activity which do not lose enzyme activity rapidly. A new mild method for whole cell immobilization, based on gel entrapment of the cells in chemically crosslinked prepolymerized polyacrylamidehydrazide, was recently developed in our laboratories [7]. The procedure, which also allows enzyme entrapment yields gels of remarkably high biological activity. It permits crosslinking of the preformed linear acrylamide polymer chains under extremely mild conditions, yielding a polymer network in which the cells or enzymes are immobilized [7-9]. The newly developed immobilization technique readily enabled the entrapment of rat hepatic microsomes containing the cytochrome P-450 system. The enzymic activity of the immobilized microsomes thus obtained was investigated and compared to that of microsomes in suspension. Materials and Methods
Chemicals. NADP, NADPH, glucose 6-phosphate, glucose-6-phosphate dehydrogenase and cytochrome c were purchased from Sigma Chemical Co., St. Louis, MO. p-Nitroanisole was purchased from Aldrich Chemical Co., Milwaukee, WI. All other chemicals were of analytical grade. Gel components. Prepolymerized linear polyacrylamide, partially substituted with acylhydrazide groups (M r = 180000; 0.75 mmol acylhydrazide groups per g) was prepared essentially as previously described by Freeman and Aharonowitz [7]. Glyoxal hydrate (trimer, Art. No. 804192) was purchased from Merck-Schuchardt, Darmstadt, F.R.G. Preparation of microsomes. Male Wistar rats, about 1 month old (120 g), were killed after light anaesthesia with ether. The livers were excised, rinsed in 0.15 M KCI until free of blood, blotted on filter paper and cut into small slices. Using a Potter-Elvehjem homogenizer with a Teflon pestle, the slices were homogenized in 7 vol. of ice-cold 0.1 M potassium phosphate buffer, pH 7.4, containing EDTA and dithiothreitol at a concentration of 0.1 mM and glycerol (20%). The homogenate was centrifuged at 12000 × g for 30 min and the precipitate was discarded. The super-
natant fraction was centrifuged at 105 000 × g for 60 min in a Beckman Model L5-50 refrigerated ultracentrifuge. The microsomal pellet obtained was rinsed with buffer, resuspended in the same medium and recentrifuged at 105000 × g for 60 min. The washed microsomal pellet was stored at - 9 0 ° C until use. All operations were carried out at 0-4°C. p-Nitroanisole O-demethylase activity, pNitroanisole O-demethylase activity was assayed by measuring the formation of p-nitrophenol. The reaction mixture consisted of NADP (2 pmol), glucose 6-phosphate (20 pmol), p-nitroanisole (2 pmol), glucose-6-phosphate dehydrogenase (3 units) and immobilized microsomes (containing 1.5-2.7 nmol P-450) all in 2 ml of 0.1 M phosphate buffer, pH 7.4. Incubation was carried out aerobically in 5 ml flasks using a constant temperature shaker at 37°C. The 3 units of glucose-6phosphate dehydrogenase were added only after preincubation of the mixture for 5 min at 37°C. A control containing all the above ingredients, except for the NADPH-generating system, was run simultaneously with each experiment. After 10 min of incubation the reaction was terminated by filtering the reaction mixture through moist filter paper. The absorbance of the gel-free clear solution was recorded at 400 nm, using the control as a blank, p-Nitrophenol concentration was determined using an extinction coefficient of 14.5 mM -1. cm -1 [13]. Determination of the demethylase activity in microsomal suspensions was carried out essentially as described above, but using microsomal suspensions instead of immobilized microsomes. The reaction was terminated by the addition of 0.1 rnl of 20% trichloroacetic acid. p-Nitrophenol was extracted from the reaction mixture into 2 ml of chloroform/ether (5: 1). The layers were separated, and the organic phase was transferred to another test tube and shaken with 2 ml of 0.005 M Na2CO 3 in 25% ethanol. Absorbance of the basic ethanol layer was determined at 400 nm, and p-nitrophenoxide ion concentration was obtained assuming a value of 18.9 m M - l - c m -1 for the molar extinction coefficient of the aromatic anion [14]. Sustained demethylase activity in immobilized microsomes. The ability of the immobilized mi-
206 crosomes to O-demethylate p-nitroanisole for several hours was investigated by placing 5 ml of the immobilized microsomal gel containing 3.6 nmol cytochrome P-450 in a column (1.5 × 20 cm) equipped with a water jacket and flow adaptor. The flow adaptor was adjusted above the gel and the column was maintained at 37°C by passing water through the jacket. Phosphate buffer (0.1 M, pH 7.4) was pumped from an open reservoir through the gel and then back to the reservoir at a rate of 15 ml/h. The total volume of the fluid in the system was 15 ml. The temperature of the reservoir, like that of the column, was maintained at 37°C. To the reservoir were added 150 ~1 of a solution of 0.1 M pnitroanisole in acetonitrile and 0.375 ml of a NADPH-generating system containing NADP (14 /xmol), glucose-6-phosphate (140 ~mol), and 100 units of glucose-6-phosphate dehydrogenase. At zero time, after 0.5 h, 1 h and thereafter at 1 h intervals, 1 ml samples were withdrawn from the reservoir, their absorbance determined at 400 nm, and the samples then returned to the reservoir. In order to maintain a relatively high concentration of NADPH, 0.375 ml of fresh NADPH-generating system was added at 1.5 h intervals. The increase in fluid volume caused by the addition of the NADPH-generating system approximately compensated for the loss of fluid by evaporation. Entrapment of microsomes. The combined microsomal pellet obtained from the livers of six rats was resuspended in 8 ml of 0.1 M phosphate buffer, pH 7.4. Aliquots (0.1 ml) of the suspension were each diluted 20-fold in the same buffer and the microsomal suspensions thus obtained were used for determination of cytochrome P-450 and protein content. Protein determination was carried out according to the method of Bradford [10]. Water-soluble linear polyacrylamide was prepared according to the procedure described previously [7]. Microsomal suspension (8 ml) was added to 40 ml of this prepolymer aqueous solution (2%, w/v), and the mixture was homogenized by gentle magnetic stirring over ice for 1 min. Aqueous glyoxal solution (2.5 ml, 5% w / v , adjusted to pH 7.0) was then added with stirring. The gel thus obtained was allowed to harden overnight at 4°C. It was then pressed through a 20 ml syringe (outlet diameter 2 mm), after which it was ground in an
Omni mixer at 7000 rpm for 0.5 min in 20 ml of ice-cold 0.1 M phosphate buffer, pH 7.5. The gel was then placed in a 1.5 × 20 cm column and washed with several volumes of the same buffer until the eluted fluid had no absorbance at 430 nm. Cytochrome P-450 and protein content that were not entrapped in the gel were determined in the eluted solution. Determination of cytochrome P-450. The content of cytochrome P-450 in the various preparations used was derived from the carbon monoxide difference spectrum according to Omura and Sato [11], using an extinction coefficient of 91 mM - t . cm-1.
NADPH-dependent cytochrome c reductase activity. The NADPH-dependent cytochrome c reductase activity was derived from the increase in absorbance at 550 nm following the reduction of cytochrome c. The reaction mixture consisted of immobilized microsomes containing 0.315 mg of protein, oxidized cytochrome c as an electron acceptor ( 5 . 1 0 5 M), KCN (1-10 3 M), and N A D P H (1 - 10 -4 M) as an electron donor in 0.1 M phosphate buffer, pH 7.4 (total volume 2 ml). Controls containing the above ingredients, except for N A D P H , were run simultaneously. Incubation was carried out aerobically with shaking in 5 ml flasks using a constant temperature shaker at 25°C. After 5 min the reaction was terminated by filtering the reaction mixture through moist Whatman No. 1 filter paper. The absorbance of the gel-free clear solution was recorded at 550 nm in a double-beam spectrophotometer, using the control as a blank. The amount of reduced cytochrome c formed was determined by assuming an extinction coefficient of 19.6mM 1.cm 1112]. Determination of the reductase activity of the microsomal suspensions was carried out by a similar procedure using approximately the same amount of microsomal protein as that in the reaction mixture. The increase in absorbance at 550 nm after 5 min of incubation was recorded directly by placing the reaction mixture in the cuvettes. A microsomal suspension was run simultaneously and used as control. It contained approximately the same amount of cytochrome P-450 and all the ingredients mentioned above, in a total volume of 15 ml. The amounts of p-nitrophenol
207
formed under conditions similar to those specified for the system using immobilized microsomes were determined according to the procedure described ahove. No degradation of p-nitroanisole in 0.1 M phosphate buffer, pH 7.4, was detected after its incubation at 37°C for 10 h.
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Results and Discussion
The cytochrome P-450 monooxygenase system is rather unstable in vitro [11]. An attempt was therefore made in the present study to stabilize the system by immobilizing it in a crosslinked gel. Rat hepatic microsomes were chosen to represent the monooxygenase system, and entrapment was effected in prepolymerized polyacrylamide-hydrazide crosslinked with glyoxal. This immobilization technique was chosen since it has been shown that it does not affect the biological activity of various microbiological and plant cells [7-9]. Under the experimental conditions employed we succeeded in immobilizing most of the cytochrome P-450 content of the microsmal suspensions used. The cytochrome P-450 which was not entrapped within the gel showed the characteristic CO-difference spectrum, thus making it possible to estimate its proportion (approx. 12% of the total) within the immobilization reaction mixture. The immobilization of approx. 88% of microsomal cytochrome P-450 in the three-dimensional gel could thus be predicted. All of the immobilized cytochrome P-450 showed full demethylase activity in the presence of a large excess of p-nitroanisole. The entrapped microsomal cytochrome P-450 preparation used in the present study contained 3.6 nmol of cytochrome P-450 and 6.3 mg protein per ml of packed gel. A constant rate of demethylation of p-nitroanisole occurred for at least 1 h in the presence of immobilized microsomal cytochrome P-450 under the conditions specified in Materials and Methods. The Irmax value for the O-demethylation of pnitroanisole was essentially the same for the immobilized and for the freely suspended microsomal cytochrome P-450 systems (2.3 nmol pnitrophenol formed/min per nmol cytochrome P450). The Michaelis constant for the immobilized system ( K m = 1.2-10 -5 M), however, was some-
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what higher than that recorded for the freely suspended microsomal system (Km = 0.8.10 -5 M). The K m values recorded for both systems seem to suggest that because of the hydrophilic character of the three-dimensional polymer network in which the microsomal system is entrapped, and the hydrophobic nature of the substrate p-nitroanisole, the concentration of substrate within the gel network at equilibrium is somewhat lower than in the surrounding medium. The pH activity curves for a microsomal cytochrome P-450 system suspended in 0.1 M phosphate buffers of different pH values, and for an immobilized microsomal system maintained under similar conditions, are given in Fig. 1. The data presented show that the pH activity curve of the immobilized system is shifted by approximately half of a pH unit towards more alkaline values. This is most probably due to the presence of some negatively charged groups on the crosslinked polyacrylamide network. Rat hepatic microsomes are known to contain cytochrome P-450 reductase in addition to cyto-
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Fig. 2. Effect of temperature on the initial rate of p-nitroanisole demethylation. Demethylation by a microsomal suspension (O). Demethylation by immobilized microsomes (O). v, rate of p-nitrophenol formed in n m o l / m i n per nmol cytochrome P-450. T, absolute temperature. A. Arrhenius plot of o vs. 1/T in the temperature range of 10°C to 37°C. B. Original data from which the Arrhenius plot was derived.
chrome P-450. The activity of the reductase was assayed by using oxidixed cytochrome c as an electron acceptor. The immobilized microsomal system reduced oxidized cytochrome c at a rate of 11.7 nmol cytochrome c per min per mg microsomal protein. Freely suspended microsomes reduced the oxidized cytochrome c at a rate of 78.0 nmol cytochrome c per min per mg microsomal protein. The reduced rate of electron transfer to cytochrome c recorded for the immobilized system undoubtedly results from the appearance of a relatively high permeability barrier to the high molecular weight cytochrome c (12 000). In this connection it is worth mentioning that the temperature variation of the demethylation of p-nitroanisole by the immobilized microsomal system was similar to that of microsomes in suspension (see Fig. 2B). Linear, almost parallel plots were obtained within the temperature range recorded in Fig. 2A, yielding apparent activation energies for p-nitroanisole O-demethylation of 5.7
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J Incubation time (hrs) Fig. 3. O-Demethylation of p-nitroanisole by immobilized microsomes (©), and by freely suspended microsomes (e). A continuous flow system (shown in the insert) was used to assay the activity of the immobilized microsomes, a, water jacket maintained at 37°C; b, immobilized microsomal gel; c, 15 ml of 0.1 M K2HPO 4 buffer, pH 7.4, containing substrate and NADPH-generating system, maintained at 37°C; d, peristaltic pump. The NADPH-generating system was added at 1.5 h intervals, as indicated by the arrows, both to the immobilized microsomal system and to the microsomes in suspension.
kcal/mol and 6.0 kcal/mol for the entrapped and freely suspended systems, respectively. The demethylase activity of immobilized microsomal cytochrome P-450 was retained considerably longer than that of microsomes in suspension. It is well known that cytochrome P-450 is rather unstable in vitro and rapidly loses its activity upon standing due to its transformation to the inactive form cytochrome P-420. The polyacrylamide gel apparently protects cytochrome P-450 from inactivation but the mechanism of this protection is not known. The addition of adequate amounts of the NADPH-generating system to the immobilized microsomes, as described in Materials and Methods, resulted in continuous p-nitroanisole demethylase
209
activity for up to 5 h, yielding 5.6% conversion of the substrate into the correspondingp-nitrophenol. This value is 3.5-fold higher than that recorded for the freely suspended microsomal cytochrome P-450 containing approx, the same amount of protein (Fig. 3). Addition of the NADPH-generating system to the microsomes in suspension at 1.5 h intervals did not increase p-nitrophenol formation beyond the initial 1 h interval (Fig. 3). This suggests that the sustained demethylase activity of the immobilized microsomes might be due to the protection afforded by the polyacrylamide gel in keeping the integrity of the microsomal structure. Orienting tests not shown in the text have shown that the immobilized microsomes in 15 ml suspension exhibited a somewhat higher activity than microsomes in suspension. However, as shown in Fig. 3, immobilized microsomes in the packed column and the circulating nature of the substrate proved to have a much higher enzyme activity. In conclusion, our results demonstrate that the immobilization of a highly active rat hepatic microsomal cytochrome P-450 system under the experimental conditions employed prevented rapid loss in activity. This immobilization technique thus enables continuous and repeated operation within a time limit of several hours. Stable immobilized microsomes might in due course be expected to serve as catalyst derivatives of choice for the oxidation of drugs, environmental pollutants and a variety of endogenous metabolites.
Acknowledgments
We acknowledge with gratitude the technical help rendered by Mr. Jacob Mozel and Mrs. Tova Blank-Koblenc. References
1 Conney, A.H., Levin, W., Ikeda, M., Kuntzman, R., Cooper, D.Y. and Rosenthal, O. (1968) J. Biol. Chem. 243, 3912-3915 2 Conney, A.H. (1967) Pharmacol. Rev. 19, 317-366 3 Gillette, J.R., Davis, D.C. and Sasame, H.A. (1972) Annu. Rev. Pharmacol. 12, 57-84 4 Conney, A.H., Levin, W., Jacobson, M. and Kuntzman, R. (1969) in Microsomes and Drug Oxidation (Gillette, J.R., Conney, A.H., Cosmides, G.J., Estabrook, R.W., Fouts, J.R. and Mannering, F.J., eds.), pp. 279-295, Academic Press, New York 5 Lu, A.Y.H. and Levin, W. (1974) Biochim. Biophys. Acta 344, 205-240 6 Baess, D., Janig, G.R. and Ruckpaul, K. (1975) Acta Biol. Med. Ger. 34, 1745-1754 7 Freeman, A. and Aharonowitz, Y. (1981) Biotech. Bioeng. 23, 2747-2759 8 Pines, G. and Freeman, A. (1982) Eur. J. Appl. Microbiol. Biotechnol. 16, 75-80 9 Galun, E., Aviv, D., Dantes, A. and Freeman, A. (1983) Planta Medica, in press 10 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254 11 Omura, T. and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378 12 Yonetani, T. (1965) J. Biol. Chem. 240, 4509-4514 13 Netter, K.J. and Seidel, G. (1964) J. Pharmacol. Exp. Therap. 146, 61-65 14 Shigematsu, H., Yamano, S. and Yoshimura, H. (1976) Arch. Biochem. Biophys. 173, 178-186