AMENQP‘YR ENE @EMETHYLASE: KENETEC EVED‘zENCE FQ-R manna MKfiegfiMAL ENZYMEg Tit-«13 he Hm Dame «:5 M. 5. RECEEGAN STATE UNIVERSITY Thomas. C. Peder-sieve: I969 E ‘ummnnmmmmmmmmnnmmnnu1‘ LIBRARY 3 1293 01008 5995 Michigan State University {r’éNflJ 3;; 1001 ABSTRACT AMINOPYRINE DEMETHYLASE: KINETIC EVIDENCE FOR MULTIPLE MICROSOMAL ENZYMES BY Thomas C. Pederson The Lineweaver-Burk plots of rat liver microsomal aminOpyrine demethylase activity are non-linear. The curve is characteristic of a reaction catalyzed by two enzymes. Pretreating animals with phenobarbital stimu- lates the demethylase activity and produces a linear re- ciprocal plot with an apparent Km for aminopyrine of 7 x 10-4M. Pretreatment with 3-methylcholanthrene causes no stimulation but increases the apparent Km for amino— pyrine by an order of magnitude or more. 3—Methylcholan- threne in vitro has no effect on the aminopyrine demethy- lase activity and the changes in kinetic behavior follow- ing 3-methylcholanthrene treatment are prevented by ad- ministration of ethionine. The inhibitor. SKF-SZSA. at a concentration of 4 x 10-5M. differentiates between the demethylase activities present in the two types of induced Thomas C. Pederson animals. inhibiting the activity found in microsomes of phenobarbital induced rats but having little effect on the activity in microsomes from 3~methylcholanthrene treated rats. These results and the results of other investigators which suggest the existence of multiple drug metabolizing activities are discussed. AMINOPYRINE DEMETHYLASE: KINETIC EVIDENCE FOR MULTIPLE MICROSOMAL ENZYMES BY Thomas C. Pederson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1969 ACKNOWLEDGEMENTS The author wishes to express appreciation to Dr. Steven Aust under whose guidance this research was con— 1 ducted. The author also wishes to thank Dr. Ronert Cook. Dr. Clarence Suelter. Dr. Charles Sweeley. Dr. William Wells. Dr. John Wilson. and the members of Dr. Aust's laboratory for their suggestions and assistance. ii TABLE OF CONTENTS AcmOWLEDGEdeN T8 O O C O O I O O O C C 0 LIST OF FIGURES O C O O O O O O O O O 0 LIST OF ABBREVIATIONS . . . . . . . . . INTRODUCTION. . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . Chemicals. . . . . . . . . . . . . . Animals. . . . . . . . . . . . . . . Preparation of Microsomes. . . . . . Aminopyrine Demethylase Assay. . . . Beanyrene Hydroxylase Assay . . . . Difference Spectroscopy. . . . . . . Synthesis of Ethylisocyanide . . . . EXPERIMENTAL. . . . . . . . . . . . . . DISCUSSION. . . . . . . . . . . . . . . REFERENCES 0 O O O O O O O O O O O I O 0 iii Page ii iv vi 10 10 12 l3 l4 14 16 39 49 LIST OF FIGURES Figure Page 1. Lineweaver-Burk plot for the N-demethylation of aminopyrine by goat liver microsomes. . l8 2. Lineweaver-Burk plot for the N-demethylation of 4-monomethylaminoantipyrine by goat liver microsomes . . . . . . . . . . . . . 20 3. Lineweaver-Burk plots for the N—demethyla- tion of aminopyrine by male rat liver microsomes . . . . . . . . . . . . . . . . 23 4. CO difference spectra of dithionite reduced rat liver microsomes . . . . . . . . . . . 25 5. Ethylisocyanide difference spectra of dithionite reduced rat liver microsomes. . 28 6. Lineweaver—Burk plot for N—demethylation of aminOpyrine by rat liver microsomes demonstrating the effect of 3—MC in vivo and in vitro . . . . . . . . . . . . . . . 30 7. Thin layer chromatograms of microsome extracts viewed under U.V. light . . . . . 33 8. Lineweaver-Burk plots for the N-demethyla- tion of aminopyrine by rat liver micro- somes showing the effect of ethionine on the changes produced by treatment with 3—MC . . . . . . . . . . . . . . . . . . . 35 9. Inhibition of aminopyrine demethylation in rat liver microsomes by SKF-SZSA . . . . . 38 iv LIST OF FIGURES (cont.) Figure Page 10. Theoretical Lineweaver-Burk plot for aminopyrine demethylation catalyzed by two enzymes with Km values of 4 x 10‘4 andZXlO'ZM.............. 41 AP i.p. 3-MC NAOH NAD (H) OD PB SKF-SZSA LIST OF ABBREVIATIONS aminopyrine intraperitoneally 4-monomethylaminoantipyrine 3-methylcholanthrene nicotinamide adenine dinucleotide reduced nicotinamide adenine dinucleotide phosphate (reduced) optical density phenobarbital 2~diethyaminoethyl-2.2—diphenylvalerate vi INTRODUCTION The endoplasmic reticulum of liver contains an enzyme system or group of enzyme systems which. in the will metabolize a large number of drugs. steroids. and carcinogenic compounds.lu3 The presence of NADPH and O2 variety of transformations: aromatic and aliphatic hydroxylation. N- and O- dealkylation. deamination. sul- foxidation. and N-oxidation. can all be visualized as . . . 4 . variations of hydroxylation.3' The requirement for NADPH + + R-H + NADPH + H + 02 a R-OH + NADP + H20 and molecular oxygen suggests that this enzyme system may be classified as a mixed function oxidase by the termin- ology of Mason.5 The incorporation of atmospheric oxygen and not water oxygen has been demonstrated to occur in the conversion of acetanilide to p-hydroxy-acetanilide and of trimethylamine to trimethylamine oxide.6'7 The demonstration of atmospheric oxygen incorporation into most other substrates has not been possible because of the rapid rate at which the incorporated oxygen exchanges with water. The identification of the oxygen activating com- ponent involved in this system began with the discovery of an unique CO binding hemOprotein in liver microsomes by Klingenberg8 and Garfinkel9 which was partially charac- terized by Omura and Sato.lo The cytochrome was called cyt. P-450. or P-450. because of its absorbance at 450 mu when reduced and saturated with CO. The role of this cytochrome in liver microsomal drug metabolism was demon- strated by COOper gt. 1.11 The microsomal flavo—protein NADPH—cyt C reductase has been implicated as part of the electron transport chain transferring electrons from NADPH to 13-450.12 A similar multienzyme oxidase system in the mitochondria of the adrenal cortex responsible for steroid hydroxylations has been separated into three components: a flavo-protein. a non-heme iron protein. and a P-450 complex.l3 Attempts to identify other components in— volved in hepatic drug metabolism have not been success— ful. Most attempts at dissociating the microsomal compon- ents destroy all activity and P—450 is converted to an in~ active form called P-420.10 A method which partially sola ubilizes the drug metabolizing enzymes. involving the use of salts and detergents in the presence of protecting agents. has been develOped by Lu and Coonl4 and modified 15 by Rikaus and VanDyke. In addition to the broad substrate specificity. another important property of the liver drug metabolizing system is its inducibility. The enhancement of liver microsomal drug metabolizing activity after treatment of the animals with polycyclic hydrocarbons such as benzpyrene and 3-methylcholanthrene was first described in 1954 by Brown. Miller and Miller.16 Subsequent investigation in- dicated that the activation was the result of increased . . . 12.17 . syntheSis of drug metaboliZing enzymes. In 1959 it was reported that phenobarbital and other drugs acted as . . . . . 18.19 .. inducers of drug metabolizing actiVity. Since then. over two-hundred drugs. insecticides. carcinogens and other chemicals have been reported to stimulate the drug . . . . . . 2 _metabolizing actiVity in liver microsomes. One of the more unusual properties of the micro- somal drug metabolizing system is its ability to carry out transformations of an extremely wide variety of struc- turally unrelated substrates which is difficult to under- stand in view of the common concept of substrate Specif— icity as found in other enzyme systems. The large number of compounds which are metabolized could mean at one extreme that there is a specific oxidase for each type of compound or at the other extreme. a singleoxidase of re~ markable nonspecificity. The first concept is difficult to accept for teleological reasons. The second concept. while supported by many general characteristics of the system. is becoming increasingly untenable. The suggestion that liver microsomes contain more than one enzyme system for the oxidation of drugs and other foreign compounds was first prOposed to explain the differential induction by phenobarbital and polycyclic hydrocarbons. Phenobarbital stimulates the metabolism of many compounds whereas. induction by 3-methylcholanthrene stimulates the metabolism of relatively few compounds.20 There are also marked species and sex differences in the metabolism of various drugs.2]'-23 Investigations of sub- strate competition by Rubin EE.§L-v24 have shown that for several compounds their Ki as an inhibitor was not the same as their Km for metabolism. and some substrates were unable to inhibit the metabolism of other compounds. The multiple enzyme hypothesis has been supported by spectral studies which suggest that liver microsomes contain more than one form of P-450. This was first pro— posed by Imai and Sato25 who found that two spectral species are detectable when the reduced cytochrome interacts with ethylisocyanide. It has also been found that the Spectral properties of 9-450 from 3-methylcholanthrene induced rats differs from that obtained with microsomes from non-induced rats. The absorption maximum of the CO difference Spectrum of reduced P-450 shifts from 450 ms to 446 mu and the ratio of the two absorption bands (455 and 430 mu) ofthe ethyli- socyanide difference spectrum shifts in favor of the absorp- . _ 26.27 . . tion at 455 mu. However. attributing these results to the existence of more than one P-4SO has been questioned by experiments which indicate that the various spectral forms . . 28 are interconvertible. Difference spectra characteristic of heme proteins are also produced by the addition of substrates of the drug metabolism system to the oxidized form of P-450. There are two types of substrate difference spectra: type one has a peak at 420-430 mm and a trough at about 390 mu. type two spectra have a trough at about 420 mu a 29030 . . and a peak at 385 mu. Most substrates examined. in- cluding aniline. phenobarbital and monomethylamino— antipyrine. have a type one difference spectra. Substrates which have a type two difference spectra include phenacetin. dihydrosafrole and aminopyrine. It is likely that these spectral changes occur as a result of the substrate bind— ing to a non-heme moiety of P—450. Similar spectra can be produced by adding organic solvents such alcohols hav- ing short carbon chains. AminOpyrine (4-dimethylamino-l. 5—dimethyl-2- phenyl-3—pyrazolone) is a drug which is frequently used as a substrate to measure N-demethylase activity in liver microsomes. It is an analgesic and antipyretic drug which at one time was used to treat the symptoms of a variety of diseases including rheumatic fever. but its occasional toxicity led to its gradual disuse.32 When the drug was administered to humans. almost all of it was altered in the body before excretion. the major metabolite being 4-aminoantipyrine (4—amino—l.5-dimethyl-2—phenyl-3- pyrazolone). The location of this transformation was first indicated by Brodie and Axelrod33 who reported that rabbit liver slices and homogenates convert aminopyrine to 4-aminoantipyrine. It was subsequently demonstrated 34 . . by LaDu 33 al.. that liver homOgenates of rabbit. rat. and guinea pig would dealkylate aminOpyrine. monomethyl— aminoantipyrine. and their ethyl and butyl analOgs to form 4-aminoantipyrine and the corre5ponding aldehyde. Both 02 and NADPH were required and the activity was located in the microsomal fraction. They also reported that the N-dealkylation activity could be inhibited by SKF-SZSA. The metabolism of aminOpyrine by liver micro- 12 somes was further characterized by Ernster and Orrenius who showed that equivalent amounts of NADPH. 0 and sub- 2. strate were used during the reaction and that the demeth- ylase activity was stimulated following induction of the metabolism of other drugs by phenobarbital. Studies by Gram. Wilson and Fouts35 have suggested that the removal of one methyl group to form monomethylaminoantipyrine occurs as a fast reaction followed by a slower reaction to form 4--aminoantipyrine. The role of P-450 in these reactions was confirmed by COOper__t__a_1_..ll who showed that the CO inhibition of demethylation could be reversed by monochromatic light at 450 mm. The aminopyrine de- methylase activity present in rat liver during different stages of growth has been studied by Soyka36 who found that newborn rats had very little activity. however,during 'the first 30 days after birth. the activity increased 3- fold. A Similar increase in aminopyrine demethylase ac— tivity occurred again in male rats at the age of puberty. This is apparently the result of induction by the sex hor- mones. testosterone and androsterone which are also . . .3 metabolized by the microsomal system.37 8 This thesis presents evidence suggesting that liver microsomes contain more than one oxidase system capable of demethylating aminOpyrine. Attempts to de- termine saturating concentrations of aminopyrine for de— methylation consistently showed that the activity con- tinued to increase as the substrate concentration became very high. Attempts to explain this non~enzymatically were without success. The subsequent kinetic analysis and study of the effects of induction and inhibition on aminOpyrine demethylase activity in rat liver microsomes support the thesis that liver microsomes contain multiple drug metabolizing systems with varying substrate Specif- icity. MATERIALS AND METHODS Chemicals Aminopyrine was purchased from K and K Labora- tories. Inc.. Plainview. N.Y. and either recrystallized or used as received since identical results were obtained. Phenobarbital was purchased from Merck and Co.. Inc.. Rahway. N.J. Benzpyrene was purchased from Aldrich Chem. Co.. Milwaukee. Wisc. SKF-SZSA was a gift of the Smith. Kline and French Laboratory. Philadelphia. P. 3-Methyl- cholanthreme. D. L-ethionine. D. L-isocitrate. NADP+. NADPH. NADH. and NADP—isocitrate dehydrogenase were all purchased from Sigma Chem. Co.. St. Louis. Mo. 4—Mono- methylaminoantipyrine was received as a gift from the Sterling WinthrOp Drug Co.. New York. N.Y. and purified by column—chromatography on silica gel~G. Carbon monoxide was obtained from the Matheson Co.. Inc.. Joliet. Illinois. Ethyl isocyanide was synthesized in our lab. 10 Animals Initial experiments were done with microsomes isolated from the livers of male goats because of their high P-450 content and N—demethylase activity. The major- ity of experiments have been done using male rats of the Holtzman strain weighing between 200 and 250 g. Animals induced with PB were given daily i.p. injections of 50 mg/kg in water for 5 days prior to sacrificing. Animals treated with 3~MC were given a single injection. i.p.. of 20 mg/kg in corn oil 24 hours prior to being sacri— ficed. Rats treated with ethionine were given injections i.p. of 500 mg/kg 60 and 30 minutes before injection of . . 2 inducer. according to the method of Alvares gt 1. 6 The ethionine was dissolved in water by raising the pH to about 10. which did cause pain when injected into the rats but did not appear to cause any lasting effects. Preparation of Microsomes The animals were exsanguished and the livers per- fused in situ by injection of 10 ml of cold 1.15% KCl into the portal vein within 1 or 2 cm of the liver. he liJer was then removed. blotted. weighed. and minced by chOpping 11 with a scissors. The minced tissue was homogenized in four volumes of 1.15%MKC1 containing 0.2% nicotinanide. added to inhibit NADP+-ase. with about 5 strokes in a Potter-Elvehjem homogenizer equipped with a teflon pestle. The homogenate was centrifuged at 15.000g for 23 minutes and the precipitate containing the nuclear and mitochon- drial fractions discarded. The microsomal fraction was isolated as a pellet by centrifuging the 15.0009 superna- tant at 105.000g for 90 minutes. The supernatant was discarded and the microsomes were resuspended in Tris- HCl buffer (0.05M; pH 7.5) containing 50% glycerol. In experiments in which the rats were not starved prior to being sacrificed. the microsomal pellet was carefully separated from the glyc0gen on the bottom of the tube by loosening the pellet in a small volume of buffer with a swirling action. The protein concentration of the resus— pended microsomes varied between 30 and 50 mg/ml. Pro- tein was assayed by the Lowry method.39 All Operations were performed at 0-5‘. The microsomes were either used immediately or stored at -15° under N2. These microsomes retained their full aminopyrine demethylase activity for several weeks providing they were kept anaerobic. l2 AminOpyrine Demethylase Assay The N-demethylase activity was assayed by measur- ing the rate at which formaldehyde was produced using the Nash method.40 In most experiments. to obtain valid ex- pressions for the rate of formaldehyde production. fixed point assays were made at two or three—minute intervals over a ten—minute period. One ml aliquots were removed from the incubation mixtures and diluted into 1 ml of 10% trichloroacetic acid. After allowing time for protein precipitation (about 5 minutes) two ml of Nash reagent (2M NH C H 0 ° 0.05 M CH 4 2 3 2, COOH: 0.02 M 2.4—pentanedione) 3 were added and the mixtures were heated at 50° for ten minutes. The assay mixtures were centrifuged at 1000g to remove precipitated protein and the 0.0. of the super- natant at 412 mu was determined using a Coleman Jr. Spec- trophotometer equipped with a flow cell. The extinction coefficient used was 7.08 OD ml"1 of assay uM_1 of HCOH. Reaction mixtures were incubated at 37° under air in a Dubnoff retabolic shaker. and unless otherwise stated contained microsomes (0.8 mg/ml). MgC12(7mM). NADPH (0.5mM). Tris HCl (0.05M: pH7.5). and the desired levels of substrates and inhibitors. 3—MC was added in 25 ul 13 of acetone to 5 ml incubation mixtures. In several ex- periments. the NADPH was provided by a generating system . . . . ' + containing: D.L-isoc1trate (2mm). NADP (0.1mM). and NADP—isocitrate dehydrOgenase (0.05 units/ml). Benzpyrene Hydroxvlase Assay The concentration of hydroxylated metabolites of ben20pyrene was determined using a method similar to that of Nebert and Gelbain.41 One ml aliquots were removed from the incubation mixtures and diluted into 1 m1 of cold acetone. The acetone sample was extracted vigor- ously with 3.25 ml of hexane. Two ml of the organic layer were removed and extracted with 3 ml of l N NaOH. The relative concentrations of the hydroxylated metabo- lites of benzpyrene in the aqueous layer was determined by measuring the fluorescence at 522 mu when excited at 396 mu. Problems were encountered in trying to obtain accurate reproducable fluoresence values from the aqueous layers. 14 Difference Spectroscopy The carbon monoxide and ethylisocyanide difference spectra of reduced P—450 were obtained with micrOSUmes re- suSpended at a concentration of 2 mg/ml in 1.0M phosphate buffer (pH 7.5) containing 50% glyceiol. The high ionic strength buffer and glycerol clarify the microsomal suspen- sion and prevent the conversion of P-450 to P—42010. Microsomes in both sample and reference cuvettes were re- duced by adding dithionate (~ < 1 mg). To obtain a CO difference spectra CO gas was bubbled into the sample cuvette until it was saturated. Lthylisocyanide differ— ence spectra were obtained by adding about as much as could be dissolved in the phOSphate glycerol buffer. The Spectra were recorded by a Coleman—Hitachi Model 124 SpectrOphotometer. Synthesis of Etnylisocyanide Ethylisocyanide was synthesized by the method of Jackson and McKusick.42 One mole of silver cyanide was added with stirring to one mole of ethyl icdide in a 3 liter. 3 necked. round bottom flask fitted with a re- fluxing condenser and sealed stirrer. The mixture was 15 heated on a steam bath and stirred for about 2 hours until a viscous. homOgeneous. brown liquid formed. The stirrer was raised above the liquid. the steam bath removed. 100 ml of water was added through the condenser. 2.75 moles of potassium cyanide in 85 ml of water was added and the mixture stirred for 10 minutes. The apparatus was re~ arranged for simple distillation. and the distillate was collected in a cooled receiver until the temperature of the solution in the distillation flash reached 115«120°. To the distillate 2.5g of NaCl was added. the aqueous layer removed and the crude product washed with two por~ tions of water. The product was dried overnight over an~ hydrous sodium sulfate and then purified by fractional distillation. The distillate coming over at 76—77° was collected and saved. Reported boiling point is 79'. The yield was 24g. 44% of theoretical (reported yield 47-55%). Caution! All Operations in which ethylisocyanide is heated should be done behind a shield to protect the Operator from the possibility of an explosion. Further~ more. the extremely vile odor and high volatility of ethylisocyanide require that all oLeraticns be performed in a hood. EXPERIMENTAL The N—demethylase activity of goat liver micro— somes at various concentrations of aminopyrine is shown in the Lineweaver~Burk plot in Figure l. The reciprocal plot deviates considerably from linearity at high sub~ strate concentrations and the activity is increased by the addition of NADH. The requirement for both NADPH and NADH for maximal activity has been reported by other investigators.1 No activity is observed with NADH alone. which indicates the effect of NADH is not due to trans~ hydrogenase activity. Since the demethylation of amino— pyrine involves the removal of two methyl groups. it was conceivable that the non—linear reciprocal plot could be explained by the existence of monomethylaminoantipyrine as a dissociable intermediate with altered kinetic par— ameters. The Lineweaver—Burk plot of MAP demethylase ac- tivity in goat liver microsomes is shown in Figure 2. The non—linear reciprocal plot. similar to that obtained with AP. indicates that the non-Michaelis-Menten kinetics must be a property of the microsomal enzymes. 16 17 Figure l.--Lineweaver—Burk plot for the N~demethylation of aminopyrine by goat liver microsomes. Velocities are given as mu moles of for— maldehyde formed min‘l. mg“1 of microsomal protein. Substrate concentration is in moles liter‘l. The microsomal protein concentration in these assays was 0.72 mg/ml. 18 H ouomwm m _ 0000. 0000 ooom oooe ooom fl _ _ _ _ 1042 + 1&0421 O Ian—<2 _.O NO md -|> 19 Figure 2.--Lineweaver—Burk plot for the N—demethylation of 4~monomethyl—aminoantipyrine by goat liver microsomes. The microsomal protein concentra- tion in these assays were 0.75 mg/ml. 20 N ouomam m OOON 000. 000. 000 O _ _ _ _ _.O NO n6 -l> 21 AminOpyrine demethylase activity in microsomes from control. PB or 3-MC treated rats. is shown in Figure 3. The curve obtained with microsomes from PB treated animals is linear and corresponds to an apparent Km of 7 x 10‘4M. in agreement with the Km of 8 x 10*4M reported by Ernster and Orrenius.12 The portion of the curve for control rat microsomes obtained at low sub— strate concentrations of AP yields a similar apparent Km. the apparent Km for aminopyrine obtained with micro- somes from 3-MC treated animals is at least an order of magnitude greater. Although AP demethylase activity is not stimulated by 3—MC treatment. benzypyrene hydroxylase activity in these microsomes is stimulated about 5—fold. The effect of 3—MC on benzypyrene hydroxylation has pre- . 43 Viously been reported by Alvares 35 al. The C0 difierence spectra of the reduced cyto- chrome P-450 present in the microsomes of the untreated. PB and 3-MC treated rats are shown in Figure 4. As was .f' 2 , . . originally reported by Alvares 2; al.. 0 there is an in- crease in the amount of P-450 present in microsomes from both PB and 3—MC treated rats and. in addition. the ab- sorption maximum of the P-450 from the 3-MC treated ani- mals has shifted to about 448 mu. The ethylisocyanide 22 Figure 3.—-Lineweaver—Burk plots for the N-demethylation of aminOpyrine by male rat liver microsomes. Rats induced with 3—MC were given a single injection i.p. of 20 mg/kg in corn oil 24 hours before being sacrificed. and. rats ins duced with PB were given daily injections i.p. of 50 ng/kg in water 5 days prior to being sacrificed. Control rats were untreated. 4|- 0.70 0.60 0.50 0.40 0.30 0 Control 0 3-MC Induced I PB Induced 1...? l l l I l 0 400 800 I 200 I 600 2000 24 Figure 4.--C0 difference spectra of dithionite reduced rat liver microsomes. The microsomal protein concentration in each case is 2 mg ml‘l. The microsomal suspension was clarified by using 1.0M phosphate buffer (pH 7.5) contain- ing 50% glycerol. A ABSORBANCY 0. I CONTROL 450 mu 3-Mc PB l l l l l 380 4:0 440 470 500 WAVE LE NGTH (mu) Figure 4 26 difference spectra of reduced P—450 are shown in Figure 5. Sladek and Mannering27 reported that the ratio of the ab- sorption at 455 mu relative to 430 mu increased in the microsomes from 3-MC treated animals. The spectra pre— sented here have absorption maximum at 455 and 435 mu which is apparently caused by the glycerol phosphate buffer. These spectral results. however. are of debat- able value since. in addition to the dependence of the spectral properties on pH and ionic strength.44 we were unable to saturate the P—450 with ethylisocyanide. Since the increase in apparent Km following treat— ment with 3—MC could be the result of inhibition by traces of 3-MC or its metabolites remaining in the iso- lated microsomes. 3—MC was added to incubation mixtures containing microsomes from untreated animals. The results. shown in Figure 6. demonstrate that 3~MC at a concentra- ting of 5 x 10-5M. failed to inhibit aminOpyrine demethyl- ation. The same results were obtained when the microsomes were preincubated with the 3-MC for five minutes. If all the 3-MC injected into the animals were still present in the isolated microsomes. the concentration in such incuba— tion mixtures would be about 7 x lO-SM. 27 Figure 5.--Ethylisocyanide difference spectra of dithionite reduced rat liver microsomes. The protein con— centration in each case is 2 mg ml‘l. The micro- somal suspension was clarified by suspension in 1.0M phosphate buffer (pH 7.5) containing 50% glycerol. The concentration of ethylisocyanide. which is not very soluble in this buffer. is about 0.35 mM. m musmwm 0.2-m me :E :E :E :6 00m mmv 000 one _ .O >oz