STUDiES ON THE .MULTiPUC-ITY 0F - MiCROSGMAL MlXEDeFUNCTIO‘N OXIDASE Thesis for the Degree of M. S. MECHEGAN STATE UNIVERSITY . JEFFREY B. STEVENS 1970 ....... HILQaé “3». m- Hagar ,-~.¢ J-Jfi‘bi‘ ABSTRACT STUDIES ON THE MULTIPLICITY OF MICROSOMAL MIXED-FUNCTION OXIDASE By Jeffrey B. Stevens The inhibition of aminopyrine demethylase activity of rat liver microsomes by dieldrin, metopirone and DDT sug- gests the presence of three activities capable of demethyl- ating aminOpyrine. Two of these systems are sensitive to inhibition by DDT, however, not equally. Only one is sen- sitive to dieldrin and metopirone inhibition. One component is extremely sensitive to DDT inhibition and is induced by pretreatment of animals with phenobarbital. The activity . of the component which is insensitive to inhibition by ei- ther of these drugs is increased preferentially by 3-methyl- cholanthrene pretreatment. Further data suggested where in the electron transport system that these drugs were in- teracting in the mixed-function oxidase system. Dieldrin and metopirone inhibition of aminopyrine demethylase has been correlated to binding the heme of the cytochrome P-450 to prevent the hydroxylation reaction occurring. DDT was found to cause inhibition by two completely Jeffrey B. Stevens different mechanisms. One of the types of inhibition can be overcome by the addition of NADH to the reaction mix- ture. Both CO- and substrate-difference spectra were used to calculate an extinction coefficient for the metopirone- heme chromophore. An extinction coefficient of 146 cm'lmgfl was found. STUDIES ON THE MULTIPLICITY OF MICROSOMAL MIXED-FUNCTION OXIDASE BY {1" kw Jeffrey BrfiStevens A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1970 ACKNOWLEDGMENTS The author wishes to express appreciation to Dr. Steven Aust under whose guidance this research was con- ducted. The author also wishes to express appreciation to Dr. John Wilson and Dr. Hyram Kitchen for their suggestions and assistance. Thanks are also given to Emerson Potter for his contribution on the metabolism of ethylmorphine, along with the other members of Dr. Aust's laboratory. ii ACKNOWLEDGMENTS LIST OF FIGURES TABLE OF LIST OF ABBREVIATIONS . INTRODUCTION . MATERIALS AND METHODS . Chemicals . Animals . Preparation of Microsomes. Aminopyrine Demethylase Assay Cytochrome C Reductase Assay Nitroblue Tetrazolium (NBT) Reductase As CO-Difference Spectroscopy Cytochrome b5 Assay. Substrate Difference Spectroscopy . . CONTENTS 0 o o m o o o o o D! ‘< CO-Oz Binding Equilibrium Experiments for P4450 . Polyacrylamide Gel Electrophoresis . . . Partial Purification of P-420 from Microsomes Optimizing Conditions EXPERIMENTAL . DISCUSSION . TABLE . . . SCHEME . . . REFERENCES . for P-450 Reductase . iii Page ii iv vii 12 12 13 14 15 16 16 17 17 18 18 19 21 22 80 94 95 96 Figure 1. LIST OF FIGURES Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes From Control (Control), Phenobarbital (PB) and 3-Methylcholanthrene (3-MC) pre- treated Animals. . . . . . . . . . Lineweaver—Burk Plots of Ethylmorphine De- methylase Activity in Microsomes From Control (Control), Phenobarbital (PB) and 3-Methy1cholanthrene (3-MC) pre- treated Animals. . . . . . . . . . Lineweaver-Burk Plots of Ethylmorphine De- methylase Activity in Microsomes From Control and Phenobarbital Pretreated Animals With or Without 6Xl0'4 M Ami- nopyrine in the Incubation Mixtures. . . Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Male and Female Control Microsomes and Animals Pre- treated With Testosterone. . . . . . . Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes From Con- trol Animals with the Inhibitors Dieldrin (200 uM), Metopirone (10 uM), and Hexo- barbitol (1. 2mM) in the Incubation Mixture. . . . . . . . . . . . . Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes from Con- trol Animals with the Inhibitors SKF- 525-A (20 mM) and SKF-8742-A (50 mM) in the IncubatIon Mixture. . . . . . . . Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes from Control Animals with the Inhibitors Benzpyrene (100 uM), Lilly 18947 (100 uM), and Testos- terone (1.4 mM) in the Incubation Mixtures. iv Page 24 26 29 31 33 35 37 Figure Page 8. Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes from 3-Methy1cholanthrene (3-MC) Pretreated Animals with the Inhibitors SKF- -525-A (20 uM), DDT (200 uM), and Metopirone (10 uM) in the Incusation Mixtures. . . 39 9. Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes from 3-Methy1cholanthrene (3-MC) Pretreated Animals with the Inhibitors SKF- -8742-A (50 uM), Hexobarbitol (1.2 mM), and Benz- pyrene (100 uM) in the Incubation Mix- tures. . . . . . . . . . . . . 41 10. Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes from 3-Methy1cholanthrene (3-MC) Pretreated Animals with the Inhibitors Testosterone (1.4 mM), and Lilly 18947 (100 HM) in the Incubation Mixtures. . . . . . . 43 11. Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes from Phenobarbital (PB) Pretreated Animals with the Inhibitors Dieldrin (200 uM), SKF- -525-A (20 uM), SKF- 8742-A (50 uM), and Lilly 18947 (100 uM) in the Incu- bation Mixtures. . . . . . . . . 45 12. Inhibition of Microsomal Aminopyrine Deme- thylase Activity by DDT. . . . . . 48 13. The Inhibition of Aminopyrine Demethylase in Microsomes from PB Pretreated Ani- mals by DDT at Various Concentrations of Aminopyrine (AP). . . . . . . . 51 13a. Lineweaver-Burk Plot of Aminopyrine Deme- thylase Activity in Microsomes from Phenobarbital (PB) Pretreated Animals at 0.1 mM DDT. . . . . . . . . . 51 14. Inhibition of Microsomal Aminopyrine Deme- thylase Activity by DDT. . . . . . . 53 15. Inhibition of Aminopyrine Demethylase Ac- tivity in Microsomes from Control (Con- trol), 3-Methylcholanthrene (3-MC) and Phenobarbital (PB) Pretreated Animals by Dieldrin. . . . . . . . . . . 56 V Figure Page 16. The Inhibition of Aminopyrine Demethylase in Microsomes from Phenobarbital Pre- treated Animals by Dieldrin at Various Concentrations of Aminopyrine (AP). . . 58 16a. Lineweaver-Burk Plots of Aminopyrine Deme- thylase Activity in Microsomes from Con- trol (Control) and Phenobarbital (PB) Pretreated Animals at 0.12 mM_Dieldrin. . 58 17. Lineweaver-Burk Plot of Aminopyrine Deme- thylase Activity in Microsomes from Con- trol (Control) and 3-Methylcholanthrene (3-MC) Pretreated Animals in the Presence of 200 uM_DDT. . . . . . . . . . 61 18. Inhibition of Microsomal Aminopyrine Deme- thylase Activity in Microsomes from Ani- mals Pretreated with 3-Methy1cholanthrene in the Presence of 200 uM DDT by SKF-525- A (1.0 mM), DPEA (0.5 mg, and Lilly . 18947 (1.0 mM). . . . . . . . . . 63 19. Reduction of Cytochrome P-450 Measured at 450 nm by NADPH and NADH with and with- out DDT (500 mM) Present in the Solution . 66 20. Inhibition of Microsomal Aminopyrine Deme- thylase Activity by DDT in the Presence of Various Levels of NADH. . . . . . 69 21. Oxidized and Reduced Substrate Difference Spectra with PB Microsomes Using Meto- pirone and Dieldrin as Ligands. . . . 72 22. CO Binding Curves for Microsomes of Control Animals and Animals Pretreated with PB, and 3-MC with and without Metopirone Present in Solution. . . . . . . . 74 23a. Metopirone Difference Spectra Obtained in the Reduced State of Microsomes from Control Animals and Animals Pretreated with Phenobarbital and 3-Methylchol- anthrene. . . . . . . . . . . . 76 23b. CO Difference Spectra in the Presence of Metopirone Obtained in the Reduced State of Microsomes from Control Animals and Animals Pretreated with Phenobarbital and 3-Methylcholanthrene. . . . . . 76 V1 AP DDT DPEA i.p. Lilly 18947 3-MC PB SKF-525-A SKF-8742-A LIST OF ABBREVIATIONS Aminopyrine l,l,1-trichloro-2,2-tris(p-chlorophenyl)ethane 2,4-dichloro-6-phenylphenoxyethylamine intraperitoneally 2,4-dichloro-6-pheny1phenoxyethy1diethylamine 3-methylcholanthrene Phenobarbital 2-ethylaminoethyl-2,2-dipheny1valerate HCl 2-ethylaminodiethyl-2,2-dipheny1valerate HCl vii MOS‘ into the This Proc metaboli1 elOPed a of these T‘ most Xe enzYme reticu ration liver ShOwn enZYI and_ Sig] the thy: S: INTRODUCTION Most of the foreign compounds which find their way into the body undergo some type of biotransformation.1 This process in general has been termed xenobiotic (or drug) metabolism. Speculations as to why such a system had dev- eloped are based upon the detoxification and/or elimination of these foreign compounds. The primary organ responsible for the metabolism of most xenobiotics is the liver. Within the liver cell, the enzyme system or group of systems, exist in the endoplasmic reticulum.2'3'4 £2 ziggg preparations of the endoplasmic reticulum are obtained by differential centrifugation of a liver homogenate and are termed microsomes. Studies have shown that the quantitative levels of activities for these enzymes are greatly dependent of the species, sex, health, and other factors generally dependent on the overall phy— siological state of the animal. It has also been shown that, in general, these activities are distributed equally throughout the microsomal fraction.5 The most unusual characteristic of this system (or systems) is its ability to transform such an extremely wide variety of compounds. This apparent lack of specificity is contrary for enzym undergo a matic andl deaminati' However, iations c Anc this SYS ber of C liver nu ment of Was fir isterec Studie torslo due tc ZYmeS rePre is,.re this Cele t“31:: find 90: an. contrary to the classical principle of substrate specificity for enzymes. The large variety of substrates can also undergo a wide variety of chemical transformations. Aro- matic and aliphatic hydroxylation, N- and O—dealkylation, deamination, sulfoxidation, and N-oxidation are but a few. However, in general all of these can be thought of as var- iations of hydroxylations.6'7 Another physiological phenomenon characteristic of this system is the inducability of these enzymes by a num- ber of compounds.8 The phenomenon of the enhancement of liver microsomal drug metabolizing activities by pretreat- ment of animals 12.3ixg_with lipid-soluble foreign compounds was first described by Brown, Miller and Miller9 who admin- istered polycyclic hydrocarbons to rats and mice. These studies were developed further by Conney and collabora- 10' 11' 12 in showing the activation was most probably tors due to an increased synthesis of the drug metabolizing en- zymes. Therefore, this increase in activity appeared to represent an increased concentration of enzyme protein and is referred to as "enzyme induction." Pharmacologically this induction is very important since it leads to an ac- celerated biotransformation of drugs ig_zizg and then al- ters the duration and intensity of drug action in animals and in man. The enhancement of enzyme activity is also considered important because of its association with drug and pesticide synergism and tolerance. All of these hydroxylation reactions require a re- duced co-enzyme (usually NADPH) and molecular oxygen. The incorporation of molecular oxygen and not oxygen from water has been demonstrated to occur in the conversion of acetan- ilide to p-hydroxyacetanilide and of trimethylamine to tri- 13'14 Other substrates used to demon- methylamine oxide. strate this phenomenon have not yielded positive results due to the rapid rate at which the incorporated oxygen ex- changes with water. By the terminology of Mason, this system (s) of enzymes is classified as a mixed-function oxidase.15 These requirements have led to the belief that mi- crosomal hydroxylations occur by a coupled redox reaction in which an "activated oxygen complex" capable of oxidizing various substrates is formed as an intermediate. Both the discovery of a CO-binding pigment by Klingenberg16 and Garfinkell7 and the partial characterization by Omura and Sato18 showing it to be a cytochrome, greatly enhanced the belief that this theory is correct. The cytochrome was called cytochrome P-450 or simply P-450 because of its ab- sorption maximum at 450 nm when a reduced microsomal CO- difference spectrum was taken. The role of this unusual cytochrome was illustrated by Ryan and Engel19 by the observation that the C21-hydro- xylation of 17-hydroxyprogesterone was inhibited by CO and that the inhibition was reversed by light. Later, 20 showed the maximum reversal of the Estabrook , gig]: , CO inhibition was obtained when the sample was illuminated at 450 nm. Omura and Sat021'22 also demonstrated that cytochrome P-450, when treated with a detergent such as deoxycholate or incubated anaerobically with phospholipase, was trans- formed into a derivative pigment, P-420, which had a nor- mal hemOprotein spectrum, but was no longer enzymatically active. Further evidence for cytochrome P-450's role in drug 23 who metabolism was presented by Orrenius and Ernster associated an increase in liver microsomal cytochrome P-450 with an increase in hydroxylase activity. One problem receiving considerable attention in this system is multiplicity of enzymes. In order for this sys- tem to carry out its vast number of chemical reactions on its numerous substrates, the system either must be totally devoid of any specificity or have within itself some form of multiplicity. The first alternative is not feasible from a biological standpoint, therefore multiplicity must lie somewhere in this system. One major group of investigators believes that more than one P-450 exists. Their argument rests primarily on data obtained from induction studies. ,Inducers of this enzyme system can be classified into two major categories: (1) compounds such as phenobarbital which induce the metab- olism of a large number of drugs, and (2) compounds that exert considerable specificity as enzyme inducers and do not stimulate many of the reactions that are stimulated by phenobarbital. This second group of inducers included polycyclic hydrocarbons such as 3-methy1cholanthrene and 3,4-benzpyrene. Drugs and other xenobiotics which are substrates or inhibitors interact with cytochrome P-450, even in the ab- sence of NADPH to give two types of spectral change.24'25' 26'27 The "type I" spectral change is characterized by the appearance of a trough 420 nm and a peak at 385 nm in the difference spectrum when compounds such as hexobarbital or aminopyrine are added to the sample cuvette. The "type II" spectral change is produced by compounds such as aniline and pyridine and is characterized by the appearance of a peak at about 430 nm and a trough at 390 nm. The intensi- ties of this spectral change are related to the concentra- tion of the substrate used. Mannering, e_1_:__31_. ,28 have shown that pretreatment with phenobarbital (PB) increased the intensity of both "type I" and "type II" spectra, but 3-methy1cholanthrene (3-MC) pre- treatment caused an increase in "type II" spectra only. These results were related to the hydroxylating activities of aniline (type II) and hexobarbital (type I). 3-MC pre- treatment enhanced aniline hydroxylation activity only, while a decrease in activity for hexobarbital was observed. There exists also a significant amount of evidence for more than one cytochrome P-450 based solely on CO-difference spectra with the three types of microsomes. Induction studies have shown that the absorbance maximum for the CO-difference spectra can be shifted from 450 to 448 nm, depending upon the extent and type of inducer used.29 Levin and Kuntzman3ohave shown a biphasic decrease of radio-active hemoprotein in liver microsomal CO binding par- ticles isolated from animals given 3H-levulinic acid. They have shown that previous administration of various inducing drugs produce remarkably different amounts of heme proteins with either fast or slow turnover rates. For example, when pretreatment was done with PB, there was much more of the 'fast' turnover particles than in control, and when 3-MC was administered, the 'slower' turnover type was more prevalent. Induction studies have provided impressive evidence for the presence of some type of multiple enzyme system. Alvares, gE_§l.,3l showed a difference in Km's for the hy- droxylation of benzpyrene by microsomes obtained from ani- 32 showed mals induced with PB and 3-MC. Conney, eE_§M., that 3,4-benzpyrene pretreatment did not induce the metabo- lism of aminopyrine or hexobarbital, but did induce the metabolism of zoxazolamine. Some other selective inducers, such as DDT and chlor- dane, had different effects on the induction of different 33 activities. DDT acted as a more selective stimulator of hepatic drug metabolizing enzymes than both PB and 3-MC. Rubin, _e_t__al_. ,34 found that a number of compounds which inhibited N-demethylation of ethylmorphine by liver microsomes were themselves metabolized by this system. This inhibition is believed to be a result of the alterna- tive substrate hypothesis, which states that if two sub- strates are competing for a common site on an enzyme, they must be competitive inhibitors of each other. For competi- tive inhibition, the Ki value of a particular compound should relate to the Km for the metabolism of that compound. However, other evidence has shown that certain in- hibiting chlorinated hydrocarbons were not metabolized.35 One possible interpretation for this latter result is that the inhibition was not competitive. Other studies have shown slight disagreement for the correspondence of Ki's with respective Km's,34'36 leading to the belief that the alternative substrate hypothesis may not be completely applicable in this system. Inhibition of drug metabolism by steroids37 led to the belief that these steroids are in fact the natural substrates for these enzymes, and that in the presence of xenobiotics become alternative substrates for a common mixed-function oxidase system. All of the above evidence has pointed overwhelmingly to the possibility of multiple enzyme oxidation systems, however, no direct evidence has been submitted. The ultimate goal of this thesis would be to directly demon- strate multiplicity in some fraction of the microsomal mixed-function oxidases. Aminopyrine (4-dimethylamino-l,5-dimethy1-2-phenyl-3- pyrazolone) was the drug substrate of choice because of the ease of enzymatic analysis. It is an analgesic and an anti- pyretic which was used to treat symptoms of a variety of diseases including rheumatic fever, but its occasional toxicity led to its disuse. The major metabolite is 4-ami- nopyrine, (4-amino-l,5-dimethyl-2-phenyl-3-pyrazolone). Brodie and Axelrod38 first located the biotransformation in liver slices and homogenates. The ethyl and butyl analogs were also shown to be metabolized to 4-aminoanti- pyrine and the corresponding aldehydes39 by the following 9 o§ ‘Nx / CH2 02 > 0\ N\N/CH2 + H20 + 2 RCHO H NADPH I-_—. CH2 H2N CH2 R2- +H+ reaction: The metabolism of aminopyrine by liver microsomes 40 who was further characterized by Ernster and Orrenius, showed the equivalent amounts of NADPH, 02, and substrate were used during the course of the reaction, and that the demethylase activity was stimulated, along with the induction of the metabolism of other drugs, by PB. The role of P-450 in the reaction was confirmed by Cooper, EEJE£.,41WhO showed that CO inhibition was reversed by monochromatic light at 450 nm. Studies by Gram, Wilson, and Fouts42 have suggested that the removal of one of the methyl groups occurs at a fast rate followed by a slower reaction to form 4-aminoantipyrine. Initial work in this area from this laboratory in- volved determining an explanation for the biphasic Line- weaver-Burk plot obtained when assaying for aminopyrine N-demethylase activity. It had been proposed that the bi- phasic nature of the plot was the result of the dimethyl and monomethyl derivatives being metabolized at different rates. However, these ideas were refuted by showing the biphasic Lineweaver-Burk plot still occurred when only the 28 Since monomethylaminoantipyrine was used as a substrate. the plots were also characteristic of a reaction catalyzed by two enzymes, the next step was to determine the point of multiplicity. Induction by PB stimulated the demethylase activity and produced a linear Lineweaver-Burk plot with an apparent Km for aminopyrine at 7X10'4 M. Pretreatment with 3-MC caused little or no stimulation of activity, but it did increase the apparent Km for aminopyrine by more than an order of magnitude. It was also found that the inhibitor SKF-525-A at a concentration of 4><10'5 M differentiated between the 10 demethylase activities present in the two types of induced animals, inhibiting the activity found in microsomes of PB induced rats but having little effect on the activity in microsomes from 3-MC treated rats. The working hypothesis for this research rests on the belief that multiplicity must exist somewhere in this system. It will be the endeavor of this research to fur- ther establish the possibility of a multiple enzyme system. Three possibilities exist for explaining the type of non- specificity present in this enzyme system. As previous evidence has tried to show, there may be the possibility of multiple cytochrome P-450's each of which has an electron transport system or they may share a common electron-trans- port system. Secondly, there may exist multiple binding sites on a single P-450, or lastly, a combination of these possibilities may also occur. The first part of this thesis presents indirect evi- dence for multiple mixed-function oxidase activities for a single substrate. With aminopyrine as a model substrate for this system, an attempt was made to kinetically show multiple demethylase activities. Based on the idea of spe- cific induction and/or inhibition of these various enzymes, multiple aminopyrine demethylase activities were shown. It has been shown that the use of specific inducers and/or in- hibitors provide extremely valuable tools in further work with these oxidases. 11 In the second part, an attempt was made to produce direct evidence for this multiplicity. The solubilization and partial purification of the cytochrome P-420 provided some evidence concerning the question of multiple cyto- chrome P-450's. MATERIALS AND METHODS Chemicals Aminopyrine (4-dimethylamino-l,5-dimethyl-2-phenyl-3- pyrazolone) and hexobarbitol (Sodium 5-(l-cyclohexen-l-yl) -l,S-dimethylbarbiturate) were purchased from K and K Labor- atories, Inc., Plainview, N.Y. and used as received as sub- strates in the assays. The two inhibitors, DPEA (2,4-dichloro-6-phenylpheno- xyethylamine HBr) and Compound 18947 (2,4-dichloro-6-phenyl- phenoxyethyldiethylamine HBr) were received from the Eli Lilly and Co., Indianapolis, Indiana. Dieldrin (1,2,3,4, lO,lO-hexachloro-6,7-epoxy-l,4,4a,5,6,7,8,8a-octahydro-l, 4,5,8-dimethanonaphthalene) was obtained from the Shell Chemical Company, New York, N.Y. and was recrystallized to a purity of 99% before use. Metopirone (2-methyl-l,2-bis (3-pyridyl)-propanone) was received from the CIBA Pharma- ceutical Company, Summit, N.J. Compounds 525-A (2-ethyl- aminodiethyl-Z,Z-diphenylvalerate HCl) and 8742-A (2-ethyl- aminoethyl-Z,2-dipheny1valerate HCl) were received as gifts from the Smith, Kline, and French Laboratories, Philadelphia, Pennsylvania. Benzo-a-pyrene (3,4-Benzpyrene), 20-methyl- cholanthrene (3-methylcholanthrene), testosterone, andros- terone, B-estradiol, progesterone, and hydrocortisone as 12 13 well as cholestrol were all purchased from the Sigma Chemi- cal Company, St. Louis, Mo. D,L-isocitrate, NADPT NADPH, NADH, and NADPiisocitrate dehydrogenase were also purchased from the Sigma Chemical Company, along with Cytochrome C, steapsin, Bee Venom, and the following Snake Venoms: Naja Naja (Hooded Cobra) and Crotalus Atrox (Western Diamond- back Rattlesnake). DDT (1,1,l-trichloro-2,2-tris(p-chlorophenyl)ethane) was obtained from Dr. Bieber who had previously recrystal- lized it to purity. Phenobarbitol (Sodium S-ethyl-S-phe- nylbarbiturate) was purchased from Merck and Company, Inc., Rahway, N.J. Oxygen and Carbon Monoxide were obtained from the Matheson Company, Inc., Joliet, Illinois. KCN was purchased from Mallinckrodt Chemical WOrks, St. Louis, Mo. Nitroblue tetrazolium was purchased from Aldrich Chemical Company. Phospholipase A was received from C. F. Boehringer and Soehne GmbH Mannheim. Animals All experiments were done with microsomes isolated from the livers of male rats of the Holtzman Strain weigh— ing between 200 and 250 grams. Animals induced with pheno- barbital were given 0.1% phenobarbital in their drinking water at least one week prior to the isolation. Animals induced with 3-methylcholanthrene were given an i.p. in- jection, of 20 mg/kg in corn oil, 24 hrs. prior to isolation. 14 Preparation of Microsomes The animals were killed by decapitation with an ani- mal guillotine and the livers were immediately perfused iglgitg_with approximately 10 mls. of ice cold 1.15% KCl containing 0.2% nicotinamide by injection into the portal vein. The livers were then removed, weighed, and minced by chopping with a pair of scissors. The tissue was then homogenized in three to four volumes of 1.15% KCl contain- 43 added to inhibit any NADPiase that ing 0.2% nicotinamide, may be present, in a Potter-Elvehjem homogenizer equipped with a teflon pestle. The homogenate was then centrifuged at 10,OOOXg (8,500 rpm, SGA rotor) for twenty minutes in a Sorvall RC2-B automatic refrigerated centrifuge. The supernatant was decanted and saved, while the pellet con- taining the nuclear, mitochondrial, and other subcellular fractions was discarded. The microsomal fraction was then isolated by centrifuging the previous supernatant in a Spinco centrifuge at 105,000Xg (30,000 rpm, 30 rotor) for 90 minutes. The pellet was resuspended in Tris-HCl buffer (0,05 M, pH 7.5) containing 50% glycerol. A protein deter- mination was done on the resuspended microsomes by the Lowry Method44 and usually ranged from 30 to 60 mg/ml. All the above operations were done at O-SOC. The micro- somes were stored at -15°C under N2 until they were to be used. Providing they were kept anaerobic, full N-demethyl- ase activity was retained for a number of weeks. 15 Aminopyrine Demethylase Assay The N-demethylase activity was assayed by measuring spectrophotometrically the rate of formaldehyde production using the Nash method.45 All assays contained: 7mM MgClz, Tris-HCl (0.05M, pH 7.5), 0.5mM NADPH or a NADPH generating system made up of NADP+(0.lmM), D,L-isocitrate (2mM), and isocitrate dehydrogenase (0.05 units/m1). The desired levels of aminopyrine and inhibitors were also added. The reaction mixtures were incubated at 370C under air in a Dubnoff metabolic shaker. The reaction was stopped by ad- dition of an equal volume of 10% trichloroacetic acid. All tubes were then allowed to stand at room temperature for 20 minutes to allow for total protein precipitation. An equal volume of Nash reagent (2M_NH4C2H302, 0.5M_CH3COOH, and 0.02M 2,4-pentanedione) was then added to each tube. The reaction product, diacetyl dihydrolutidine (DDL) has a yellow color and was read spectrophotometrically at 412 nm in a Coleman Jr. Spectrophotometer equipped with a flow cell. The extinction coefficient used was 7.08 cm-l, uflfl HCHO. The inhibitors: dieldrin, DDT, metopirone, benzpy- rene, hexobarbitol, Lilly 18947, SKF-SZS-A, and SKF-8742-A were all added to the incubation mixture in a minimum amount of acetone, prior to the addition of any of the enzymes. The acetone was then subsequently removed by blowing N2 gas into and over the incubation mixture. All of the steroids were also added in acetone. 16 Cytochrome C Reductase Assay Incubations were in 0.05M KPO4 buffer, pH 7.3 contain- ing 10'4M_EDTA. Protein was mixed with 0.2 ml of cyto- chrome C stock solution (36nM) and the above buffer to a total volume of 1.0 ml. The reaction was initiated with 10 ul NADPH (lOmM). The activity was followed by reading the absorbance increase at 550 nm. Reduced cytochrome C has an extinction coefficient of 27.7 cm'l, mel. Nitroblue tetrazolium (NBT) Reductase Assay Protein (0.1 ml) was added to 0.5 ml of NET stock solution (1.0 mM) and enough 0.05M KPO4 buffer, pH 7.3 to make a final volume of 1.0 ml. The reaction was initiated by addition of 10 ul of NADPH (10 mM) and followed at a wavelength of 580 nm. One unit of activity corresponded to absorbance change of 1.0 per minute. CO-difference Spectroscopy The carbon monoxide difference spectra were obtained by resuspending the protein in 0.05M Tris-HCl buffer, pH 7.5. The solutions in the reference and sample cuvettes were reduced by the addition of dithionite (<1 mg). To obtain the CO difference spectra, the sample cuvette was bubbled with deoxygenated CO until it was saturated. The CO gas was deoxygenated by Fieser's Deoxygenating Solution which contains: concentrated KOH (2.7M), Na—anthraquinone- B-sulfonate (0.068M), and Na-dithionite (0.81M). Spectra 17 were recorded with either a Beckman DB or a Coleman 124 spectrophotometer and a Sargent SRL recorder. CO difference spectra were used to analyze for both cytochrome P-450 and cytochrome P-420. The extinction coefficient used for P-450 1 was 91 cm-l, mel and for P-420 111 cm-1, me as described by Omura and Sato.46 Cytochrome b5 Assay Cytochrome b5 assays were also accomplished by dif- ference spectroscopy. Equal concentrations of protein was added to the reference and sample cuvettes just as in the CO-difference spectra procedure, with dithionite (<1 mg) only being added to the sample cuvette. A reduced vs. oxi- dized difference spectrum was taken between 440 nm and 400 nm. The absorbance difference between 424 nm and 409 nm was measured. The extinction coefficient between these 1 1 two wavelengths is 163 cm- , me . Substrate Difference Spectroscopy Substrate difference spectroscopy was a technique used to determine Type I or Type II binding curves for various microsomal preparations (see Introduction). DDT (100 HM), aminopyrine (25 mM), Lilly 18747 (100 uM), DPEA (100 uM), dieldrin (100 mM), and metopirone (33 uM) were used as ligands. Substrates were added to 1.0 mg/ml mi- crosomes, diluted with Tris-HCl buffer, pH 7.5, with or without dithionite for reduction of P-450. The spectra were recorded on a Cary Spectrophotometer. 18 CO-02 Binding Equilibrium Experiments for P-450 Microsomes were diluted to a final concentration of 1.0 mg/ml with 0.05 M Tris-HCl buffer, pH 7.5. Both cu- vettes were then reduced with dithionite. CO was added by addition of a known volume of Tris-HCl buffer saturated by CO (assumed to be 930 HM), and the absorbance change between 450 nm and 485 nm was taken. Metopirone (33 mM) and DDT (100 uM) were added to both cuvettes to determine their effects upon to amount of P-450 reduced. Polyacrylamide Gel Electrgphoresis The disc gel electrophoresis system which was used was a modification of the method by Takayama.47 The gels were prepared by mixing stock solutions A and B with tetra- methylethylenediamine in prOportions, 3:1:0.02 (%). Stock solution A consisted of 6 grams of acrylamide, 0.16 grams N,N'-methylene bisacrylamide, 12 grams urea, 28 m1 glacial acetic acid and water to make 60 ml final volume. Stock solution B contained 12 grams urea, and 0.3 grams ammonium persulfate in 20 ml water. Stock solution A could be stored up to 6 months if kept refrigerated, however, solution B was made fresh before each experiment. The buffer system used was 10% acetic acid, at both the anode and cathode. Polymerization of the gels was carried out in a water bath at 47°C for 15 minutes. A thin layer of water was placed on each gel prior to incubation to insure a flat surface 19 on the top of each gel. The gels were then covered with a solution containing 5M_urea in 75% acetic acid and pre- electrophoresed for one hour at 5 milliamps per tube to remove the ammonium persulfate. All protein samples were dissolved in a mixture con- taining phenol, acetic acid, and water (2:1:1) to a final concentration of 1.0 mg/ml and from 0.1 to 0.5 mg applied to each gel. Only running gels of 7.5% acrylamide, 5.5 cm in length were used. Electrophoresis was routinely per- formed at room temperature for one hour with a constant current of 5 milliamps per tube. The gels were stained for a minimum of one hour in Coomassie Blue (0.05% in 12.5% TCA) and destained by dif- fusion in distilled water for at least 30 minutes. Partial Purification of P-420 from Microsomes Microsomes, isolated from phenobarbital treated ani- mals, were diluted to a final protein concentration of 10 mg/ml with 0.05M KPO4 buffer, pH 7.5. 0.072% (5) powdered steapsin was dissolved into the solution. It was then transferred to Thunberg tubes and made anaerobic under N2. The tubes were placed in a Dubnoff metabolic shaker- waterbath at 37°C and allowed to incubate for 60 minutes with occasional shaking. The incubated solutions were then pooled and centri- fuged at 105,000g (40,000 rpm, 40.2 rotor) for 60 minutes. 20 Both the supernatant and the pellet were assayed for cyto- chrome P-420 and cytochrome b5. The supernatant contained 75-100% of the cytochrome b with only 25% of the cyto- 5 chrome P-420. The pellet was resuspended in 0.05M Tris-HCl buffer, pH 9.5 to a protein concentration of 0.5 mg/ml. Powdered snake venom (Naja Naja) was dissolved into the suspension at 0.5 mg/ml. The suspension was then incubated with con- tinuous shaking in air at 37°C for 20 minutes. The incubated solution was then centrifuged at 196,000g (50,000 rpm, 50 rotor) for 30 minutes. The super- natant contained 90-100% of the P-420. It was cooled in an ice bath and fractionated by ammonium sulfate precipitation. The fraction precipitating between 60-75% saturation contained essentially all the P-420 present in solution. Upon centrifuging at 3,8009 (5,000 rpm, SGA rotor) in a Sorvall centrifuge, a yellow, floating pellet was obtained. It was removed by the use of a spatula and redis- solved in 0.05M Tris-HCl, pH 9.5. If the P-420 solution was kept refrigerated for at least 48 hours, a precipitate resulted along with high turbidity. This precipitate has also been reported by another investigator48 as micro- tubules. 3-MC microsomes were treated exactly by the same procedure, except 0.01% (g) steapsin was used. Both solu- tions gave typical reduced CO-difference spectra for P-420 with a maximum absorption at 420 nm. 21 Optimizing Conditions for P-450 Reductase Assay Whole microsomes were diluted with 0.1 MKPO4 buffer, pH 7.5 until a change in absorbance (450-485 nm) was equiv- alent to 0.2 when a P-450 CO-difference spectra was taken. Glycerol concentrations, varying from 0 to 25%, in buffer, were used to dilute whole microsomes and then the rate of reduction under anaerobic conditions was followed. Ex- cessive levels of NADPH were added to start the reaction which was followed at 450 nm on a Hitachi spectrophotometer. Glycerol concentration was then held at 15% and the pH of the enzyme solutions was varied from 6.0 to 9.0 using 0.5 increments. The pH optimum for this particular reduc- tase was found to be 7.0. EXPERIMENTAL Lineweaver-Burk plots of aminopyrine demethylase activity from microsomes isolated from untreated male rats (control microsomes) were found to be non-linear (Figure 1). Non-linear Lineweaver-Burk plots are characteristic of multiple enzymes with varying Km values for a single sub- strate.48 However, similar plots of aminopyrine demethyl- ase activity from microsomes isolated from animals pre- treated with phenobarbital (PB microsomes) were linear (Figure l). Phenobarbital pretreatment resulted in an in- duction of an activity with an apparent Km for aminopyrine of 5><10u4 M. 3-methylcholanthrene (3-MC microsomes) pre- treatment of animals resulted in greater activity at high substrate concentrations (>2mM) resulting in an apparent Km for aminopyrine of 2X10.3 M. Similar experiments were performed using ethylmor- phine as the substrate. No differences in Km's were found for the different types of microsomes when values were extrapolated from 2.5 mM substrate concentrations or lower and concentrations above 20 mM resulted in substrate in- hibition of activity in all three types of microsomes (Fig- ure 2). 22 23 .HmHoE mum mcoflu umuucowcoo mamuumnzm .cflmuoum Hume .HIGHE mohsmpame (How m0 215 GA ma wuflooHo> .mHmEHcm omumwnumnm Auzlmv mcououomflosoasgumsum 6cm Amie Hmosoumnooozo .Aaosu scoov Houucoo Eoum moEOmouoflE as >ua>fluom mmmamsumeU mcflummocflfim mo muon stmlum>mm3mcflqll.a .mflm 24 \m cemp coop P mm .0255 U2- Onwm .—\ > 25 .HmHoE mum mcowumuu Icmocow opmnumnsm .cwmuoum Hume .HIcHE momnmpameuom m0 238 CH ma muflooam> .mHmEHCM pmuwmuumum Auzlmv mcmnzpcmaonoamnumfinm pom Ammv Hmufinumnocwcm .Aaonu Icouv Houucoo Eoum mmEomouan CH hpwbfiuom onwahnumfimn mcH£QHOEHhcpm mo muoam Musmnum>mmsmcfiqnl.m .mHm 26 __\m oom— oo_o_ 2.3 o — l 1.1 1 E IN. IV. oz...” 10. _ozcou rm. Fo.— u—|> 27 Ethylmorphine N-demethylase in both control and PB microsomes has been plotted with a constant level of amino- pyrine on a double reciprocal plot. Upon analysis it had become evident that phenobarbital had induced an activity which was not common to both demethylase substrates. The two activities were additive over a wider range in PB mi- crosomes (Figure 3). Lineweaver-Burk plots of aminopyrine demethylase activity from microsomes isolated from untreated female animals were also found to be non-linear, but their activity was less than the activity from male animals. Female ani- mals treated with testosterone (40mg/kg) showed non-linear activity with increase in enzymatic activity occurring only at lower levels of substrate (Figure 4). Male animals treated with testosterone showed no effects in metabolism with this substrate (Figure 4). The following compounds were tested for the inhibi- tion of aminopyrine in control, PB, and 3-MC microsomes: SKF-SZS-A, SKF-8742-A, Benzpyrene, DDT, Testosterone, Lilly 18947, Hexobarbitol, Metopirone, and Dieldrin (Fig- ures 5-11). In control and PB induced microsomal activity Lilly 18947, SKF-525-A, and Benzpyrene gave linear Line- weaver-Burk plots when they were used as inhibitors indi- cating that only one activity was being inhibited. How- ever, when 3-MC microsomes were used, the non-linearity of the reciprocal plots remained. From Figures 5 and 6 it 28 .HmaoE mum mcowumuucmocoo mumuumndm .cflmuoum Hume .chflE momnmUHmEuom mo_mafi CH MM wuflooa 10> .mmusuxwe coflumnsocfl Gnu CH mafiummocflam z vloaxm usonufl3 Ho nufl3 mamfiflcm pmummuuwnm Hmuflnwmnocwnm pom Houucoo Eoum moEOmOHOHE ca mua>wuom mmmahsumsmo mcH£QHOEHS£um mo muon xnsmnnw>mmzmcHAII.m .mHm 29 d1<+2m _ot.:ou < 30 .HMHOE mum Mcoflumuucmocoo mwmnumnsm .cflmuoum Hume .HICHE opanmpamfiuom MO 218 GA ma xufloon> .mcououmoumwu :ufl3 pmumwwu (mum mHmEHcm pom mmEOmOHOHE Houucoo mamfiwm was mama cw mufi>fluom mwmamnumEmp mcfiummocflfim mo muoam stmIHm>mmzmcflAII.v .mHm 31 w oow; oo_o_ _ own ocotoemofiohknu .1 H v .1 _o:c0UkO 1n\\ 1111 039.2330... m .920 U m 32 .CMHOE mum MCoHumHquOCoo mumnumnzm .CHmDOHm HImE .HICHE mmemUHmEHom mo 22E CH mH quoon> .musuxHE CoHumnCoCH mCu CH $5.: 3.133%me oom . :13 o: 883302 I? 88 :2336 muoanHCCH on» CuHB mHmEHCm HouuCoo Eoum moEOmouoHE CH muH>Huom wmmesumEmp mCHHmQOCHEm mo mpon xnsmuum>mmszqul.m .mHm 33 m \_ 00m — 00.9 0mm 0 _O.=:OU n \u .1 .2mwunoxo1 .N 19.9.3232 -V 5.205 34 .umHOE mum lmCoHumuquocoo mumupmnsm .CHmuoum Hume .HnCHE momCmonEuow H0 218 CH mH lmuHUOHm> .musu 1st coHumnsoaH may cH A2: omv « :meem mew new 12: one «1mmmnmsm muoanHCCH on» CDH3 mHmEHCm HouuCoo EOHH mmEOmouoHE CH muH>Huom mmMHmnqumo mCHHHQOCHEm Ho muon xusmuum>mwszHA11.m .mHm 35 00M— coo— 11.11 _o.:cou .mmusuxHE COHumnsoCH 0:» CH ASE v.HV 283833 9:... .3: 8: $2: HHHHC .9: 83 83323 muoanHCCH 0C» CHHB mHmEHCm HOHuCoo EOHH mmEomouoHE CH huH>HHom mmmHmCumEmU mCHHHQOCHEm Ho muon xHCmIHm>mm3mCHAII.h .mHm 37 00.3 00.0— .9 .cou «1 11.11 :82 2.: 9.0.2333 occidncom .—|> 38 .HCHOE mum mCOHumHuCooCoo oumuum (Cam .CHmuoum Hume .HICHE 00AMoonEHom Ho 21E CH mH thoon> .mmnsuxHE COHumndoCH OCH CH A2: oHV mCOHHQOHmE UCC .AZC oowv Boo .Azz omv Cummmnmmm muoanHCCH may CuH3 mHmEHCm pmumwuu (mum Auzumv maHCuCMHOCOHSCumEIm EOHH mmEOmouoHE CH muH>Huom ommHmnuoEmp mCHHHQOCHEm Ho muon xusmlnm>mm3mCHnul.m .mHm 39 00m— 000p 950572302 .mmHCHxHE CoHummCoCH map CH Hz: OOHV mCmHmmnCmn pCm .AEE N. HV HouHQHmno -xon .121 omv HanHm mMm muoquHnCH mag Cqu mHmchm ooummuu loud Auznmv oCoHCHCMHOCoHHCuoEIm Comm mmEOmOHOHE CH >HH>Huom mmmHmsumEmp OCHHHQOCHEC mo mHon xnsmnum>mmszqul.m .mHm _a . I w .. ..__ 5;”... :1.” ._ , ...... . 1.. 1.4.... .. I . . W _ . ., . m .....__ . _ .3. .y. H H 1. 1|: _ 41 00m— .—\V’ coop 00m ocoidncom .2553 oxo I <1N§mi¥m INF .—1:> 42 .HmHoE who mCOHumnu ICwOCoo mumuumnsm .CHmuoum Hume .HICHE spammuHmEHom mo_mafi CH mH HuHooHo> .monsust coHumnsoaH on» cH 12; oOHV vamH HHHHH pCm .Amfi v.Hv mCoumumoummu muouHCHCCH may CuH3 mHmEHCm pmummuu Iona Huxlmv mCmHCquHOCoHHCHmsnm Scum meOmouoHE CH muH>Huom mmMHmzqump mCHumm0CHEC mo muon stmlum>mm3oCHH11.OH .mHm a. 7‘14" A M“ 11%. E 43 00% — 00.9 0mm U2 m :2: 2.: 0:03.330» 44 .HCHOE mum mCOHumuquoCoo oumuumnsm .Cku loud HImE .HICHE wmmnmpHmEHom Ho_mafi CH mH hywoon> .mmusuxHE CoMumnsoCH on» CH ASCIOOHV hvmmH hHHHH pCm .Azn omv Cnmwbmumxm .Az: omv CummmlmMm .Az: oomv CHHUHme muouHCHCCH 0:» nqu mHCE IHCC woumwnumum Ammv HmuHQHmCOCow Eoum mmfiomOHOHE CH >HH>Huom mmmHmCumEmU mCHHMQOCHEm Ho muon xnsmlnm>mm3mCqul.HH .mHm 45 OOm — coo— .11 I ma 5&2 3.: <-mum-u_v_m <-N§miv_m 5 -m— 46 seemed that in control microsomes dieldrin and metopirone inhibited activity at low substrate levels. When 3-MC microsomes were used, SKF-8742-A exhibited a greater ex- tent of inhibition than other two types of microsomes (Fig- ures 6 and 9). However, in PB induced microsomes, dieldrin was a better inhibitor than SKF-8742-A (Figure 11). The inhibition of aminopyrine demethylation in con- trol, PB, and 3-MC microsomes at increasing concentrations of the inhibitor DDT at a constant substrate level is shown in Figure 12. The data was presented as percent con- trol microsomal activity in absence of inhibitor. The curves are not hyperbolic as classical kinetics predicts49 but rather seem to be made up of three linear segments which divided the total demethylase activity into three components, one not inhibited by DDT, one moderately in— hibited by DDT, and a third which was extremely sensitive to DDT inhibition. In PB microsomes the component which was most sensitive to DDT inhibition was induced to an ac- tivity 10 times that in control microsomes. This component was essentially completely inhibited at 50 uM_DDT. The second component, also sensitive to DDT, was slightly more active in 3-MC microsomes only, and it was inhibited from 50-150 uM DDT. The activity which was insensitive to DDT inhibition does not contribute significantly to the overall activity of control microsomes at this substrate level (2mM'amin0pyrine). 47 .86 m mm3 CoHumHquoCoo OCHHHQOCHEC .Auzlmv oCmHCquHOCloCquIm oCm Ammv HCHHCHCQOCmCm CuH3 poummuu noun mHmEHCm Eoum UCm HHouuCouv mHmEHCm HoupCoo Scum omCHmuno mums mmEOmonon .HoanHCCH Ho moCmmnm mCu CH AmooHv MHH>Huom HouuCoo Ho quouwm mm ommmmumxo mH huH>Hu04 .Boo an >HH>HHOC wmmHmnumEmU mCHHHQOCHEm HCEOmOHoHE Ho COHHHCHCCH1|.NH .mHm 48 So 212.. N. — smug-<1- u1_o::ou #221” H mm d< SEN ICON Dov 1oalNoa 1° "4: 49 At increasing substrate levels, the contribution of the DDT-insensitive component to the total activity seemed to increase significantly relative to the other two compo- nents (Figure 13). At concentrations of DDT over 150 uM both of the DDT-sensitive components would be completely inhibited, and therefore it was possible to study the DDT- insensitive component. As can be seen in Figure 13 and 13a, the activity of this component was related to the substrate concentration as described by the Lineweaver-Burk plot. If the activi- ties at various levels of substrate and 200 uM DDT are plotted in a reciprocal plot, a Km of 1.3><10'2 M was ob- tained (13a), which was about an order of magnitude greater than the Km obtained by extrapolation of the Lineweaver- Burk plot for 3-MC microsomes shown in Figure 1. Figure 14 shows the effect of increasing levels of DDT on the aminopyrine demethylase activity in microsomes prepared from control rats (male and female) and rats pre- treated with testosterone. Testosterone induced the DDT- insensitive component in female control microsomes to a level equivalent to that in male microsomes, yet testoste- rone had no effect on male control microsomal activity. When dieldrin was used as the inhibitor of aminopy- rine demethylation in PB microsomes, only two components were evident. At substrate concentrations above 2 mM, de— methylase activity at increasing levels of dieldrin did 50 .HmHoE mum mCoHumuquoCoo mumuumndm .CHououm Hume .HuCHm moanmonEHom mo moHOE:E mm commoumxo mH huH>Huo< .Baa 2E H.o um mHmEHCC poumouu 10mm Ammv HMHHQHCQOCMCQ Eoum moEOmOHOHE CH muH>Huom mmmHmnu (damp mCHHHQOCHEm mo uon xuzmnuo>mm3mCHHI|.mmH .mHm .Amdv mCHHHCOCHEm Ho mCOHumuquo (Coo mCoHHm> um ago an mHmEHCm woumwupmum mm EOHH mofiomOHoHE CH mmmHmCHmEmp mCHHmCOCHEC Ho CoHanHCCH mCBII.MH .mHm 51 pan 2 .0? 1.. m o .1 1 ._< in. u 1 /o 1 1 ._< Em. m 1 o w ., ._< 2:... o. c U W W '- ooo. oom \— o .omM — b 1 IA .o. .8 00.. 52 .mE m mm3 CoHumuquoCoo mCHHHQOCHE< .mCoumumoummu CuH3 ooummuumnm mmHmEmm Eoum UCC HouuCoo mHmEmm UCC mHmE Scum meHmu Ino mumB mmEOmOHOHE .HoanHCCH mo moCmmnm map CH AwOOHV Houu (Coo mHmE Ho HCmoHom mm pwmwmnmxw mH huH>Huo¢ .900 an AHH>HHUC ommHmzuoEmo mCHHmQOCHEm HMEOmOHOHE Ho CoHuHaHCCHII.¢H .mHm 53 41 | 2 1. . .lilllafl .00 v.0—x m P _ .N - __. _ T .otEou m ION 0.5053030... m o/o 071 1.11 111 m. .otEouku 10.» m4 m... M I00. 100 00.. 54 not give a hyperbolic curve but seemed to be divided into two linear portions representing two components (Figure 15); one having a low Ki for dieldrin and the second not in- hibited by dieldrin. Repeating an experiment like that shown in Figure 13, using dieldrin as the inhibitor at concentrations greater than 100 uM, a non-linear Line- weaver-Burk plot was obtained (Figure 16 and 16a). When metopirone was used as the inhibitor, quite similar results were obtained. Comparison of the relative amounts of each component in control, PB, and 3-MC microsomes suggest that the diel- drin and metopirone sensitive component was that which was induced by PB (Figure 15). The dieldrin and metopirone sensitive activity therefore must be made up of two compo- nents, that which was moderately sensitive to DDT inhibi- tion and that which was DDT-insensitive. Thus the PB in- duced component was not the same as the low Km component in control microsomes. The Km of the PB induced component could only be estimated assuming that the low Km component of control microsomes would not contribute significantly to the activity in "highly" induced PB microsomes. Using this assumption a Km for aminopyrine of 5><10"'4 M was ob- tained. Pretreatment of animals with 3-MC consistently de- creased the maximum degree of inhibition obtainable by either DDT or dieldrin (Figures 12 and 15). These results 55 .m& m mm3 CoHumHquOCoo mCHH>QOCHEC .HOUHQHCCH mo moCmmnm mCu CH muH>Huom HonuCoo Ho “Cmouom mm pommmumxm mH >HH>Huo< .CHHUHmHU an mHmEHCm pmummunmnm .mm. HmanHCQOCmCm ch auxin. 0C0HCHCMH0C0H>CHmE|m .AHouuCoov HouuCoo Scum mmEOmonoHE CH huH>Huom mmmHanuoEmU oCHHHQOCHEC mo CoHanHCCHII.mH .mHm 22.0.3.0 << 0..x w v ..O~...ZOU M11 9 . 1.1 1.1 T. 0.2..m c O O 56 1 oumoa 1o °Io 00w 57 .HMHOE mum mCoHumuquoCoo wumuumnsm .Ckuoum Hume .HICHE mphzopHmEHOH Ho me0218 mm pmwmmumxm mH HHH>HH0C .CHHG 13:. as 3.0 pm 222... ooummuuoum 3.: HSHfimnoqosm on... .HouuCouv HouuCoo Scum mmEOmOHOHE CH >9H>Huom mmmHHCHmE Imp mCHHHQOCHEC Ho muon xusmlum>mm3mCqul.mmH .mHm .ACH. mCHHHQIOCHEC Ho mCoHumHquoCoo mCoHHm> um CHuonHU an mHmEHCm pmummuumum HMHHQHCQOCmCm COMM mmEOmouoHE CH mmmHmCumEmU mCHHHQOCHEm Ho COHHHQHCCH mnaul.mH .mHm q hyfi-C-va-I-Jéé'wr _ . 58 22.315120. N — O _ _ A 1. 1 a1 19 1 U1 1 17 m \. _O:COU ma. 00— O All/\llDVo/ 59 could be explained if 3-MC pretreatment induced the synthe- sis of the DDT-insensitive component. This was investigated by comparing the aminopyrine demethylase activity in con- trol and 3-MC microsomes in the presence of 200 0M DDT. The results (Figure 17) showed a two-fold increase in maximum velocity with no change in the Km. Inhibition of the high Km component for aminopyrine demethylase was seen using SKF-SZS-A, DPEA, and Lilly 18947. DDT (200 mM) was present in all assays to insure complete inhibition of the two DDT-sensitive components. All three types of classical inhibition: competitive, non-competitive, and uncompetitive were seen using these three inhibitors, respectively (Figure 18). Experiments were then designed to study the mechanism of inhibition of some of these inhibitors, in hopes of learning the nature of the multiplicity of aminopyrine de- methylase. Inhibition studies such as those shown in Figure 12 and Figure 14 were performed in a pure oxygen atmosphere to determine if any reversal of inhibition could be obtained. A pure oxygen atmosphere resulted in a slight additional in- hibition of all activities rather than a reversal of inhi- bition. Increasing NADPH concentrations resulted in only a slight reversal of inhibition at extremely high concen- trations (28 mM). However, a Km for NADPH for this partic- ular reaction was found to be 9.ZXl0'5 M, 60 .umHoE mum mCOHumHquOCOU mpmuumndm .CHmu Amum Hume .HICHE momnmonfiuom Ho mmHoznfi CH mH muHoon> .500 2: com Ho moCmmon map CH mHmEHCC ompmouumum .Uzlm. 0C0HCHCC IHOConCumEIM oCm .HouuCoo. HonuCoo Comm mmEomouoHE CH muH>Huom mmMHwnumEmo mCHHHCOCHEm mo uon xnsmnum>mm3mCann.nH .me “a?! .7 «Ii " --'! . , (a 4.; . .3... , pmmu-j 61 o? .—\m 0mm 5.10 .2200 #00 5.3.8... .2 ._\ > 62 .umHoE mum mCmHumHuCooCoo mumuquCMI .CHmuoum Hume .HICHE mohmonmEHom Ho Ems CH mH muHUOHm> .AZE ohH. hvmmH aHHHH 0C8 ..25 m.o. damn ..zE o.H. «(mamumxm an son 2: com mo mOCwmmum 0CH CH maHCqu (HOCoHanumEIm CHHB ooumwuuoum mHCEHCm Comm mmEOmOHOHE CH muH>Haom omMHxCumEop mCHHHQOCHEC HCEOmOHoHE Ho COHanHCCHII.mH .mHm 63 00m .. 000 — . _ .—\"’ 0mm So 2.1 oou .2230 3.2 <1 .8 M Alt-‘4'“ ..:"~.¢-¢' 64 KCN was also tested as an inhibitor of the demethyl- ase system. However, the results were almost identical to the effect of DDT when the KCN concentration was varied from 0 to 4.0 mM. Since the DDT and KCN inhibition studies seemed related, the next set of experiments were done to determine if DDT had affected the reductase activity, a logical choice since it took so high a KCN concentration that it would seem reasonable to assume that it wasn't binding to the heme group of P-450. The effect of metopi- rone and dieldrin was also investigated. To test this hy- pothesis, cytochrome C- and nitroblue tetrazolium-reduc- tases were chosen as reductases since they might be in- volved in microsomal electron transport for aminopyrine demethylase. However, none of the above compounds were effective inhibitors of either reductase. The requirement for both NADPH and NADH for maximal aminopyrine demethylase activity has been reported by other investigators.50 NADH alone supported no aminopyrine demethylase activity, however, in the presence of NADPH, NADH does seem to stimulate activity suggesting that it supplies reducing equivalents. Therefore, it was reasoned that if a relationship between an inhibitor and NADH could be shown, the mechanism of inhibition might involve the electron transport system. NADH was found to reduce P-450 but at a slower rate than NADPH (Figure 19). DDT was found to stimulate the reduction of P-450 by either NADH 65 l .COHuC 0m may cH Hammoum .2: com. Boo usonsHs new nqu mon can mmonHHn EC omv um pmusmmoe omvnm oEOHCoogho mo CoHuoCCQM1I.mH .me 6 6 .Ee. 2:: 0— m 0 v F _ _ — — — _ _ IDHuom HonuCoo Ho quoqu mm pmmmmumxm mH >HH>HHOC am m.o mHCmmmumoH 0 pCm .m m.o mqummu lawn 0 .fl H.o mqummHQmH m .CoHuMHquOCoo madz oo.o mqumwume C uon .madz Ho mHm>mH mCoHHm> mo GUCmmmHm may CH 900 an muH>HHom mmmHHCquop mCHHHCOCHEC HmEOmOHOHE mo CoHuHQHCCH11.om .mHm 200 69 o fifi. ~13- .— D D 11 -~ 2 T 2 X 1 H» D u m < o I] o O O (ouuog go °/o 70 (Figure 21). Dieldrin gave an atypical type I difference spectrum in the oxidized state and a maximum at 450 nm with reduced microsomes (Figure 21). It was then reasoned that these inhibitors could be effecting the affinity of P-450 for 02 thus causing inhi- bition of activity. CO binding experiments were therefore performed on P-450 using a CO saturated buffer to intro- duce a specified concentration of CO to compete with the metopirone already present. It was found that DDT had no effect upon the binding of CO to microsomal P-450. However, when the inhibitors metopirone and dieldrin were added, a general decrease in the amount of P-450 available for binding CO was found (Figure 22). It had been previously found that metopirone and dieldrin had strong P-450 binding spectra (Figure 21) indicating some type of interaction with the heme group of P-450. The extent of this inhibition of CO binding by metopirone or dieldrin varied depending upon the type of microsomes used. PB microsomes showed the greatest inter- action between metopirone and CO while 3-MC pretreated microsomes resulted in the least interaction (Figure 23). When difference spectra with metopirone and dieldrin were obtained using all three types of microsomes at equal P-450 concentration, it was found that PB microsomes had exhibited the largest metopirone (446 nm) peak and 3-MC microsomes the smallest peak. Dieldrin gave only a slight 71 .mocmmHH mm Isouuon. cHuoHoHo UCC .mou. mConHmoumE mCHmC mmEomouoHE mm CHH3 mupommm ooCmHoHHHo mumuumnsm omospmu oCm UoNHponII.HN .mHm 22.0.5.0 0mm omv .xO .3. 72 uZOEdOhmE 73 Fig. 22.--CO binding curves for microsomes of control animals (middle) and animals pretreated. with PB (top), and 3-MC (bottom) with and without metopirone present in solution. O.D. was measured. at 450 nm. A00 .081 .04- .081 .04- 74 .L + v JC+ l l I 1 20 4O .1. A + 20 40 At __.__ . 20 40 fl 75 .0C0uCuC0HononxuoE1n 1:: Hmanumn0C0Cm nuH3 U0u00uu0um mHCEHCs 0C0 uHsEHCs Heubcoo Eoum mmfiomouoHE Ho oumum 000300» onu CH pochuCo 0C0CH13435 Ho 00C000nm 0:» CH wuuomdm 00C0C0HHHU 0011.3mm .3H1 .0C0HCHC0HOCUHACuoeum 0C3 HnquCsCoca:; :HH3 errcrugsC; mHmeHCm UCC mHCEHCC Houucoo Eouw masomoCUHE Ce :Lch $512001 03» CH meHmuno muuommm mucouawwHo SCOCHAGHGSII.CHN .?.1 76 19:on oz-..” one g H 0mv oo .6 one 1 + one omm 11/1 _ 11 1. 1 057m /I1/ 11/ 1.1 11111 1 1 /I\1 soaHZOU /\.I..I.l+( 77 spectral change with each type of microsome. An extinction coefficient for the interaction of metopirone with the heme of P-450 was obtained by calculating the amount decrease in absorbance at 450 nm in a CO difference spectrum with PB microsomes and assuming that this amount of P-450 was re- sponsible for the difference in absorbance from 446 nm to 480 nm in the metopirone difference spectrum. An extinction coefficient at pH 7.3 in KPO4 buffer of 146 cm'lmM'l was calculated. A procedure was then developed for the solubilization and partial purification of P-420, the soluble cytochrome product of P-450 in hopes of using this product for further studies on the multiplicity of microsomal oxidases. The first question concerned the multiplicity of P-450 itself while the second involves the possibility of using P-420 as a substrate for NADPH-cytochrome P-450 reductase and for obtaining difference spectra. Steapsin (0.072%) solubil- ized all of the NADPH-cytochrome C and 80-90% of the b5 present while only 25-30% of the P-420 was solubilized. It was found that a 37°C incubation for 60 minutes at a pH of 7.5 under anaerobic conditions was optimum for this solu- bilization step. The next step involved solubilization of the P-420 with snake venom (Naja Naja; 0.5 mg/ml). The optimum con- ditions for this step were 20 minutes at 37°C, pH 9.5 in an aerobic atmosphere. This procedure solubilized all of the cytochrome P-420. 78 The final step involved a 0-75% ammonium sulfate fractionation of the venom-solubilized P-420. Essentially all of the solubilized P-420 was precipitated out of solu- tion, and could be separated and redissolved. The final concentrations in the prep were generally 6-8 nmoles P-420/mg protein without any cytochrome b5 present for PB microsomes, and 2-4 nmoles P-420/mg protein when either 3-MC or control microsomes were used. Electrophoretic gels have shown that a substantial purification had taken place, but some impurities still remained. The solubilized P-420 was then tested in the same manner as P-450 for its CO binding ability with or without metopirone. Both metopirone and dieldrine had an identical effect on the binding of CO to the soluble P-420 as compared to whole microsomal P-450. The absorbance peak had shifted from.420 nm to 416 nm when metopirone was present. Studies were then done on NADPH reduction of the solubilized P-420 in hopes of showing specific reductases for each P-450. P-420 was isolated from PB pretreated animals and reduced by NADPH with whole microsomes as the source of reductase from all three types of animals (con- trol, PB, and 3-MC). It was found that all three types were capable of reducing P-420 without any preference as to the extent and rate of reduction. Optimum conditions for P-450 reductase were found for further investigation of multiple reductases. These 79 conditions were 0.05 M P04 buffer with 15% glycerol, pH 7.0. It was found that the initial rate of P-450 reduction was directly proportional to the glycerol concentration found in solution. Fifteen per cent glycerol was chosen due simply to the fact that it produced a rate which could easily be studied. DISCUSSION The discovery that many drugs were metabolized by a microsomal mixed-function oxidase, coupled with the dis- covery of cytochrome P-450, led to the belief that this single oxidase system was responsible for all of the mixed- function oxidase reactions carried out by the liver micro- somes. Many studies were carried out to show that all com- pounds which were substrates for the system were competitive inhibitors of the metabolism of each other. For example, ethylmorphine demethylation was competitively inhibited by other drugs and steroids.49 Lipid peroxidation was in- hibited by drugs which were metabolized.50 However, evi- dence then started to accumulate which said all of the re- actions were not catalyzed by the same oxidase. Induction by phenobarbital or 3-methylcholanthrene gave microsomal enzymes with altered kinetic parameters. Then it was dis- covered that PB and 3-MC induction resulted in two differ— ent cytochrome P-450's. These results suggested that mul- tiplicity of microsomal mixed-function oxidases must exist. The best evidence for multiplicity of aminopyrine demethylase activity is the fact that Lineweaver-Burk plots of demethylase activity in control microsomes is not linear. 80 81 These results are suggestive of at least two activities with differences in Km for aminopyrine. Therefore, it is impossible to determine whether other compounds undergoing oxidation can competitively inhibit aminopyrine demethyla- tion. Lineweaver-Burk plots of control activity is not linear and the addition of inhibitors introduce another degree of complexity depending on which activity is in- hibited. The data published by Wada and coworkers,51 is very good evidence for more than one enzyme for the hydroxyla- tion of aniline. Essentially linear Lineweaver-Burk plots were found for the hydroxylation of aniline; however, non- linearity was evident when the inhibitor prednisolone was included in the incubation mixture. The Km's of the two enzymes appear to be similar, however, the differences in inhibition by prednisolone show up by kinetic evaluation. Alvares g£_gl.52 have shown a difference in Km's for the hydroxylation of benzpyrene by microsomes obtained from animals induced by phenobarbital and 3—methylcholan- threne. The results of this study, and of Pederson and Aust29 suggest that the aminopyrine demethylase activity at low substrate concentrations can be induced by PB while the activity at high substrate concentrations can be in- duced by 3-MC. A similar investigation of ethylmorphine demethylase activity did not reveal large changes in kinetic parameters 82 upon induction with PB or 3-MC. However, substrate compe- tition experiments with ethylmorphine and aminopyrine sug- gested that phenobarbital induction resulted in less compe- tition between the two substrates. PB must induce some enzyme(s) which are specific for only ethylmorphine or aminopyrine. Microsomes from female rats consistently show less aminopyrine demethylase activity. It has been suggested that this involves differences in hormonal balance. Ac- tivity in male rats is thought to be induced by such hor- mones as testosterone, because this androgen is much more prevalent in the male than in the female. It was also thought that the differences in aminopyrine demethylation in male and female rat microsomes could involve kinetic parameters. That is the low Km component may contribute less to the overall aminopyrine demethylase activity, re- sulting in lower activities, especially at low substrate concentrations. However, Lineweaver-Burk plots of amino- pyrine demethylase in female microsomes were similar to that in microsomes from male rats. Pretreating female rats with testosterone increased activity at low concen- trations of substrate, however, activity was still less than activity in microsomes from male rats. Testosterone had no effect on activity in microsomes from male rats. Gillette, M. ,53have shown that the state of pregnancy affects the kinetic parameters of the metabolism of a 83 number of substrates. Other investigators have reported similar results for the NADPH-linked electron transport system and the terminal oxidase.54 Others have reported variations of the detoxification enzymes during develop- ment.55 The differences in inhibition of aminopyrine deme- thylation in control, PB- and 3-MC-microsomes is also sug- gestive of multiple activities with different suscepti- bility to inhibition. Lineweaver-Burk plots of aminopy- rine demethylase activity in all three types of microsomes in the presence of various inhibitors indicated some simi- larities among inhibitors. The extent of inhibition by DDT, metopirone and di- eldrin varied depending upon the type of microsome used. These compounds inhibited aminopyrine demethylase activity far less in 3-MC microsomes than in either control or PE microsomes. They were among the most potent inhibitors for aminopyrine demethylase activity in PB microsomes, sug- gesting that these compounds were inhibiting a component which was responsible for less of the total activity in 3-MC microsomes and a far greater percentage in PB micro- somes. SKF-525-A and SKF-8742-A, on the other hand, were much more potent inhibitors of the N-demethylase activity in 3-MC microsomes. Takayangi, e_1_:__a_L,56 have shown that one particular inhibitor, SKF-SZS-A, and a few other drugs 84 were not only competitive, but also noncompetitive and mixed-type according to the substrate used and to the species of animals used. Hexobarbitol, which was not a good inhibitor of activity in control microsomes, was a strong inhibitor of aminopyrine N-demethylase activity in both types of induced microsomes. Other studies showed that structural analogs of some compounds did not inhibit N-demethylase activity in the same manner.57 These results have been reported for other substrates also. Due to the variability of inhibition in the three types of microsomes, DDT, metopirone and dieldrin were then chosen for further studies in this system. A plot of activity vs. inhibitor concentration (DDT) was not hyperbolic as classical kinetics would predict,58 but instead yielded three linear segments dividing the to- tal aminopyrine demethylase activity into three components based on their affinity for DDT. In practice this plot of activity vs. the concentration of inhibitor was useful, in that for a system of multiple activities, at any substrate level it was possible to estimate the contribution of any one particular activity to the total activity. The fact that complete inhibition could not be ob- tained at very high concentrations of inhibitor could not be explained by partial competitive inhibition, as de- scribed by Palmer.59 In partial competitive inhibition 85 the fraction of activity remaining at high inhibitor con- centrations would be independent of enzyme concentration. In this system, however, the fraction of total activity re— maining at high inhibitor concentrations was altered by in- duction; increased by 3-MC induction and decreased by PB induction. This fact applies to any argument for only one enzyme with multiple sites for the demethylation of amino- pyrine. The only alternative would be that the induced enzyme is not a different enzyme but rather one in a dif- ferent environment, which affects the kinetic parameters. Such a system seemed unlikely and might still be considered separate enzymes. The suggestion that these activities involve sepa- rate.proteins with different specificity came from the fact that the individual activities can be preferentially induced by PB or 3-MC and the activities induced by these compounds have completely different kinetic properties. Upon induction by PB pretreatment of animals only one com- ponent seemed evident in a Lineweaver-Burk plot because of its low Km and high activity. However, upon the addition of increasing levels of DDT to the incubation mixture, three components were evident. One, preferentially induced by PB, was extremely active and easily inhibited by DDT, dieldrin and metopirone. A second, present in all types of microsomes, was responsible for the majority of the activity in control microsomes and was moderately sensitive 86 to DDT and insensitive to either metopirone or dieldrin. The third component was preferentially induced by 3-MC pretreatment and was insensitive to all three of these inhibitors. The later component had a high Km for amino- pyrine so it could only be observed at high substrate con— centrations. This activity seemed to be present in all aw types of microsomes. The Km of the phenobarbital induced component (5><10"4 M) can be fairly accurately estimated from a Line- weaver-Burk plot of activity in "highly" induced PB micro- somes because of its low Km and high activity. The Km of the DDT-insensitive component can be obtained from a Line- weaver-Burk plot of aminopyrine demethylase activity in the presence of sufficient DDT to completely inhibit the other components. By this method a Km of l.3><10"2 M was obtained. The Km of the third component can only be es- timated by plotting a double reciprocal plot from activi- ties obtained at a DDT level between 50 and 150 uM, By using this method a Km of 4.2><10"4 M was obtained. There- fore, the total aminopyrine demethylase activity involved three enzymatic activities, two with low Km's and a high Km activity. By the use of increasing levels of the inhibitor DDT it was possible to show that the degree of induction of aminopyrine demethylase cannot be accurately assessed by only assaying for total activity. Since total activity was made up of multiple activities, it was necessary to 87 find.which activity had been induced. For example (Fig- ure 12), phenobarbital induction caused about a four-fold increase in total activity (2mM_aminopyrine), however, in actuality it had induced a ten-fold increase in one par- ticular component. Dieldrin and metopirone separated the total amino- pyrine demethylase activity into two components; one sen- sitive to inhibition and one insensitive. The insensitive fraction could be shown to consist of two activities by a Lineweaver-Burk plot in the presence of 100 uM_dieldrin. Such a plot was biphasic presumably because of the presence of two activities with large differences in their Km for aminopyrine. The fact that PB slightly induced the high Km component was also evident. The dieldrin and/or metopirone sensitive component could be shown to be induced by PB by comparing the v vs. [I] plots for both control and PB microsomes. This latter finding also confirmed that the PB-induced component was present, although difficult to observe in control and 3-MC microsomes. Inhibition studies on the DDT insensitive component by other drugs indicated that only one activity existed. All three types of classical kinetic inhibition were also shown to exist for this component. Since both metopirone and dieldrin gave similar inhibition of this system, the assumption was made that the 88 mechanism of inhibition for these compounds was the same. Substrate difference spectra suggested that both metopirone and dieldrin bind to the heme group of P-450 in both the oxidized and reduced states of the P-450 particle. How- ever, Imai and Sato60 have suggested that substrates com- bine with a site in P-450 close enough to interact with the activated oxygen molecule on the heme group, and that the spectral changes induced by substrates are due to a secondary effect of the substrate binding to the non-heme moiety of P-450 on the ligand state through changes in conformation. Other investigators61 have also shown ligand binding was dependent upon type of induced microsome used. Two possible mechanisms could explain these results. The inhibitors could prevent the binding of substrate to the P-450 or they could prevent oxygen binding. A pure oxygen atmosphere did not overcome dieldrin inhibition; however, metopirone and dieldrin did decrease the binding of CO. If the binding of CO and oxygen are related, per- haps these compounds inhibit by preventing the formation of the "activated oxygen complex." Correlating the decrease in binding of CO, the loss of demethylase activity and ligand binding (metopirone, measured at 446 nm) suggested that this compound binds the heme which had been prefer- entially induced by PB. The fact that not all of the ami- nopyrine demethylase activity was lost and/or total in- hibition of CO binding were not obtained, provides 89 evidence that at least one other heme protein is present in this microsomal mixed-function oxidase system. Murphy, 9:53;. ,62 have shown the appearance of more than one P-450 fraction upon subfractionation of rat liver microsomes by use of rate-zonal centrifugation. PB and 3-MC induction were found to have a very distinctive effect on the distribution of cytochrome P-450 within the micro- somal vesicles. The altered distribution observed with 3-MC induction was inhibited by pretreating the animals with thioactamide and ethionine. These results suggest that the P-450 produced after treatment with 3-MC is pre- ferentially formed in, or bound to a vesicle distinct from that which contains the P-450 in the normal or phenobarbi- tal-induced animal. By making the assumption that the extent of P-450 lost in the CO binding experiment paralleled the increase in the metopirone binding spectrum at 446 nm, an extinction coefficient for metopirone-P-450 was calculated to be 146 cm'1 mMIl. Previous studies on the induction of P-450 by phe- nobarbital and 3-methylcholanthrene have shown that the absorbance maximum for P-450 in a CO-difference spectrum depended upon which inducer was used.63 Control micro- somes had an absorbance maximum at 449 nm, which could be shifted to 448 nm upon induction with 3-MC or to 450 nm with PE induction. Investigators therefore argued for the 90 existence of two microsomal cytochromes; one induced by 3-MC which absorbed at 448 nm and another absorbing at 450 nm which was induced by PB. If metopirone binds to the P-450 induced by PB such that it cannot bind CO, the P-450 remaining should be cy- tochrome P-448. Therefore, this inclusion of metopirone in both the sample and reference cell should cancel out all P-450 and leave P-448 to obtain a CO-difference spectrum at 448 nm. However, the maximum occurred at 451 nm. Esta- brook, 9.2.11.- ,64 reported that this maximum occurred at 454 nm. The CO-difference spectrum of 3-MC microsomes in the presence of metopirone showed a slight decrease in absorbance without any shift in the maximum. Estabrook proposed the existence of a P-448 - P-454 interconvertible cytochrome form based upon their reaction with CO. They had concluded that metopirone changed the equilibrium es- tablished between the two functionally different forms of cytochrome P-450. They further had shown that a decrease in one form appears as an "inhibition" of aminopyrine or hexobarbitol metabolism, while the concomitant increase of the other form paralleled the "stimulation" in the ring hydroxylation of acetanalide. Solubilized P-420 also gave a difference spectrum with metopirone, however the maximum occurred at 416 nm. Increasing the pH of a suspension of whole microsomes from 7.0 to 9.0 resulted in the loss of P-450 with a concomitant increase in P-420. The same type of result was obtained 91 with metopirone difference spectra. The peak at 446 nm was lost with the appearance of a peak at 418 nm. There- fore, just as P-420 is the breakdown product of P-450 with CO-difference spectra, P-418 is the breakdown product of P-446 with metopirone difference spectra. Unlike P-450, however, the presence of metopirone caused a shift in the CO-difference spectrum of solubilized P-420 to a maximum at 416 nm. The mechanism of inhibition of aminopyrine demethyl- ase by DDT appears to be different than the mechanism by which metopirone and dieldrin inhibit. No spectral evi- dence for binding was obtained with DDT nor did DDT affect the binding of CO by P-450. When KCN was used as an in- hibitor for aminopyrine demethylase activity, a pattern very similar to DDT inhibition was obtained. Total activ- ity was divided into three components. Although KCN inhi- bition generally involves cytochromes, because of the ex- cessive amounts of KCN needed to obtain this particular inhibition (4mM) and the fact that no difference spectrum was obtained,18 the assumption was made that KCN inhibition was not due to the binding to the heme group. Therefore, the inhibition might involve the electron transport sys- tem. However, no reversal of DDT inhibition by excessive levels of NADPH was obtained; either DDT bound irreversibly or this was not the mechanism by which DDT inhibited. DDT had no effect on cytochrome C or nitroblue tetrazolium re- ductase indicating that neither of these reductases were '5' 15!. . - T' 92 involved in the microsomal mixed-function oxidase system or that the point of inhibition by DDT occurred at a later step in the electron transport system. It has been known that both NADPH and NADH are needed for maximal aminopyrine demethylase activityzg'65 however, the mechanism is not known. NADH does not support demethylase activity alone or in the presence of NADPT in- dicating no transhydrogenase activity. In addition to the reduction of P-450 by NADPH, NADH will also reduce this cytochrome but at a slower rate. Both reactions are bi- phasic. NADH had no effect on the rate of reduction of P-450 by NADPH. This biphasic reduction of P-450 has been reported by other workers.51 Both rates were found to be dependent upon pH and glycerol concentration. Usually the faster reduction rate is too fast to accurately measure, however, this rate could be observed by the inclusion of 25% glycerol in the reaction mixture. DDT had only a slight effect on the reduction of P-450 by NADPH, however, DDT stimulated the fast reduction of P-450 by NADH at least three-fold. Since DDT acted as an inhibitor of aminopyrine demethylase, the effect seen in these reductase experiments did not indicate the mechanism of DDT inhibition. However, it was found that NADH could reverse DDT inhibition of the moderately DDT-sensitive aminopyrine demethylase activity. The component which was highly sen- sitive to DDT, metopirone and dieldrin inhibition was not 93 substantially affected. This therefore, led to the belief that DDT inhibited the two activities by two separate me- chanisms. The two activities must also have different electron transport systems. It would appear that the two components seen in Lineweaver-Burk plots of aminopyrine demethylase activity in control microsomes are made up of the two enzymes as postulated earlier.29 However, the low Km component does not seem to be a single activity, induced by PK. Two low Km activities seem to exist, and one is induced by pheno- barbital. The high Km activity (induced by 3-MC) does not seem to be primarily responsible for the demethylation of aminopyrine but is capable of doing so at high substrate concentrations. The low Km activity which is induced by PB is also highly sensitive to DDT, metopirone, and dieldrin inhibi- tion. The inhibition by both metopirone and dieldrin is presumably a mechanism of P-450 heme binding preventing the formation of the "active oxygen complex." The mecha- nism of DDT inhibition of this component is not yet under- stood, however, the DDT inhibition of the other low Km com— ponent seems to involve the electron transport system. It is therefore believed that these aminopyrine demethylase activities, in addition to involving a multiplicity in P-450, have multiple reductases, perhaps one reductase system for each activity seen. 94 TABLE l.--A summary of the properties of the aminopyrine demethylase activities in rat liver microsomes. Property Component of Aminopyrine Demethylase* l 2 3 Km 5x10‘4M 4-2x10’4l‘l 1.3x10'2M Induction by: PB ? 3-MC Stimulated by NADH - + — DDT inhibition + + - Dieldrin inhibition + - — Metopirone inhibition + - - KCN inhibition + + _ P-450 Involved 450 ? 448 _L *The three components correspond to: (1) highly DDT sensitive; (2) moderately sensitive to DDT; (3) DDT insensitive (see Figure 12) 95 Scheme for Microsomal Mixed-function Oxidase 4T-“"FAD -x k£///’ red cyt.C P- 450 (ox. ) NADPH P- H450 (red. ) p-4so (red.) P- -450 02 (red.) (1) Possibility for 3 P-450's. (2) NADH involved with only one P-450. 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