Characterization of the purified microsomal FAD-containing monooxygenase from mouse and pig liver

Chem.-Biol. Interactions, 51 (1984) 125--139

125

Elsevier Scientific Publishers Ireland Ltd.

C H A R A C T E R I Z A T I O N OF THE P U R I F I E D MICROSOMAL FADCONTAINING MONOOXYGENASE FROM MOUSE AND PIG L I V E R

P A T R I C K J. S A B O U R I N

and E R N E S T H O D G S O N

Interdepartmental Toxicology Program, North Carolina State University Raleigh, NC 27695-7613 (U.S.A.) (Received April 22nd, 1984) (Revision received June 11th, 1984) (Accepted June 11th, 1984)

SUMMARY

The FAD~ontaining monooxygenase (FMO) has been purified from both mouse and pig liver microsomes by similar purification procedures. Characterization of the enzyme from these t w o sources has revealed significant differences in catalytic and immunological properties. The pH optimum of mouse FMO is slightly higher than that of pig FMO (9.2 vs. 8.7) and, while pig FMO is activated 2-fold by n-octylamine, mouse FMO is activated less than 20%. Compounds, including primary, secondary and tertiary amines, sulfides, sulfoxides, thiols, thioureas and mercaptoimidazoles were tested as substrates for both the mouse and pig liver FMO. Kin- and Vmax-values were determined for substrates representative of each of these groups. In general, the mouse FMO had higher Kin-values for all of the amines and disulfides tested. Mouse FMO had Kin-values similar to those of pig FMO for sulfides, mercaptoimidazoles, thioureas, thiobenzamide and cysteamine. Vmax" values for mouse FMO with most substrates was approximately equal, indicating that as with pig FMO, breakdown of the hydroxyflavin is the rate limiting step in the reaction mechanism. Either NADPH or NADH will serve as an electron donor for FMO, however, NADPH is the preferred donor. Pig and mouse FMOs have similar affinity for NADPH (Kin = 0.97 and 1.1 pM, respectively) and for NADH (Km = 48 and 73 ~M, respectively). An antibody, prepared by immunizing rabbits with purified pig liver FMO, reacts with purified pig liver FMO b u t not with mouse liver FMO, indicating structural differences between these two enzymes. This antib o d y inhibited pig FMO activity up to 60%.

Abbreviations: FMO, FAD-containing monooxygenase ; NOA, n-octylamine ; IgG, immune globulin. 0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

126

Key words: Flavin~ontaining monooxygenase -- FAD-containing monooxygenase -- Microsomal monooxygenase -- Mouse liver -- Pig liver INTRODUCTION The microsomal FMO (EC 1.14.13.8) is responsible for the oxidation of m a n y nitrogen and sulfur containing drugs, pesticides and other xenobiotics [1--4]. Studies, using microsomes or whole cell homogenates, indicate that this enzyme is present in most mammalian species [1,5] and exists mainly in the liver, though also in the kidney and lungs [1,6]. The enzyme has been purified from pig [7], rat [8], and mouse [9] liver, however, only the purified pig liver enzyme has been further characterized. Pig liver FMO oxidizes secondary and tertiary, but not primary amines, although the turnover varies considerably among different substrates [1[. Although originally classified as an N,N-dimethylaniline oxidase, sulfur containing compounds were f o u n d to be even better substrates for this enzyme [1,4]. Oxidation of nitrogen or sulfur containing compounds generally leads to the addition of one oxygen atom on the N or S atom, respectively [1]. However, Hajjar and Hodgson [10] have shown that some phosphonodithioates, such as fonofos, can be converted to the corresponding oxon by this enzyme and studies in progress have shown that pig liver FMO can oxidize the phosphorus atom of compounds containing neither sulfur nor nitrogen (B.P. Smyser and E. Hodgson, unpublished data). The capability of this enzyme, as well as the cytochrome P-450 dependent monooxygenase system, to oxidize a broad range of chemicals, drugs and pesticides is important when considering the metabolic detoxification or activation of these compounds. Oxidation by these two monooxygenases may lead to different products [11]. The metabolic fate of compounds may depend on the relative amounts of these two enzymes in a tissue and their relative affinities for the substrate. Extrapolation of the studies with the pig FMO to other mammals, including humans, requires further characterization of this enzyme in other species. The cytochrome P-450-dependent monooxygenase system and its genetic control have been extensively characterized in the mouse. Since this laboratory has purified cytochrome P-450 isozymes from the liver of uninduced mice and has reconstituted active cytochrome P-450-dependent monooxygenase systems [12], we felt that characterization of the mouse liver microsomal FMO for comparison was appropriate. We have purified the FMO from both mouse and pig liver microsomes by similar purification procedures [9]. The characterization of these two enzymes, reported herein, reveals significant differences in kinetic properties and apparent differences in structure. MATERIALS AND METHODS

Chemicals Cyclohexylamine,

t-butylamine,

2-chloro-6-methylaniline,

N-methyl-

127 aniline, N,N
Enzyme preparation Pig livers were obtained immediately after slaughter from the Jesse Jones Sausage Plant, Garner NC. Only livers with high FMO activity were selected for use [7]. Mouse livers were obtained frown 25--35 g female Dub:ICR mice (Dominion Laboratories, Dublin, VA). Microsomes were isolated from mouse or pig liver by differential centrifugation and the FAD-containing monooxygenase was purified from microsomes as previously described [9]. For analysis of kinetic constants for NADPH and NADH, purified mouse liver FMO was dialyzed against 10 mM potassium phosphate, 0.1 mM EDTA (pH 7.5} to remove NADPH remaining from the last purification step.

Assays With all substrates except thiobenzamide, FMO activity was measured by monitoring NADPH oxidation at 340 nm using an Aminco DW-2 double beam spectrophotometer. Both sample and reference cuvettes contained buffer (0.1 M Tricine/KOH, 0.1 mM EDTA (pH 8.1)unless indicated otherwise} and 0.1 mM NADPH. The final volume was 1.0 ml. The enzyme was added to the sample cuvette and the endogenous rate (i.e. the rate in the absence of substrate) of NADPH oxidation by FMO was measured. The substrate (in 10 gl H20 or acetonitrile) was then added to the sample cuvette and the substrate
128 with standard deviations were obtained using the KINFIT program of Knack and R o h m [14]. This program uses curve-fitting techniques to determine the kinetic constants which best fit the data to the Michaelis-Menton equation. Protein was measured by the fluorescamine technique of Bohlen et al.

[15]. Preparation of an tibody Pig liver FMO, purified as described [9], was precipitated with 20% polyethylene glycol [7] and resuspended in 0.9% sodium chloride ('antigen'). Three adult white New Zealand male rabbits were injected at four intradermal sites along the back with a primary dose of 0.8 mg of this antigen emulsified with an equal volume of Freund's complete adjuvant. On days 8, 22 and 50 after the initial injection, 0.8 mg of antigen was again administered, this time in Freund's incomplete adjuvant. Further immunizations were given every 4 weeks and sera were collected 8--12 days later. The immunoglobulin (IgG) fractions obtained from rabbits before and after immunization were purified by ammonium sulfate precipitation followed by DEAE-ceUulose chromatography [16]. The purified fractions are referred to as the pre-immune IgG and anti-pig FMO.

Ouchterlony immunodiffusion Ouchterlony double immunodiffusion [17] was performed as described [6] except that the agarose gels contained 0.2% Emulgen 911 instead of 0.2% sodium deoxycholate and microsomes were solubilized with 3% Emulgen 911 (1 mg Emulgen 911/mg protein). Gels were stained with Coomassie Blue as described [ 16 ]. RESULTS The FAD-containing monooxygenases from mouse and pig liver were purified by similar procedures and their properties were compared. The mouse and pig FMO activities were linear with protein concentration up to the highest amounts tested (16 t~g and 80 t~g protein, respectively) in either potassium phosphate buffer (pH 7.6) or Tricine buffer (pH 8.1) (Fig. 1). In the absence of substrate, the FMO can oxidize NADPH producing NADP ÷ and H20: [18]. This reaction will be referred to as the 'endogenous' NADPH oxidation. Below pH 8.1 the endogenous rate of NADPH oxidation by the mouse FMO is negligible compared to the substrate-dependent rate and was therefore ignored in all calculations. However, pig liver FMO displays significant endogenous oxidation (Fig. 1B). The subtraction of this endogenous rate of NADPH oxidation from the rate in the presence of substrate is n o t necessarily valid [ 19]. Poulsen and Ziegler [ 18] have shown that the oxygenated flavoprotein reacts much faster with N,N-dimethylaniline than it decomposes to give H202. At saturating or near saturating concentrations of substrate the rate of NADPH oxidation is probably due entirely to oxidation of the substrate. For these reasons it is incorrect to

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Fig. 1. Variation in mouse and pig liver FAD-containing monooxygenase activity with protein concentration. Mouse (A) and pig (B) liver FMO activities were measured by monitoring NADPH oxidation as described in Methods, using 2 mM N,N-dimethylaniline as substrate. The reaction was carried out in either 0.1 M Tricine, pH 8.1 (o) or 0.1 M potassium phosphate, pH 7.6 (:). Solid and dashed lines indicate NADPH oxidation in the presence and absence of substrate, respectively. All points are the average of two determinations.

subtract endogenous rates from rates obtained in the presence of substrate in order to calculate the substrate
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Fig. 2. Effect o f pH and n-octylamine on mouse and pig liver FAD-containing monooxygenase activity. Mouse (A,B) and pig (C,D) liver FMO activities were measured by monitoring NADPH oxidation as in Methods using 2 mM N,N-dimethylaniline as substrate. Assays were carried out in either 0.1 M Tricine (w,c) or 0.1 M glycine ( * , : ) adjusted to the designated pH with potassium hydroxide. Reactions shown in B and D contained 3 mM n-octylamine. Open symbols indicate NADPH oxidation in the absence of substrate. Solid symbols indicate NADPH oxidation in the presence of substrate. All points are the average o f two determinations.

presence of substrate for pig and mouse liver FMO, respectively. N-Octylamine activates pig liver FMO about 1.6--2.0-fold but does not stimulate mouse FMO more than 20% at any pH value {Fig. 2 and Table I). A selection of compounds was tested as substrates for both the mouse and pig liver FMO and their apparent Kin- and Vmax-values determined at pH 8.1 {Table I). Poulsen and Ziegler [18] have shown that the Kin-value of the pig liver FMO for oxygen varies with pH and that, at pH > 7.8, the oxygen concentration is rate limiting. The kinetic constants reported herein were determined at pH 8.1 and therefore at a limiting oxygen concentration. As mentioned previously, the subtraction of endogenous NADPH oxidation from total NADPH oxidation in the presence of substrate is not valid, at least not at saturating levels of substrate. However, at rate limiting concentrations of substrate, endogenous NADPH oxidation may be occurring to some extent. Therefore, the apparent kinetic constants for pig liver FMO have been calculated with and without subtracting the endogenous NADPH oxidation. The actual kinetic constants will lie between these two values. Endogenous NADPH oxidation was negligible with mouse liver FMO even at substrate concentrations well below the apparent Kin-value.

131 NADPH oxidation in the presence of methimazole and 2-mercaptobenzimidazole showed an initial 'lag phase' of 5--10 min before maximal velocity was achieved (unpublished observation). This 'lag phase' was prevented by the addition of 1 mM glutathione to the reaction mixture. Kinetic values for methimazole and the mercaptoimidazoles were, therefore, determined in the presence of 1 mM giutathione. Compounds which previously have been shown not to be substrates for the pig FMO [7], primary amines, 45 mM) or thiobenzamide (>0.2 mM) inhibited purified mouse and pig liver FMO. Kinetic constants for these two substrates were determined using levels of substrate below the inhibitory concentration. The rate of formation of thiobenzamide S-oxide by FMO or microsomes from pig liver decreases with time. Even during the first minute, formation of the S-oxide is non-linear. Initial rates were determined by drawing a tangent to the curve at zero time. The conversion of thiobenzamide to the S-oxide by FMO or microsomes from mouse liver was linear for at least several minutes. The Vmax-Value of pig FMO for all substrates should be equal since the rate limiting step in the reaction mechanism is the breakdown of the pseudo base, C(4a)-hydroxyflavin [20]. In the absence of n-octylamine the Vmaxvalues of pig and mouse FMO were approx. 0.45 and 1.50 ~mol/min/mg protein, respectively, for most of the substrates tested. Twelve of the 25 substrates tested, however, had significantly lower Vmax-Values with the pig FMO. In contrast, only 7 of the substrates had significantly lower Vmaxvalues for mouse FMO. The variation in Vmax-Values may be due to the stimulation of FMO by lipophilic substrates b u t not other substrates in the same manner as n-octylamine [1]. Mouse FMO may show less variation in Vmax-values due to the fact that it is n o t susceptible to this kind of stimulation as evidenced by the poor stimulation by n-octylamine {only a 17% stimulation and slight inhibition by 1 mM and 5 mM n-octylamine, respectively). Pig FMO, however, was stimulated 36% and 76% by 1 mM and 5 mM n-octylamine, respectively.

34 N 6

D i m e t h y l sulfide Dimethyl sulfoxide Thioanisole

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N

N

7 74 284 N

27 1670 c 1650

Imipramine Benzphetamine Ethylmorphine

343 11 19 29 44

N

1060 105 148 120 144

N-Methylaniline N,N-Dimethylaniline + 1 mM NOA + 5 mM NOA N,N-Diethylaniline

N N N N N 617 1090

Pig

Benzonitrile 4-Dimethylaminoantipyrine

2340 2890

Nb N N N N

Mouse

K m (~M)

Trimethylamine Triethylamine

Aniline Cyclohexylamine t-Butylamine 2-Chloro-6-methylaniline 1-Naphthylamine

Compound

14 N 4

N

N

9 105 413

521 15 28 46 61

884 1840

N N N N N

Piguncoupled a NADPH oxidation

1.37 N 1.46

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1.39 1.45 1.70 1.37 1.39

1.38 0.85

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0.39 N 0.33

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Piguncoupled a

Assays were carried o u t b y m e a s u r i n g N A D P H o x i d a t i o n as d e s c r i b e d in M e t h o d s , using 1.7 ug a n d 8.8 ug o f m o u s e a n d pig liver FMO, respectively. Where i n d i c a t e d , 1 m M or 5 m M n - o c t y l a m i n e ( N O A ) was i n c l u d e d in t h e assay m i x t u r e . K i n e t i c c o n s t a n t s were d e t e r m i n e d as described in M e t h o d s . T h e s t a n d a r d d e v i a t i o n s o f these values were always less t h a n 10% o f t h e value r e p o r t e d .

K I N E T I C C O N S T A N T S O F M O U S E A N D PIG L I V E R F A D - C O N T A I N I N G M O N O O X Y G E N A S E

TABLE I

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> 800c

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8 0.97 48

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16

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78 108 296

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NA NA

NA f

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> 800 c

21

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1.42

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1.35

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0.98

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1.56 0.22 c N

0.26 0.29

0.34

0.40 0.45 0.45 0.34

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0.46

0.35

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0.38 0.46 N

NA NA

NA

0.39 0.45 0.37 0.29

0.37 c _

0.38

0.28 0.35 0.14

0.45

0.32

0.13

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a T h e rate of N A D P H o x i d a t i o n in t h e a b s e n c e o f s u b s t r a t e ( e n d o g e n o u s ) was s u b t r a c t e d f r o m t h e rate in t h e p r e s e n c e o f s u b s t r a t e b e f o r e d e t e r m i n i n g a p p a r e n t Kin- a n d Vmax-values. bN, n o d e t e c t a b l e rate at a c o n c e n t r a t i o n o f 2 m M . ':Kinetic analysis carried o u t w i t h t h e s u b s t r a t e c o n c e n t r a t i o n m u c h less t h a n t h e Kin-value d u e t o p o o r s o l u b i l i t y . T h e r e f o r e , kinetic c o n s t a n t s s h o w n are o n l y e s t i m a t e s a n d are s u b j e c t t o large ( 5 0 - - 1 0 0 % ) error. --, i n d i c a t e s t h a t a l t h o u g h t h e c o m p o u n d c a u s e d s o m e N A D P H o x i d a t i o n , kinetic c o n s t a n t s c o u l d n o t be d e t e r m i n e d d u e t o p o o r s o l u b i l i t y . d K i n e t i c analysis carried o u t in p r e s e n c e of 1 m M g l u t a t h i o n e . CMonitor t h i o b e n z a m i d e S-oxide f o r m a t i o n in t h e p r e s e n c e o f 0.1 m M t h i o b e n z a m i d e . fNA, n o t applicable.

NADPH e NADH e

Thiobenzamide e

Thioacetamide Thiourea Phenylthiocarbamide Thiocarbanilide

Methimazole d 2-Mercaptobenzimidazole d 2-Methylmercaptobenzimidazoled

262 330 847

65

Cysteamine

Bu t a n e t h i o l Benzyl mercaptan Dithiothreitol

1310

> 50 c

2 51 c N

4,5-diol

trans-o-Dithiane-

B e n z y l disulfide

B e n z y i m e t h y l sulfide D i p h e n y l sulfide Diphenyl sulfoxide

134 Apparent Vmax-values in some cases may be low due to depletion of oxygen in the assay mixture by the substrate. In this regard, note that sulfhydryl containing compounds such as butanethiol, benzyl mercaptan and dithiothreitol give lower Vmax-values than dimethylaniline. Glutathione inhibition of FMO may also be due to oxygen depletion. Due to the very low Km-value for NADPH, this value could n o t be determined by measuring NADPH oxidation. At NADPH concentrations near the Kin-value, initial rates could not be determined due to the rapid depletion of the NADPH. In the presence of an NADPH-regenerating system, a constant NADPH concentration can be maintained throughout the reaction and the conversion of thiobenzamide to thiobenzamide S-oxide can be measured spectrophotometrically [13]. The Km-value for NADPH is similar for both mouse and pig FMO, as is the Km-value for NADH. NADPH, however, is the preferred cofactor for both of these enzymes. Vmax-Values were the same with either cofactor. No thiobenzamide oxidation was observed in the absence of NADPH or NADH. Ouchterlony double immunodiffusion analysis gave a single band indicating cross-reactivity between purified pig liver FMO and the anti-pig FMO (Fig. 3). A single immunoprecipitin band was also obtained with solubilized pig liver microsomes. Lack of spur formation indicates that these two bands are due to the same antigen. No bands were observed in any of the experiments with pre-immune IgG. Purified mouse liver FMO reacted very weakly with the antibody. A very faint band appeared between the mouse liver FMO and anti-pig FMO but this band was n o t equivalent to the major precipitin band formed between pig liver FMO and the

6

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4

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4

Fig. 3. Ouchterlony double diffusion analysis of mouse and pig liver FAD-containing monooxygenase. Ouchterlony gels w e r e p r e p a r e d as d e s c r i b e d in Methods. The c e n t e r well contained 20 ~l (72 ~g) anti-pig FMO (A) or 20 ~l (31 ~g) pre-immune IgG (B). Sample wells contained 20 ~g pig FMO (1,5), 510 ~g solubilized pig microsomes (2), 510 ~g solubilized mouse microsomes (3), 20 ~g mouse FMO (4) or Buffer (6).

135 antibody. This light band was also seen with the pig liver FMO and may indicate a c o m m o n , but weak, antigenic determinant. Anti-pig FMO inhibited oxidation of thiobenzamide (Fig. 4B) and N,Ndimethylaniline (unpublished observation) by pig liver FMO. Anti-pig FMO was pre-incubated with the enzyme, in a cuvette containing the complete assay mixture (minus substrate) for 5 min at 37°C (Fig. 4, solid lines). Thiobenzamide was then added to the cuvette and the formation of thiobenzamide S-oxide measured spectrophotometrically. Complete inhibition by increasing amounts of the antibody could not be attained. Increasing the time of preincubation of the antibody with the enzyme to 20 min had no effect on these results. Maximum inhibition of the purified pig liver FMO was 60% with thiobenzamide and 70% with N,N-dimethylaniline as substrate. Anti-pig FMO inhibition of thiobenzamide oxidation by pig liver microsomes reached a plateau at 30% inhibition. The loss of FMO activity in the presence of anti-pig FMO may be due to either direct blockage of the

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mq IqG Fig. 4. Inhibitory effect of anti-pig liver FAD-containing monooxygenase antibody on mouse or pig liver FAD-containing monooxygenase activity. Microsomes (re,D)or purified FMO (o,o) from mouse (A) or pig (B) liver were preincubated with either the antipig FMO antibody (solid symbols, solid lines) or pre-immune IgG (open symbols, dashed lines) in the thiobenzamide assay reaction mixture (see Methods) for 5 min at 37°C. The antibody or pre-immune IgG was added to both the sample and reference cuvettes. After preincubation, 0.1 mM thiobenzamide was added to the sample cuvette and formation of thiobenzamide S-oxide measured as in Methods. All points are the average of two determinations.

136 active site by the antibody or due to formation of multiple antibody-FMO complexes which eventually form a precipitate. Since 100% inhibition could not be achieved, the latter possibility is favored. Purified mouse liver FMO was only slightly inhibited by the anti-pig FMO reaching a m a x i m u m at approx. 20% inhibition (Fig. 4A), however, the antibody had no effect on mouse liver microsomal thiobenzamide oxidation. Preincubation of either pig or mouse liver FMO or microsomal preparations with pre-immune IgG had no significant effect on thiobenzamide oxidation (Fig. 4, dashed lines). DISCUSSION The results presented herein clearly establish the presence of species differences in the FAD-containing monooxygenase. Mouse liver FMO has a slightly higher pH o p t i m u m than pig liver FMO. Under identical assay conditions the affinity of the mouse and pig liver FMOs for various substrates are different. Mouse FMO has lower Kin-values for some substrates, such as thiourea and methimazole. The Kin-values of mouse FMO for all of the nitrogen containing substrates tested, however, is as much as 10 times higher than that of the pig FMO. Although pig FMO is stimulated approx. 2-fold by n-octylamine, mouse FMO is stimulated <20%. This may indicate that a 'regulatory' site, present in pig FMO, is absent in mouse FMO. n-Octylamine activates pig liver FMO by increasing the breakdown of the C(4a)-hydroxyflavin, the rate limiting step in the reaction [20] and also inhibits reduction of the flavin by NADPH [21]. However, even when inhibited, the reductive step is 10 times faster than the rate limiting step of the reaction. Possibly, in mouse liver FMO the rate of flavin reduction is closer to that of the rate limiting step (hydroxyflavin breakdown) and thus the inhibitory role of n-octylamine on reduction might then affect the overall rate. There are also differences in the structure of the mouse and pig liver FMO as determined immunologically. An antibody to the pig liver FMO has been used for identification of the FMO in other species [5]. The lack of cross-reactivity between pig and mouse liver FMO illustrates that some caution is necessary when these types of studies are carried out. The rate limiting step in the FMO reaction mechanism is either the attack of the nucleophile by the C(4a)hydroperoxyflavin or the breakdown of the pseudobase, C(4a)-hydroxyflavin [20]. Most of the substrates tested in this study, despite differences in nucleophilicity, give similar Vmax-values. Therefore, the breakdown of the C(4a)-hydroxyflavin is more likely to be the rate-limiting step in both mouse and pig liver FMO. Sulfoxides of thiobenzamide, thioacetamide, dimethyl sulfide and some other sulfur containing compounds can be further oxidized to the sulfone [3,21--25]. This reaction can be mediated by either the c y t o c h r o m e P-450dependent monooxygenase system [25,26] or the FMO [3,21,22,24].

137 Oxidation of thiobenzamide sulfoxide in rat liver microsomes is inhibited 70% by methimazole, a competitive inhibitor of FMO, but not by cytochrome P-450 monooxygenase inhibitors, such as SKF-525A and N-octylimidazole [22]. Thioacetamide S-oxide oxidation, however, is inhibited by SKF-525A and metyrapone [25], indicating that the c y t o c h r o m e P-450dependent monooxygenase system is at least partially responsible for this oxidation. The Kin-value of the liver FMO for these sulfoxides is usually much higher than the Kin-value for the parent c o m p o u n d , as seen with the two sulfoxides tested in this study. Diphenyl sulfoxide and dimethyl sulfoxide elicited no detectable NADPH oxidation at a concentration of 2 raM. Poulsen [3] reported a Kin-value of 90 mM for dimethyl sulfoxide using pig liver FMO. Other sulfoxides, such as thiobenzamide sulfoxide ( K m = 0.25 mM for pig liver FMO), have much lower Kin-values [3], which are however, still higher than the Kin-value of the parent compound, (i.e. thiobenzamide has K m = 8 and 3 gM for mouse and pig liver FMO, respectively). Before characterization of the FAD-containing monooxygenase, the NADPH-dependent oxidation of compounds by microsomes was generally attributed to the c y t o c h r o m e P-450
Paper No. 9171 of the J o u m a l Series of the North Carolina Agricultural Research Service, Raleigh, NC. This investigation was supported, in part, by Grants ES-00044 and ES-07046 from the National Institute of Environmental Health Sciences, U.S. Public Health Services.

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