Sulfoxide reductase activity of liver aldehyde oxidase

86

Bwchtmwa et Bwphyswa Acta, 747 (1983) 86-92 Elsewer

BBA 31690

SULFOXIDE REDUCTASE ACTIVITY OF LIVER ALDEHYDE OXIDASE K TATSUMI, S KITAMURA and H YAMADA

Institute of Pharmaceutwal Sciences, Htroshtma UnwersJty, School of Medwme, 1-2-3, Kasumt, Mmamt-ku, Htroshtma 734 (Japan) (Recewed March 22nd, 1983)

Key words Aldehyde oxtdase, SulfoxMe reductase, Xanthme oxldase, Electron transfer, (Rabbit hver, Buttermdk)

The present study provides evidence that guinea pig and rabbit liver aldehyde oxidase (EC 1.2.3.1) in the presence of its electron donors such as aldehydes or N-heterocyclic compounds functions as a sulfoxide reductase towards sulindac and other sulfoxide compounds. In addition, the study shows that a combination of liver aldehyde oxidase and milk xanthine oxidase also exhibits suffoxide reductase activity in the presence of xanthine, an electron donor of xanthine oxidase. Based on these facts, we propose a new electron-transfer system consisting of these two flavoenzymes.

Introduction

Sulfoxtde compounds are easily reduced to the corresponding sulfides in ammal bodies [1-7]. Such sulfoxade reduction is responsible for activation, inactivation and alteration of action of sulfoxlde compounds; e.g., the pharmacological activmes of sulindac ( czs-5-fluoro-2-methyl-l-[p-(methylsulfinyl)benzylidenyl]indene-3-acetic acid), one of the nonsteroidal anti-inflammatory agents, are attributable to the reduction product, sulindac sulfide (5-fluoro-2-methyl- 1-[ p-(methylthio)benzyhdenyl] mdene-3-acetic acid) [8]. However, httle has been known about the nature of mammalian sulfoxtde reductase. Recently, Anders et al. [9,10] suggested the involvement of thioredoxln m the sulfoxade reduction of suhndac by cytosohc enzyme(s) of rat liver and ktdney. Independently, we demonstrated that suhndac reductase acuvaty occurs m guinea pig hver cytosol, and that some flavoenzymes such as rmlk xanthane oxadase and hver rmcrosomal NADPH-cytochrome c reductase supplemented with their electron donors exhibit the reductase activity m the presence of 'soluble factor' in guinea pig liver cytosol [ 11,12] In our prehmlnary communication [13], more 0167-4838/83/$03 00 © 1983 Elsevier Soence Pubhshers B V

recently, we provided the evidence for the reduction of suhndac by gumea pig and rabbit hver aldehyde oxadase. We wish to describe here m detad the involvement of liver aldehyde oxidase m sulfoxlde reduction. Materials and Methods

Chemicals. NADPH, NADH, FAD, methyl vlologen, dlcoumarol, allopurinol, qulnacrlne dihydrochlonde, fl-estradiol, chlorpromazine hydrochloride and alumina gel C ~, were purchased from Sigma Chemical Co. Proplonaldehyde, benzaldehyde, menadlone, potassium cyanide, p-chloromercuribenzoic acid, sodium arsemte, dlthlothreltol and xantlune were obtained from Nakaral Chemicals, Ltd. 2-Hydroxypyrimadme hydrochloride, N~-methylnicotmarmde chloride, butyraldehyde, phenothtazme, diphenyl sulfoxlde, diphenyl sulfide, dlphenyl sulfone, dibenzyl sulfoxlde, dlbenzyl sulfide, L-metbaomne, L-methaonlne sulfoxade and amidol were obtained from Tokyo Chermcal Industry Co, Ltd. (+)-Biotin was purchased from Wako Pure Chermcal Industry, acetaldehyde from Merck Co, bowne serum alburton (Fraction V from bovine plasma) from Amour Pharmaceutical Co, calcmm phosphate gel

87 from Blo-Rad Laboratones and DEAE-cellulose (DE-52) from Whatman Ltd., respectively L[methyl-3H]Methionme (200 mC1/mmol) was purchased from New England Nuclear Corp. Suhndac and suhndac sulfide were kandly donated by Merck Sharp and Dohm Research Laboratories. Phenothiazlne sulfoxade was synthesized by oxidation of phenothiazlne with hydrogen peroxide according to the method of Beckett et al. [14]. (+)-Biotin (+)-sulfoxade methyl ester was synthesized from (+)-biotin methyl ester, which was prepared by methylatlon of (+)-biotin with diazomethane, according to the method of Melwlle [ 15]. L-[methyl- 3H]Methiomne sulfoxlde (3 3 m C i / mmol) was synthesized by oxadatlon of L-[methyl3H]methlonlne with hydrogen peroxade according to the method of Sysak et al. [16]. Its radlochenucal purity was ascertained to be over 99.9% by thin-layer chromatography. Enzymes Buttermilk xanthine oxadase (× 4500 chromatographically purified) and bowne liver catalase were purchased from Sigma Chemical Co. Male albino rabbits weighing 2.0-2.5 kg and male guinea pigs weighing 350-400 g were used for the following enzyme preparation. Rabbit hver aldehyde oxldase was purified according to the method of Rajagopalan et al. [17]. Subcellular fractions of guinea pig livers were prepared as follows: animals were killed by decapitation and their hvers were removed The tissue was homogenized m 4 volumes of 1 15% KC1, the homogenate was centrifuged for 20 nun at 9000 × g, and the 9000 × g supernatant fraction was centrifuged for 60 nun at 105000 × g. The mlcrosomal fraction was washed by resuspenslon in the KC1 solution and resedlmentatlon for 60 rnin at 105000 × g The 105000 × g supernatant (cytosol) was subjected to ammonium sulfate fractionation, and proteins wtuch were precipitated between 0 and 30%, 30 and 45%, and 45 and 60% ammomum sulfate saturation were collected, respectively. Assays of sulfoxtde reductase acttvtty A typical incubation mixture consisted of 0 4 #mol of a sulfoxade compound, 2/~mol of an electron donor and liver preparation in a final volume of 2.5 ml of 0.1 M phosphate buffer (pH 7.4). Anaerobic incubation was performed using a Thunberg tube. The side arm contained the substrate and the body

contained all other components. The tube was gassed for 3 min with nitrogen, which was passed through a deoxygemzang solution consisting of 0.5% sodium dlthionite and 0.05% of the sodium salt of 2-anthraquinonesulfonlc acid in 0.4% NaOH, evacuated with an aspirator for 5 mln and again gassed with nitrogen. The reacUon was started by mixang the components of the side arm and the body together, and continued for 30 nun at 37°C. In aerobic experiments, the incubation was performed m an open vessel. In the reduction of suhndac, the incubation mixture was extracted twice w~th 2 volumes of ethyl acetate, and the combined extract was evaporated to dryness in vacuo. The residue was then apphed to a slhca gel plate (Kleselge160F-254, Merck) and developed with CHC13/ethyl acetate/ acetic acid (16 : 5 : 1, v/v). The areas correspondlng to sulindac (R F 0.31) and suhndac sulfide (R F 0.61) were scraped and eluted separately with 3 ml of 28% ammonia water/methanol (1.49, v/v). The amount of each compound was deternuned with a Hitachi 320 spectrophotometer at 327 nm (the absorption maximum of suhndac) and 344 nm (the absorption maximum of suhndac sulfide), respectwely. In the reduction of diphenyl sulfoxtde, dibenzyl sulfoxade or (+)-biotin (+)-sulfoxide methyl ester, the incubation nuxture was twice extracted with 2 volumes of ethyl acetate and the combined extract was evaporated to dryness in vacuo. The residue was then apphed to a Hitachi 163 gas chromatograph equipped with a hydrogen flame-ionization detector. The instrument was fitted with a 1 m × 3 mm i.d. glass column packed with 1.5% OV-17 on Chromosorb W. The operatlon conditions were as follows: injection port and detector temperature, 180°C for diphenyl sulfide (&phenyl sulfoxide), 210°C for dibenzyl sulfide (&benzyl sulfoxide) and 290°C for (+)-biotin methyl ester (( + )-biotin (+)-sulforade methyl ester); column temperature, 150°C for dlphenyl sulfide (dlphenyl sulfoxide), 180°C for dibenzyl sulfide (&benzyl sulfoxlde) and 240°C for (+)biotin methyl ester (+)-biotin (+)-sulfoxlde methyl ester); internal standard, dlbenzyl sulfide for &phenyl sulfide (diphenyl sulfoxtde), &phenyl sulfone for chbenzyl sulfide (dibenzyl sulfoxlde) and fl-estra&ol for (+)-biotin methyl ester (( + )biotin ( + )-sulfoxide methyl ester); flow rate of N 2

88 career gas, 20 m l / m l n . Retentmn ume: dlphenyl sulfide 2.8 nun (diphenyl sulforade 15 1 mm), dlbenzyl sulfide 2.6 min (dibenzyl sulfoxlde 12.2 mln) and (+)-biotin methyl ester 5.9 nun ( ( + ) biotin (+)-sulfoxide methyl ester 21.5 nun). In the reductmn of phenotluazine sulfoxtde, the incubation nuxture was twice extracted with 2 volumes of ethyl acetate and the combined extract was evaporated to dryness m vacuo. The residue was then apphed to a Toyo Soda HLC-803A lugh-pressure hqmd chromatograph eqmpped with a UV-8 ultraviolet absorpuon detector. The instrument was fitted with a 30 cm × 4 mm 0 d ) LS-410K reversed-phase column (Toyo Soda). The mobile phase was 0.1 M K H z P O 4 / C H 3 C N (1"1, v/v). The chromatograph was operated at a flow rate of 1 m l / m i n at ambient temperature and at a wavelength of 210 nm Elution ume: phenothiazane 24 min (phenothiazane sulfoxade 5 nun). In the case of L-metluonine sulfoxtde, the mcubaUon nuxture consisted of 0.4 /zmol of its 3H-labelled compound, 8 /~mol of acetaldehyde and rabbst liver aldehyde oxldase (1 unit) m a final volume of 0.8 ml of 0.1 M phosphate buffer (pH 7.4). The incubation was carried out for 30 nun at 37°C using a Thunberg tube under anaerobic conditmns as described above. After incubation, 2 volumes of ethanol were added to the mixture and then the mixture was centrifuged for 10 nun at 20000 rpm. The supernatant (100 #1) was apphed to a cellulose plate (DC-Alufohen Cellulose F254 0.1 mm, Merck) and developed with b u t a n o l / a c e t o n e / w a t e r / p y r l d l n e (10" 10" 5 : 2, v / v ) The areas corresponding to L-methlonme sulfoxlde (R F 0.08) and L-mettuonme (Rf 0.37) were scraped and the radloacuvlty of these samples was determined on a Packard hquld scmtdlatlon spectrometer (Model 3255) with automatic external standardization. Assay of aldehyde oxMase The assay was performed according to the method of the rabbit hver enzyme assay which was described by Felsted et al. [18], measurmg the increase m absorbance at 300 nm wluch accompames the oradatmn of N i methylmcotlnanude to the 2- and 4-pyrldones. Assay of xanthme oxMase. The assay was performed by the method of Nakamura and Yamazalo [19], measunng the increase m absorbance at 292 nm which accompames the oxadatmn of xanttune to uric aod.

Determmatwn of protem Protein was determined by the method of Lowry et al. [20] w~th bovine serum albumin as a standard or by measuring absorbance at 280 nm DEAE-cellulose column chromatography. The protein that precipitated between 30 and 45% ammomum sulfate saturation from guinea pig hver cytosol (equivalent to 10 g of hver) was collected, redlssolved m 5 ml of 10 mM phosphate buffer (pH 7.4) containing 1 mM dlthmthreltol and &alyzed against 1 liter of the same phosphate buffer for 12 h. The dialyzed solution was adsorbed on a column (1.5 × 12 cm) of DE-52 wluch was eqmhbrated with the phosphate buffer Elutlon was carried out by a hnear gradient method of mcreasmg NaC1 concentration m the phosphate buffer up to 0.2 M concentration. Fractions (5 ml) were collected at flow rate of 25 m l / h . Results

Guinea pig hver 9000 × g supematant alone extublted sulfoxide reductase actwlty towards suhndac, especially under anaerobic conditions. The reductase actwlty was slgmficantly inhibited by mena&one, an mlubltor of aldehyde oxldase, but not by aUopunnol, that of xanthlne oxldase On the other hand, the reductase actwlty was strikingly stimulated by acetaldehyde, whereas N A D P H or N A D H showed httle sUmulatory effect on the reductase activity. These facts strongly suggested that aldehyde oradase m the hver preparation is revolved m the reduction of suhndac When the hver 9000 × g supernatant was separated to cytosohc and nucrosomal fractions, the acetaldehyde-linked activity occurred m the former fracUon, but not in the latter. Furthermore, when the hver cytosol was fracuonated with ammomum sulfate as described m Materials and Methods, a protem with the acetaldehyde-hnked acuwty was exclusively precipitated between 30 and 45% ammonmm sulfate saturation. The ablhty of the preopltate to reduce suhndac was again examined in the presence of aldehydes, N-heterocychc compounds or reduced pyndme nucleoUdes under anaerobic conditions As a result, the preop~tate showed a slgmficant sulfoxlde reductase activity in the presence of propionaldehyde, butyraldehyde, benzaldehyde, N l-methylmcotlnanude or 2-hy-

89 TABLE I E F F E C T OF V A R I O U S C H E M I C A L S ON S U L F O X I D E R E D U C T A S E A N D A L D E H Y D E O X I D A S E ACTIVITIES OF A M M O N I U M S U L F A T E P R E C I P I T A T E (30-45%) F R O M G U I N E A PIG LIVER CYTOSOL In the assay sulfoxlde reductase, the incubation nuxture consisted of 0 4 #mol of suhndac, 2 / t m o l of acetaldehyde, an mlubltor and 0 5 ml of the a m m o n m m sulfate precipitate (eqmvalent to 0 08 g of hver) m a final volume of 2 5 ml of 0 1 M phosphate buffer (pH 7 4) The incubation was earned out under anaerobic c o n d m o n s Each value represents the mean of four experiments Activity ~s expressed as percent of control Ad&tlon

Concentratmn (M)

Sulfoxade reductase actlwty

Aldehyde oxldase acUv~ty

N o n e (control) Menachone Chlorpromazme Anudol Potassmm cyamde S o d m m arsemte p-Chloromercunbenzoic acid Qumocnne Dlcoumarol Allopurmol

-5 2 2 25 1

10- s 10- 4 10- 4 10 - 4 10 - 4

100 2 22 3 6 5

100 0 19 0 9 18

1 • 10 - 4 1 10 - 4 1 10- 5 2 10 - 4

3 41 66 93

9 37 67 100

droxypyrm'ndme m varying degrees, but not m the presence of N A D P H or NADH. These aldehydes and N-heterocychc compounds as well as acetaldehyde are known as electron donors of aldehyde oxadase, winch ts one of the liver cytosohc enzymes. Boiling the ammonium sulfate precipitate prior to incubation completely abohshed ~ts abihty to reduce sulindac. Next, the comparative abdlty of some chemicals to minint the aldehyde oxidase and sulfoxide reductase activities of the 30-45% ammomum sulfate precipitate was exarmned. As shown m Table I, both acttvltles were stmtlarly susceptible to mhlbttlon by all of these chemtcals except allopunnol Furthermore, the ammonium sulfate precipitate was chromatographed on a DEAE-cellulose column. As shown in F~g. 1, the peak postuon of aldehyde oxldase free of xanthme oxtdase was entirely tdentical with that of sulfoxlde reductase. These facts led us to conclude that gumea pig hver aldehyde oxadase m the presence of tts electron donor can catalyze the reduction of suhndac to suhndac sulfide To confirm tins conclusion, we deternuned whether rabbit hver aldehyde oxldase shows sulfoxade reductase activity or not. In the present

study, the enzyme was purified from rabbit ltver according to the method of Rajagopalan et al. [17]. As shown m Table II, the purified enzyme exInblted a s~gmficant suhndac reductase acUwty m the presence of acetaldehyde In addttion, the enzyme can also catalyze the reduction of some sulfoxlde compounds besides suhndac m varying

o0z o

,

_

.,

Q02~E/~

I0

ZO

uJ

0 =L// =

~

o-

30 40 Fret,on number

F18 I Chromatography of sulfoxlde reduetase (O), aldehyde ox~dase (e) and xantlune oxJdase (&) m ammomum sulfate precipitate (30-45%) from guinea pig hver cytosol on a DEAE-CeIIuIos¢ column In the assay of su|foxlde reduetas¢, the incubation rmxture conslste..d of 0 4 /~mol of sullndac, 2 pmol of acetaldehyde and 0 5 ml of each fracUon m a final volume of 2 5 ml of 0 I M phosphate buffer (pH ? 4) The incubation was camcd out under anaerob=c condlUons

90

degrees The sulindac reductase activity of the enzyme was markedly inhibited by menadlone (data not shown), like the guinea pig hver enzyme described above. On the other hand, N t-methylnlcotmamide and 2-hydroxypyrinudine as well as acetaldehyde were effective as electron donors for suhndac reduction (data not shown). Previously, we demonstrated that flavoenzymes such as xanttune oxadase and NADPH-cytochrome c reductase supplemented with their electron donors showed sulfoxlde reductase activaty m the presence of the 30-45% ammonium sulfate precipitate from guinea pig liver cytosol, which was temporarily designated'soluble factor' [ 11,12]. In the present study, the ammonium sulfate precipitate or xantbane oxldase by itself showed no sulfoxtde reductase activity in the presence of xanthane, an electron donor of xantbane oxadase However, when the ammonmm sulfate preopltate was combined with xantlune oxadase, a sigmficant sulindac reductase activity (33 nmol/30 rmn) was observed in the presence of the electron donor. The ammonium sulfate preopltate was subjected to DEAE-cellulose column chromatography as described above. The elutIon peak of the soluble

REDUCTION OF SULFOXIDE COMPOUNDS BY RABBIT LIVER ALDEHYDE OXIDASE The incubation nuxture consisted of 0 4 /tmol of a sulfoxade compound except for L-metluonlne sulfoxade, 2 #mol of acetaldehyde, 0 1 unit of aldehyde oradase (0 3 mg of protein), 3 mg of bowne serum albunun and 30 ~tg of catalase m a final volume of 2 5 ml of 0 1 M phosphate buffer (pH 7 4) In the case of L-metluomne sulfoxade, the incubation nuxture conslsted of 0 4 #moi of its 3H-labelled compound, 8 #mol of acetaldehyde and 1 umt of aldehyde oxldase (3 mg of protein) m a final volume of 0 8 ml of 0 1 M phosphate buffer (pH 7 4) The mcubatmn was carried out under anaerobic conditions Each value represents the mean of four experiments

Sulmdac Phenotluaune sulfoxade Dlphenyl sulfoxtde Dzbenzyl sulfoxade ( + )-Bmtm ( + )-sulfoxlde methyl ester L-Metluomne sulfoxade

~

03

o

c

~oI

~

~

/

.0 Z

004>,

o~

o o3 ..:.__-.

0~I

o01

10

20

30 40 Fr~mctton number

Fig 2 Chromatography of nulk xanthme oxldase-llnked sulfoxade reductase (O), aldehyde oxadase (o) and xanthane oxadase (A) in ammonmm sulfate precipitate (30-45%) from guinea pig hver cytosol on a DEAE-cellulose column In the assay of nulk xanthlne oxldase-hnked sulfoxlde reductase, the mcubatmn nuxture consisted of 0 4 #tool of suhndac, 2 #mol of xantlune, 1 ml of each fraction and 0 5 unit of nulk xanthme oxldase in a fmal volume of 2 5 ml of 0 1 M phosphate buffer (pH 7 4) The mcubatlon was camed out under anaerobsc conditions

factor, which was assayed by its capacity to support sulfoxade reduction by xanthine oxldase supplemented with xanthme, occurred at a 0.125 M TABLE III SULFOXIDE REDUCTASE ACTIVITY OF A COMBINATION OF RABBIT LIVER ALDEHYDE OXIDASE AND MILK XANTHINE OXIDASE

TABLE II

Compound

0,,

Sulfoxade reductase acuwty (nmol/30 nun per mg protein) 715 754 629 290 79 8

The mcubaUon rmxture conszsted of 0 4 #mol of suhndac, 2 pmol of xantbane, 0 2 umt of aldehyde oxidase (0 6 mg of protein), l umt of xanthane oxadase, 0 25 /tmol of FAD or methyl wologen, 3 mg of boxane serum albumin and 30 #g of catalase m a final volume of 2 5 ml of 0 1 M phosphate buffer (pH 7 4) The incubation was camed out under anaerobic con&tlons Each value represents the mean of four experiments Enzyme

Aldehyde oradase (I) Xanthme oxtdase (II)

I plus II

Addmon

Sulforade reductase actlwty (nmol/30 nun)

Xantlune

0

Xantlune Xanthme, FAD Xanthme, methyl xaologen Xantlune Xanttune, FAD Xantlune, methyl wologen None

0 0 0 149 317 1549 0

91

concentration of NaC1. This peak position was identical with that of aldehyde oxtdase, winch was separated from xanthine oxidase (Fig. 2). This fact indicates that guinea pig liver aldehyde oxidase functions not only as sulfoxade reductase m the presence of its electron donor as described above, but also as the soluble factor which can cooperate with xanthine oxadase in sulfoxade reduction. Supporting this view, the purified rabbit liver aldehyde oxldase described above, like guinea pig liver enzyme, showed no sulfoxlde reductase activity in the presence of xanthlne, but when the enzyme was combined with xanthlne oxadase, a significant sulfoxlde reductase activity (149 nmol/30 nun) occurred. The activity was stimulated by FAD or methyl xaologen up to 317 or 1549 nmol/30 min, respectively (Table III).

molybdenum-contaimng flavoenzymes and the mechanisms for their catalytic reactions seem to be very similar to each other [21]. In drug metabolism, for example, both enzymes function as nltroreductases towards some orgamc nitro compounds [22-25]. However, the present study indicated that there is an apparent difference between the abilities of both enzymes to reduce sulfoxide compounds. In addition, our recent work [26,27] showed that guinea pig and rabbit hver aldehyde oxidase in the presence of its electron donor can also catalyze the reduction of N-nitrosodlphenylarmne and some cychc nitrosarmnes to the corresponding hydrazine derivatives, but not xanthine oxadase. These facts suggest that aldehyde oxldase and xanthine oxldase have different specificity patterns for each other to reduce substrates.

Discussion Acknowledgement The present study provides evidence that hver aldehyde oxidase is involved m sulfoxlde reduction in two ways; (1) the enzyme by itself exhibits sulfoxide reductase activity in the presence of its own electron donor, and (2) the combination of the enzyme of xanthine oradase also extubits the activity m the presence of xanthlne, an electron donor of xanthine oxldase. Based on these facts, we propose here a new electron-transfer system consisting of aldehyde oxldase and xantlune oxtdase as shown m Scheme I. In the system, FAD or methyl v~ologen seems to function as an electron carrier between these two flavoenzymes Both aldehyde oxidase and xanthine oxldase are

Electron donor of

e

Xanthlne oxtdase

xanthme oxadase

Aldehyde oradase

References i Dlstefano, V and Borgstedt, H H (1964) Sctence 144, 1137-1138 2 Kolb, K H , Jamcke, G , Kramer, M., Schulze, P M and Raspe, G (1965) Arznelm Forsch 15, 1292-1295 3 Mestu, T , Yoslukawa, M and Sato, Y (1970) Blochem Pharmacol 19, 1351-1361

\ /

Electron donor of

We are grateful to Miss. M. Hayashl for her skilled technical assistance. This work is supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education of Japan, which is acknowledged.

FAD or methyl vtologen

e

~ Sulfoxlde compound

aldehyde oxldase Scheme I Involvement of aldehyde oradase in sulforade reducuon

92 4 Duggan, D E , Hare, L E, Dltzler, C A , Lel, BW and Kwan, K C (1977) Chn. Pharmacol Ther 21,326-335 5 Duggan, D E, Hook, K F and Hwang, S S (1980) Drug Metab Dlspos 8, 241-246 (1980) 6 Mazel, P, Katzen, J, Skolmck, P and Shargel, L (1969) Fed Proc 28, 546 7 DeBaun, J R and Menn, J J (1976) Science 191, 187-188 8 Duggan, D E, Hooke, K F, Rasley, E A, Shen, T Y and Van Arman, C G (1977)J Pharmacol Exp Ther 201, 8-13 9 Anders, M W, Ratnayake, J H, Hanna, P E and Fuchs, J A (1980)Blochem Blophys Res Commun 97, 846-851 10 Anders, M W, Ratnayake, J H, Hanna, P E and Fuchs, J A (1981) Drug Metab Dlspos 9, 307-310 11 Katamura, S, Tatsuml, K and Yoshamura, H (1980) J Pharm Dyn 3, 290-298 12 Katamura, S, Tatsuml, K, Hlrata, Y and Yoshamura, H (1981) J Pharm Dyn 4, 528-533 13 Tatsuml, K, Katamura, S and Yamada, H (1982) Chem Pharm Bull (Tokyo) 30, 4585-4588 14 Beckett, A H, A1-Sarraj, S and Esslen, E E (1975) Xenoblotlca 5, 325-355 15 Melvdle, D B (1954)J Blol Chem 208, 495-501

16 Sysak, P K Foote, C S and Chang, T-Y (1977) Photochem Photoblol 26, 19-27 17 Rajagopalan, K V , Fndoxach, I and Handler, P (1962) J Bxol Chem 237, 922-928 18 Felsted, R L, Chu, A E -Y and Chaykln, S (1973) J Blol Chem 248, 2580-2587 19 Nakamura, M and Yamazakt, I (1982) J Blochem 92, 1279-1286 20 Lowry, O H, Rosebrough, N J, Farr, A L and Randall, R L (1951) J Biol Chem 193, 265-275 21 Bray, RC (1975) m The Enzymes, 3rd Edn (Boyer, P D , ed ), pp 299-419, Acadermc Press, New York 22 Wolpert, M K, Althaus, J R and Johns, D G (1973) J Pharmacol Exp Ther 185, 202-213 23 Taylor, J D , Paul, H E and Paul, M F (1951) J Blol Chem 191,223-231 24 Wang, C Y, Behrens, B C, Ichakawa, M and Bryan, G T (1974) Blochem Pharmacoi 23, 3395-3404 25 Tatsuml, K, Katamura, S and Yoshamura, H (1976) Arch Blochem Blophys 175, 131-137 26 Tatsuml, K, Yamada, H and Katamura, S (1983) Chem Pharm Bull (Tokyo) 31,764-767 27 Tatsuml, K, Yamada, H and Katamura, S (1983) Arch Blochem B~ophys m the press