262

Btochtmica et Biophy~h'a Acla, lel~l (1991)262-266 © 1991ElsevierSciencePublishers B.V (BiomedicalDivision)000S-27S0/91/$03.50 ADONIS f100527609100081E

Role o:~ the side chain of lanosterol in substratc recognition and catalytic activity of lanosterol 14a-demethylase

(cytochrome P-45014DM) of yeast Y u r l A o y a m a ~, Y u z o Y o s h i d a 1 Y o s h i k o S o n o d a 2 a n d Y o s h i h i r o S a t e 2 ! FacuRy of Pharmaceutical Sciences. Mukogawa Women's University, Nrshinomiya and " Kyoritsu Collage of Phar~cy. Mmato-ku. Tukyo (Japan)

(Received lS June 1990) Key words: Cytochrome P-450subslrate recognition; Lanosterol side chain; Dihydrolanosterol; Lanostero114wdemethylase; Lanosterol derivative;(Yeast mierosome) The 14~-demethylatlon af 24,2~-dihydrolanosterol (DHL) derivatives having trimmed side chains, 27-nor-DHL 26,27.diner-DHL, 25,?~27-trinor-DHL, 24,25,26,27.tetranor.DHL, 23,24,25,26,27-pentanor-DHL and 22,23,24,25,26, 27-hexanor-DHL, was studied with the reconstituted lanosterol 14a-demethylase system consisting of eytochrome P-45014DM and NADPH-eytochcame P-450 reduetase both purified from yeast microsomes. The demethylase catalyzed the 14n-demethylation of the derivatives having the side chains longer than tetranor but the activities Io¢ the trinor- and tetranor.derivatives were lower. Kinetic analysis indicated that affinity of the trinor-derivative tot the demethylaso, was considerably higher than that of DIEL. The affinities of the 27-nor- anti dlnor-derivatives were increased by this order and were the intermediates of DHL and the trinor derivative. On the other hand, Vm~ values of the demethylnse for the DHL derivatives were decreased depending on their side-chain lengths, and the substrate.dependent reduction rate of cytochrome P-45014[, M was also decreased in the same manner. Based on these observations, it was condeded that interaction of the side chain of lannsterol especially C-25, 26 and 27 with the substrate site of lanosterol 14a-demethyl. ase was necessary for enhancing the catalytic activity of the enzyme. However, this interaction was considered not to be essential for substrate binding. Intreduetinn kanosterol 14a-demethylase (cytochrome P-4501aDM) [1,2] is a species o1 cytochrome P-450 which catalyzes the oxidative removal of the 14-methyl group (C-32) of lanostero[ (lanosta-8,24-dien-3fl-ol) and 24,25-dihydrolanosterol (lanost-g-en-3~-ol, DHL). This enzyme is known to occur in yeast [1-3] and mammalian liver [4] and is considered to be one o1 the key enzymes of sterol biosynthesis by these organisms because the 14a-demethylation is the first step of sterol biosynthesis from lanosterol. Although cytochrome P-45014DM has been

Abbreviations: DHL, 24,25-dihydrolanosterol;dinor-DHL, 26,23-dinor-DHL; trinor-DHL, 25,26,27-trinor-DHL; tetranor-DHL, 24.25,26,27-tetranor-DHL; pentanor-DHL, 23,24,25,26.27-pentanorDHL; hexanor-DHL, 22,23,24,25,26,27.hexanor-DHL; GC-MS, gas chromatography-mas~ spectrometry; TMS, trimethylsilylresida~ Cnrrespondence: y, Yoshida. Faculty of Pharmaceutical Sciences, Mukogawa Women's University, 11-68 Koshien-Kyuban-cho, Nishinorfdya663,Japan.

purified from rat liver [4], extensive studies on its molecular and catalytic properties have been made on the purified preparation from yeast ( S a c c h a r o m y c e s cerevisiae) [1-3, 5-12], In the recent years, we have made a series of investigation to elucidate the necessary structure of substrate for substrate recognition and catalytic activity of lanosterol 14a-demethylase of yeast. In the preceding papers [7.8], we revealed that the S-double bond and the 3-hydloxyl group were essential for substrate recognition by the enzyme. Base on these findings, we assumed that the substrate site of lanosterol 14a-demethylase had a contact with the ,~-surfaca of the substrates by recognizing the 8-1anostene conformation, and the 3-hydroxyl group was essential for orienting the substrates to the correct direction by a hydrogen bond formation with an amino acid residue in the substrate site. Recently, Fischer et al. [13] reported that the 8-lanostene structure was essential for substrate recog. nition by rat liver microsomal lanosterol 14a-demethylase too, suggesting that our assumption was applicable to the mammalian enzyme. Lanosterol 14~-demethylas¢ showed higher affinity

263

a

a

4

a 6

T

Fig. 1 Structural formula of DHL derivatives used in this work. I. DHL; 2. 27-nor-DHL: 3, dinor-DHL: 4. tfinor-DHL: 5, tetranorDHL: 6. pentanor-DHL; 7. hexanor-DHL

for lanosterol than D H L [12,13]. It was also reported that some D H L derivatives having trimmed side chains showed lower affinity for the partially purified rat liver enzyme [14]. T h e ~ facts suggested that the side chain might play some role in substrate recognition and catalytic activity of lanosterol 14a-demethylase. To obtain more detailed information for the role of the side chain in substrate recognition, we examined metabolism of several D H L derivatives having trimmed side-chains (Fig. 1) by the reconstituted lanosterol 14a-demethylase system consisting of eytochrome P-45014DM and NADPH-cytoehrome P--450 reduetase both purified from yeast microsomes [2]. Results indicated the terminal part of the side chain consisting of C-24, 25, 26 and 27 was essential for the catalytic activity of lanosterol 14a-demethylase. Materials and Methods Cytochrome P-450t.,r~ M was purified from semianaerobically grown cells of S. cerevisiae by the method of Yoshida and Aoyama [1]. NADPH-cytochrome P-450 reductase were purified from the same material according to the method of Aoyama et al. [15] with the following modifications. The dialyzed crude reductase preparation after ammonium sulfate fractionation [15] was subjected directly to a DE-52 column chromatography [15]. The reductase containing eluate from the DE-52 column was concentrated and the medium was replaced with 10 mM potassium phosphate buffer (pH 7.5), containing I mM EDTA, 0.5% sodium cholate and 1 p,M each of F A D and F M N in an Amicon ultrafihrator. The concentrated reductase was adsorbed on a column of 2'5'-ADP-Sepharose 4B equilibrated with 10 m M potassium phosphate buffer (pH 7.5), containing l m M EDTA and 0.5% sodium eholate. The column was washed with the equilibration buffer containing l .aM F A D and 1 .aM F M N and the reductase was elated out of the column with 1 mM N A D P in the washing buffer. The reductase preparation thus obtained was electrophoreticaly homogenous and stored as the purified preparation. Lanosterol and D H L were isolated from a commercially obtained mixture as described previously [16]. The

D H L derivatives shown in Fig. 1 were gynthesized by the published methods [17]. Other chemicals and hipchemicals were guaranteed reagents obtained from com~ilercial sources. Lanosterol 14~-demethylasc activity of the reconstituted system consisting of ¢ytochrome P-45014DM and NADPH-cytochrome P-450 reductase was assayed according to the previous method [2] with the following reaction mixture (1.0 ml): 0.06 nmol eytochrome P45014ora, 1.0 unit NADPH-cytochrome P-450 reductase, 0.15 mM N A D P H , l 0 mM glucose 6-phosphate, 0.l unit glucose-6-phosphate dehydrogenase, 8 to l 0 nmol sterols dispersed with 25 p g dilauroytphosphatidylcholine and 0.1 M potassium phosphate buffer (pH 7.5). Reaction was carried out at 3 0 ° C for 5 to 20 min and sterols were analyzed with gas-liquid chromatography as described previously [2,51 except that OV-I7 fused silica capillary column (0.32 m m × 30 m) was employed. All D H L derivatives and their 14-demethylated products could be determined by this method as described in Results and Discussion (see Fig, 2), The demethylase activity was calculated from gas chromatographically-determined convertion ratio of a substrate to the corresponding product and the initial amount of the substmte [2]. The rate of cytoehrome P-4501aDM reduction in the reconstituted system was assayed by the previous method [2]. The reaction mixture (2.0 ml) was contained 0.34 nmol eytochrome P45014txx1, 1.5 unit NADPH-eytochrome P-450 reductase, 0.15 m M N A D P H . 16 to 20 nmol sterol substrates dispersed with 50 ~g dilauroylphosphatidylehollne, glucose-glucose oxidase-eatala~ deoxygenizing system [2] and 0.1 M potassium phosphate buffer (pH 7.5). Reduction of cytochrome P-459~4ma was followed spectrophotometrically at 30 ° C under carbon monoxide [2], Results and

Discussion

Identification of the products formed from the DHL dericatioes by the incubation with the reconstituted lanosterol 14a-demethylase system A series of D H L derivatives having the trimmed side chains (Fig. 1) were incubated with the reconstituted lanosterol 14a-dcmethylase system as described in Materials and Methods and the sterols were analy~,ed with gas chromatography (Fig. 2) and G C - MS (Table 1) after trimethylsilylation. As shown in Fig. 2, gas chromatographically-detectable products (peaks P of chromatograms 1 through to 5) were formed from 27-norD H L (peak S of chromatogram 2). dinor-DHL (peak S of chromatogram 3), trinor-DHL (peak S of chromatogram 4) and tetranor-DHL (peak S of chromatogram 5) as well us from D I l L (peak S of chromatogram 1). A trace product was formed also from pentanor-DHL but no detectable product was formed from hexanor-DHL (not shown). Table I summarizes the mass spectra of the

264 products from D H L , 27-nor-DHL, dinor-DHL, tr~norD H L and tetranor-DHL, together with those of their parent compounds (substrates) with respect to M + and the three characteristic fragments; M + - C H 3, M + TMSOH and M * - TMSOH - C H 3. Molecular weights of all products assumed from the m / z values of M + were decreased by 16 from those of the corresponding substrates, indicating that one carbon and four hydrogens were removed from the substrates by the metabolism. The mass spectra also showed that intensity of M ~ relative to M + - CH~ was significantly increased in all products to compare with their substrates, and M + - T M S O H was detectable only in the products. The product formed from D H L by the reconstitwed demethylasc had been identified as its 32-nor-14-unsaturated derivati~,e I5] and the above-mentloned characteristic differences observed between the mass spectra of D H L and the product were due to the absence of the 14a-methyl group in the product [2,5,12]. Consequently, the mass spectra shown in Table I indicated that all of the products were formed by the 14a-demethylation which was accompanied by introduction of the 14-double bond as in the case of DHL. It can thus be concluded that yeast lanosterol 14a-demethylase could mediate the 14a-demethylation of 27-nor-DHL, dinorDHL. trinor-DHL and tetranor-DHL with the same manner as that for its normal substrates, D H L and lanosterol.

14a-Demethylating activity of the reconstituted system for the D H L derioatives The 14¢~-demethylating activity of the reconstituted lanosterol 14a-demethylase for the D H L derivatives was dependent on their side-chain length. The activities

for 2%nor-DHL and dinor-DHL were comparable to that for DHL, but the activities for trinor-DHL and tetranor-DHL were markedly decreased and no activity was observed on pentanor-DHL and hexanor-DHL (Table 11). As descrived previously [2,5-7,11,16], significant enzymatic reduction of cytochrome P-45014DMwas observed only in the presence of the substrates, the intermediates or certain substrate analogues. The substrate-dependent reduction rates observed in the presence of 27-nor-DHL and dinor-DHL were comparable to that observed in the presence of D H L . but the rate was decreased by further trimming of the side chain (Table II). This effect of the side-chai n length on the reduction rate was comparable to that on the demetbylase activity. These observations indicated that the sidechain length of the D H L derivatives modified their efficiency as substrates for lanosterol 14ot-demethylase.

Effect of the side-chain on the affinity for the demethylase K m of the reconstituted demethylase for D H L was about 3-times higher than that for lanosterol (Table Ill). This higher K m was lowered by the removal of the terminal part of the side chain as shown by the K= values of 27-nor-DHU dinor-DHL and trinor-DHL (Table III). These three D H L derivatives inhibited the D H L demethylation by 40 to 50~ (Fig. 3) as lanosterol did [6], while D H L inhibited the 14a-demethylation of these D H L derivatives only by 10 to 20 (data not shown). The inhibitory effects of D H L derivatives having side chains shorter than tetranor were lower, but more than 10% of inhibition was still observed on hexanor-DHL (Fig. 3). These lines of evidence indicated that the most part of the side chain was not essential for the substrate binding but the terminal part of the side

TABLE1

Mas.~spevtra af DHL, it.~derwatweshaving trinmwdside chains and the productsformed from them by the recenJtituled lanastero114.clemethylase The trimethylsilyiatedsterols separated by gas chromatograph (peaks S and P of Fig. 2) here analyzedwith a JEOL LMS-DX363integrated gas chromatograph-mass speclromelerequipped with a JMA DAh000data praetor. Ionization ener~ was 70 eV. Subslrate DttL

S P

M+ ,n/: 500 484

27-nor-DHL

S P

486 470

16.1 71.4

471 455

30.1 10.2

380

S P

472 456

18.0 67.1

459 441

31.6 9.5

366

S P

458 442

16.3 57.2

a~.3 427

273 8.0

352

S P

444 428

22.3 63.0

429 413

31.3 8.9

338

Dinor-DHL "1rinor-DHL Tetranor-DHl,

Peak ~'

q~ 13.1 59.3

M' -CH3 m/z 485 469

q~ 21.8 7.3

M* - TMSOH m/: % n.d. h 394 24.8

s and P arc corresponded to peaks S and P in Fig. 2. b These fragmentswere not detectable.

n.d. b

M+ - C H a - TM$OH m/z 385 100 379 100

35.1

381 365

100 IIKI

30,9

367 351

100 1oo

25.8

353 337

100 100

27.0

339 323

t(30 100

n.d. ~ n.d. b n.d. n

265 TABLE II The demerh~ia¢e actwJty and the nzte of sub.rtraw-dependem P-450tj~ f reduction tlf the reconstituted fan~terol 14a.demethylme for DHL and DH L d¢'rl~allt~ having trimmed std¢ chmns

S

S 1 s

DHL 27-nor-DHL Dinor-DHL Trinor-DHL Telranor-DHL Pen~nor-DHL Hexanor-DHL None '~

Demethylase activity

P-45O~eduction

Inmol/min per nmol P450) 5.04 4.96 3.'70 0.98 O24 0 0

(%)

(rain"t )

(%1

100 98.4 73.,1 19.4 4.8 0 O

21 17 20 ~0 5.1 2.4 L2 0.1~6

100 81 98 48 24 ll 5.7 0.3

Only dilauroylphosphatidyleholine micelles were added.

lanoslerol t)y r e d u c i n g t h e steric h i n d r a n c e by the rem o v a l o f o n e o r t w o t e r m i n a l m e t h y l g r o u p s (Table I l l ) . I n a d d i t i o n to such sterie factor, s o m e c o n t r i b u t i o n of or-electrons of t h e 24-double b o n d m a y be considered. H o w e v e r . t h e c o n t r i b u t i o n of the ¢r-electrons seems to be insignificant, because affimties of t h e e n z y m e for the D H L derivatives h a v i n g t r i m m e d side c h a i n s w i t h n o w-electron were r a t h e r h i g h e r t h a n t h a t f o r lanosterol (Table I l l ) .

,t~

I

i

Time after Injection

{ mln I

Fig. 2. Gas chromatographic detection of reaction products from the DHL delivatives having trimmed side chains. DHL derivatives shown in Fig. 1 were incubated with the r~onslituted lanosterol 14a-demelhylase system as described in Materials and Methods. Sterolg cxlraCtod from each reaction mixture were Ifimethylsilylated and separated with an OV-17 fused silica capillary column {a.32 mm x 31) m) el 2550C. Traces 1, 2, 3, 4 and 5 represent chromatograms of stetols from the reaction mLxtures using D H L 27-nor-DHL, dinorDHL, fd1)or-DHL and lelranor-DHL, respectively, as substrates. S and P denote the sterol peaks identified ~ substrate and product, r~1),:ctively.

c h a i n consisting o f C-24, 25, 26 a n d 27 critically affected the affinity of t h e s u h s t r a t e for t h e demethylase. C o n f o r m a t i o n of t h e t e r m i n a l p a r t of t h e side c h a i n c o n s i t i n g o f C-24, 25, 26 a n d 27 o f lanosterol is flat d u e to t h e 24-double b o n d , while t h a t o f D H L is n o t flat a n d m o r e bulky. Accordingly, if t h e local c o n f o r m a t i o n of t h e substrate site w h i c h c o n t a c t s w i t h this p a r t is fitted for t h e flat c o n f o r m a t i o n o f lanosterol, affinity o f D H L m u s t b e r e d u c e d by steric h i n d r a n c e . T h i s possibility is s u p p o r t e d b y *.he fact t h a t t h e h i g h e r K m o f D H L w a s l o w e r e d to t h e c o m p a r a b l e value t o t h a t o f

Effect o f the side chain on the catalytic activity of the demetl~vlase V ~ . values of t h e reconstituted d e m e t h y l a s e for lanosterol a n d D H L w e r e essentially identical (Table I l l ) . H o w e v e r , Vm~ values for t h e D H L - d e r i v a t i v e s h a v i n g t r i m m e d side c h a i n s were considerably lower t h a n t h o s e for D H L a n d lanosterol (Table I l l ) a n d t h e d e m e t h y l a s e s h o w e d n o activity for t h e derivatives having e x t r e m e l y s h o r t side c h a i n s (Table It). F u r t h e r m o r e , the s u b s t r a t e - d e p e n d e n t e n z y m a t i c r e d u c t i o n rate of c y t o c h r o m e P-450~aD~4 w a s also lower for t h e substrates h a v i n g t r i m m e d side c h a i n s (Table II). T h u s , t h e intera c t i o n of t h e side c h a i n especially C-25, 26 a n d 27 with t h e s u b s t r a t e site seems to be necessary for t h e active

TABLE Ill gmerw puramet¢~v of the r~on~tituted laaostt,rol 14a ~temetln'l~ for Dill. DilL d,~ivala,es having srimmed .fide chains and lanr~sl¢~L Kinetic paramelers were determined by the double recip~al plols Sulxstrale

K~ UtM)

V=~ (nmol/min per nmol P-450)

Lanosterol DHL 27-nor-DHL Dinor-DHL Tlinor-DHL

6.25 16.7 5.88 4.00 2.22

16.0 16.5 I 1.1 6.90

1.92

266

' l l l l 5 6

i I

;0

°'*

Relative

Activity

i0

°'~ ,;*

Finally, it m u s t b e p o i n t e d o u t t h a t the partially p u r l ; l e d r a t liver lanosterol 1 4 a - d e m e t h y l a s e s h o w e d lower affinity for 2 7 - n o r - D H L a n d d i n o r - D H L t h a n for D H L , a l t h o u g h t h e Vm. , values f o r these c o m p o u n d s were n o t significantly d i f f e r e n t f r o m t h a t for D H L [14]. This f i n d i n g seems to suggest t h a t in the r a t liver e n z y m e t h e t e r m i n a l p a r t of sterol side c h a i n play a n i m p o r t a n t role in substrate b i n d i n g of t h e e n z y m e a n d its c o n t r i b u t i o n to t h e catalytic t u r n o v e r of the e n z y m e is n o t so significant. I f this is t h e case, t h e m o d e o f r e c o g n i t i o n of t h e lanosterol side c h a i n b y the r a t liver e n z y m e is c o n s i d e r e d t o b e d i f f e r e n t f r o m t h a t by t h e yeast e n z y m e . T h i s is a n i n t e r e s t i n g p r o b l e m b u t f u r t h e r studies w i t h h i g h l y purified rat liver c y t o e h r o m e P 450141)~a a n d m o r e r e f i n e d r e c o n s t i t u t e d system m u s t be n e e d t o r e a c h t h e conclusion.

(~ )

Fig. 3 Inhibition of DHL ]4wdemethyinhon by DHL derivatives having trimmed side chains. DHL 14a-flemethylase activity of the reconstituted lanosterol 14a-demethylase was assayed as de~ibed in Materials and Methods with 7.8 ~M DHL as the substrale in Ihe presence of the following DHL derivatives as inhibitors. 1, no inhibitor; 2, 27-nor-DHL (9.0 gM); 3. dinor-DHL (8.6 pM); 4, tllnor-DHL (8.0/zM); 5, tetranor-DHL (10,0 /xM); 6, pentanor-EIHL (10.4/tM); 7. hexanor-DHL (11.0 /tM). The values in parenthesis were the concemrations of the inhibitors detenmned gas chromatographically after extraction from the unincubated reaction mixtures. catalytic t u r n o v e r o f t h e demethylase. Since t h e I/mas value (Table 11I) a n d t h e s u b s t r a t e - d e p e n d e n t r e d u c t i o n rate [2] for lanosterol were n o t substantially d i f f e r e n t f r o m t h o s e for D H L (Tables I I a n d 111 a n d Refs. 5 a n d 6), t h e 24-double b o n d o r t h e c o n f o r m a t i o n of t h e t e r m i n a l p a r t of t h e side c h a i n m a y n o t affect t h e catalytic t u r n o v e r of t h e e n z y m e .

General consideration T a k e n all together, it is c o n c l u d e d t h a t the side c h a i n of lanosterol interacts w i t h t h e substrate site of yeast lanosterol 14c~-demethylase especially by its t e r m i n a l p a r t consisting or C-25, 26 a n d 27. T h i s i n t e r a c t i o n e n h a n c e s t h e catalytic t u r n o v e r of t h e e n z y m e , w h i l e this i n t e r a c t i o n m a y n o t b e essential for s u h s t r a t e b i n d ing. T h e local c o n f o r m a t i o n o f t h e e n z y m e r e s p o n s i b l e for this i n t e r a c t i o n m a y fit for t h e flat c o n f o r m a t i o n of t h e t e r m i n a l p a r t of the lanosterol side c h a i n c o n s i s t i n g of C-24, 25, 26 a n d 27 a n d affinity of D H L m a y h e reduced by steric h i n d r a n c e d u e to t h e n o n - f i a t a n d m o r e bulky c o n f o r m a t i o n of its side c h a i n t e r m i n a l .

References 1 Yoshida, Y. and Aoyama, Y. (1984) J. Biol. Chem. 259.1655-1660, 2 Aoyama, Y., Yoshida, Y. and Sato, R. (1984) J. Biol. Chem. 259, 1661-1666. 3 Aoyama. Y.. Oklkawa. T. and Yoshlda. Y. (1981) Biochim. Biephys Acta. 665, 596-60l. 4 Tzzaskos~ J.. Kawala, S. and Gayly-, J.L. (1986) J. Biol. Chem. 261,14651-14657. 5 Aoyama, Y., Yoshida, Y., Sonoda, Y. and Salo. Y. (1987) J. Biol. Chem. 262,1239-1243. 6 Aoyama, y., Yoshida. y., Sonoda, Y. and Sato, 3(. (1989) J. Biol. Chem. 264. 18502 18505. 7 Aoyama, Y., Yoshida, Y., Sonoda. Y. and Sato. Y. (1989) Biechim. Biophys. Acla 1001.1%-20q. 8 Aoyama, Y. Yoslada, y.. Sonoda, Y. and Sato, Y. 0989) Bi~ chlm. Biophys. Acla 1006, 209-213. 9 Ishida, N . A0yama, Y.. Hatanaka. IL, Oyaraa. Y.. Imaj0, S.. lshiguro, M., Oshima, "L, Nnhazalo. H., No$uchi, T., Maitra, U.S.. Mohaa. V.P., Sprinson, D.B. and Yoshida~ Y. (1988) Biochem. Biophys. R¢~. Commun. 155, 31"/-323. 1O Kalb, V.F, Woods, C.W., Tuff. T.G., Dey. CR.. Suuer. T.R. and Loper. J.C, (1987) DNA 6. 529-537. 11 Aoyama, Y, ~ d Y~hida. Y. (19781 Biochem, aiophys. Res. Conunun. 82, 33-38. 12 Aoyama, y. and Yoshida. Y. (1978) Biochem, Biophys. Res. Commun, 85, 28-34. 13 Fischer. R.T., Stare, S,H., Johnson, P,R.. Ko, S.S.. Magolda, R.L.. Gaylor, &L. and Trzaskos, J.M. (1989l J. Lipid Res, 30.1621-1632. 14 Sonoda, y.. SekiRawa, y. and Sato. Y. (1989) Chem. Pharm. Bull. 37, 718-'722 15 Aoyama. Y., Yoshida, Y., Kubota, S., Kumaoka, H. and Furumiehi. A. (1978l Arch. Biochem. Biophys. 185, 362-369. 16 Aoyama, Y., Yoshida, Y., Sonoda~ y. ann Sat(:*. Y. (1987) nieclaim. Biophys. Acta 922. 270 277. 17 Sato, Y. and Sonoda, y. (1981) Chem. Phaxm. Bull. 29. 356-365.