Catalytic properties of cytochrome P-450scc from bovine and porcine adrenocortical mitochondria: Effect of tween20 concentration

Z Ste~id .Biaehem. Molee. BioL Yd. 3~ No. ~ pp. ~7-731, I~I

0960-07~]91 $3.00+ 0.00 Copyright © l~l Pergamon Pressple

P~im~ ~ Great Bdtain. All ~ t s r e s e ~

CATALYT~ PROPER~ OF CYTOCHROME P~5~ FROM BOVINE AND PORCINE ADRENOCORTICAL MITOCHOND~A: EFFECT OF TWEEN20 CONCENTRA~ON KAZ~O

~ A X A S ~ , i* MOTON~I T S O ~ , ~

AKIgA M ~ A ~ : SmOETOSm MWRA, ~Y~m Hoso~w~ a~ Y~m~ Icm~w^ ~ iDfpartme~ ~ ~ h o l l 2 i g ~ 2g~a~h ~u~mem ~n~r ~ d ~Departm~t ~ Ne~opsyc~at~, ~wa M~ ~ h o ~ K ~ w a 761-07 ~d 4Ba~ Resea~h ~ m t o ~ , ~meji I n s t ~ ~ Tec~ology, ~ m ~ ~ogo ~1-22, Ja~n (Received 10 September 1990)

Snmmary--Cho~cr~ side~hain c~avagc acfifitics of cytochrome P-450ssc purified ~om bo~nc adrenccortic~ mitochondda were measured for various substra~ including cho~stcrol, 20[S]-hydroxycholesterol, 22[R~hydroxycho~s~r~ and 2~R~ 2~R~dihydroxycholes~rol, in the ~consfitu~d enzyme system at various Twcen20 concentration~ The ~de~h~n ~cavage activity for cholcsmr~ showed rno~ than 10-fo~ enhancement upon addition of ~ 1% Tween2~ compared with that without the detergent. Ad~tion of Tween20 ~d not cau~ any enhancexncnt of the sid~ch~n cleavage activities for 2~S]-hydroxycholesmr~ and 22[R]-hydroxycholesterol; ratheL R resul~d in an inhibition of the acfififies. The fide-ch~n c~avage actifity for 20[R],22[R]-dihydroxycholesterol showed a very high value even without the dc~rgcnt. As the sfiraulatory cffc~ of Tween20 was only specific for cholesterol, Twecn20 ~emed to enhance the rate of access of choles~rol to cytochrome P-450scc. The~ results are consis~nt with the suggestion that a Wans~r of subs~a~, cho~s~rol, in mitochonddal inner membrang to the subs~ate-binding site of cytochrome P-450scc ~ the ram~imifing step in the ch~es~r~ fidc-ch~n cleavage ~acfio~

INTRODUCTION The c h o ~ e r o l ~ d ~ c h ~ n c~avage enzymatic sys~m h common to a v a r i f y of steroidog~n~ tissues (adren~ c o ~ e ~ ovar~ ~stis and placenta), and Rs product, pregnenolone, ~ the common precu~or of various steroid hormones. Fu~hermorc, the conversion of cho~smrol into pregnenolone [1] ~ a rate-~mifing step in the s ~ r o i d o g e n e ~ and control of the rate of th~ reaction ~ m o ~ impo~ant for an acute regulation of ~eroid hormone ~v~s. This cho~smrol ~ d ~ c h ~ n cleavage system c o n ~ s of three component: an iron-sulfur protein (adrenodoxin); an NADPH-speofic flavoprotdn containing a finOe F A D (adrenodown reductase); and a heme b~ontaining hemoprotein (cytochrome P-450sc~. 3mol each of mo~cular oxygen and N A D P H are required for the conver~on of cho~s~rol to pregnenolone [2]. Hydroxylations occur scquen*To whom correspondence shoed be addressed. ~Present address: Basic Reseamh Laborato~, Himeji ~sfit~e of Technology.

t i ~ at the 22[R] and 20[S] positions ~ ~eld, 22[R]-hydroxy and 20[R],2~R]-dihydroxycholcs~rol, ~spcctiv¢l~ b o t h intermediates b ~ n g tightly bound to the enzyme during the ~quentim ~acfion~ The thkd o~dafion ~action ~ s~m ~ the cleavage of the 20-22 carbon-carbon bond to ~ d prcgnenolone. The ~ d e ~ h ~ n c~avage enzyme shows ~ f i v d y ~ w ~ actifi~ for chole~eroL compared with the smrdd l lflh y d r o x y h ~ ~ M y z c d by a similar mitochondfi~ enzym~ ¢ytochrome P ~ 5 0 1 1 ~ with a turnover rate of 100-120 min-1) even c o n s ~ ing the number of steps rvquired for the chdest ~ side~hain c~avage ~ a c t ~ possibly due to the extreme ~ s ~ u ~ y of Rs substrate, cho~sterol, ~ w~er[2]. To ove~ome t~s problem, many p ~ o u s ~vestigators used var~ u s d ~ g e n t a n d ~ r a~ificial phosphofi~d v ~ i c l ~ to profide a ~ s of ch~esmrol to the c~ochrome. In the p m ~ n t ~udy, we added various concemmfions of detergent, Tween20, ~ t o the mconstituted enzyme s~tem to ~vestig~e Rs eff~t morn precise~ on the side-chain cleavage 727

728

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Bo~ne ~ r o m e P~5~cc, ~ r e n ~ o x i n and NADPH-adrenodoxin ~ u c ~ were p~p a r d from a ~ n o c o ~ i c ~ ~ o n ~ a by pubfished ~ o ~ r e s ~ . P o ~ n e cou~erpa~s were pufifi~ from ~ r e n ~ ~ o n d f i a u~ng p u b ~ d ~ o ~ d u r ~ ~, ~. Tween20 and ~rol w~e obt~ned ~om N a c ~ ~ s q u e Inc. ~ y ~ o , ~ p a ~ . ~ ] ~ o x y c h o l e s t e r o l and ~ d r o x ~ h ~ r o l were p ~ c h a s ~ ~om S i v a ~ ~ u i s , M ~ . ~ R ~ h ~ droxycho~s~r~, ~ e ~ r e d by me method of M o ~ a ~ et ~ ~ , was ~ n ~ y d o n a l d by Dr ~ F~o at T o ~ o ~ of T ~ o l o g y , Toky~ Japa~ The ~ r i ~ was e x ~ i n e d on a gas chroma~gmphy-m~s spe~rome~y ~ C M ~ to ~ a ~n~e peak. T ~ e~ymadc a c t ~ ~eno~ne ~ marion from sub~ratO was meas~ed ~ the method ~ M o ~ a ~ et ~ ~ . 100 #g Each of s ~ s ~ e ~olesterol or ~s ~ o ~ a t e d d~ fivafives such as 2 ~ S ~ h y d r o x y c h ~ r ~ [R~hydroxychoks~roL ~ d 2 ~ R ~ 2 ~ R ~ hy~ox~ho~s~roD, dissolved ~ 50#1 of d~em~a~ was ~ d e d ~ 2 ~ of • e recons~m~ e ~ e sys~m at various Tween~ con~mrat~ns. A~er 10 ~ n of prei n c u ~ n of the ~ c o n s d ~ d e ~ e ~em at 37°C, 0A ~ ~ NADPH-~neradng s ~ e m was ~ d ~ ~ ~a~ me reaction and me reaction was continued ~ ~ c ~ y 5 ~ n ~ ~ . ~he e n ~ a t i c acti~ty was finear ~ r ~ Last 10 ~ n ~ r HI s u ~ e x ~ i n e ~ ~ e NADPH~neradng ~ s ~ m con~sted ~ 0.5 ~ ~ ~ mM NADPH, ~02 ~ of ~ o ~ a ~ ~ h y ~ o ~ n a s e ~AD+) ~ D , ~ 1 5 ~ of0.1M M g ~ and 0~Sml of 0.1 M VL-iSo~a~ ~ 20raM ~ t a s ~ u m ~ o s ~ a t e b ~ e r ~ H 7.~ with a ~ v~ume of 2.87 ~ . T ~ fin~ v ~ e of the ~acdon m ~ r e was 2.4 ~ , w~ch ~ m ~ n e d : • 2#M c~ochrome P~5~cc, 10#M ~renodo~n, 0.14#M N A D P H - ~ n o ~ n reductase a ~ various concenffa~ons ~ Tween~ ~ 20 mM p o t a s ~ phosphate b ~ e r OH 7A~ The reaction was ~ a t e d ~ a ~ o n of 5 ml of ~ c ~ o r o m ~ n e con~ 2.0#g of [ l ~ l ~ l ~ l - ~ p r e g n e n o l o n e p r e ~ r ~ by the method of M o ~ a ~ et ~ ~ , as me ~ stan~ ~w~ by ¼gorous a ~ o n ~m a vortex mixer for 30 s. A ~ r ~ a ~ g ~ r ~ v ~ ~ n m e ~ ~ ~yer of ~c~orom~hane was

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extractS. The ~ o n was repea~d o n ~ more ~ m 5 ml ~ dichloromem~e but ~ o ~ t ~ ~ t e ~ gandard. The ~ m ~ d extrac~ were dried und~ r~uced p r e s ~ Afar tfimet~ation, the p r e ~ o l o n e content was measured ~ the GC-MS memod ~ the ~ t ~ ~ n m o ~ r i n g mode using t ~ ~ t e m ~ standard as d ~ c f i ~ d by M o ~ a ~ et aL ~ . ~UL~ The effec~ of Tween20 concentration on the side-chin cleavage reaction catalyzed by bo~ne cytochrome P-450~c for cho~s~r~ and ~s hydroxylated derivatives are shown ~ Fi~ 1 (A, B). The enzymatic acd~ty of cytochrome 2O E

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Fig. I. Enzymaticacfiv~s of the reconstitut~ bo~n¢ cytochrom¢ P-450scc systemat va~ousTween20concen~ations for (A) cholesterol(C)) and 20[S~hydroxycholes~rol (~) and (B) 22[R~hydroxychol~s~rol(C)) and 20JR~22[R]-dihydroxycholesterol( ~

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P-450sec for choks~rol was on~ 0~ nmol of pregnenolone formed/min/nmol P-450sec without detergent but showed more than 10-fold enhancement upon addition of 0.1% (v/v) Tween2~ Howeve~ further addition of Tween20 caused a decrease of the enhancement of the a c ~ t y as pre~ously repor~d [9, 1~. The addition of Tween20 did not cause any apprechble enhancement of the acfi~fies for 20[Skhydroxycholesterol and 22[R]-hydroxychoks~rol; ~ resulted in an inhibition of the acti~t~s. The activity for 2 ~ R ] ~ R ~ dihydroxycholestcrol showed a very high value (135nmol/min/nmol P-450scc) even without the detergent and exhibi~d a macmum (260 nmol/min/nmol P-450sec) at ~02% Tween20. This optimum concen~afion for 20[R~22[Rkdihydroxycholes~rol ~ ckarly different ~om that for choks~rol. In Table 1, enzymatic activity ratios of P-450sec at the optimum concentration of Tween20 and in the absence of the detergent are tabuhted. It ~ ckafly demonstra~d that Tween20 showed a con~derable activating capability (12.5-fold enhancement) only for cho~gerol. For other hydrox~a~d derivatives its enhancement capability ~ at most 2-fold. To obt~n a more detailed in~ght into the enzymatic acti~ties of cytochrome P-450sec, we compared the enzymatic acti~ties of bo~ne and porcine enzymes for various substra~s in the presence of ~1% Tween20. We chose 0.1% Tween20 as the optimum concentration for the reconstituted enzyme sy~em, ~nce ~ afforded a mafimum activity for cho~eroi. In Table 2, both of the reconstituted enzyme sys~ms showed remarkable ~mfiarities in the acti~ties for each of the substra~s examined. It seemed there was no spedes difference in catalytic prop-

PdS0sc¢ ~ the mean f f 3 t ~ g

erties between these two. The turnover rates (at saturating substra~ concentration) we~ ~gheg for 20[R],22[R]-dihydroxychol~ter~ and ~west for cholester~. When p~gneno~ne formation was measured u~ng 22[R]-hydroxych o k s ~ r ~ or 20[S]-hydroxychd~terd as subs~at~ the turnover rate showed an interme~a~ v~ue. These observations were in complete accordance with the ~suRs obtained ~om Fi~ 1 (A, 8). D~CUS~ON

P~fious~ severM groups have measured the enzymatic acfififies of the reconstituted cytochrome P-450~c system urine nonio~c d e ~ gents ~uch as Tween20 or Emulgen91~ or artifid~ phospholi~d v e ~ c ~ Takikawa et aL [9] ~po~ed that the reconstituted bo~ne cytochrome P-450sec sys~m showed a maximum actifity in the p ~ n c e of 0.3% Tween20 for cho~s~r~ as substrate [3]. The V ~ (measured ~ the turnover rate) of converfion of chol~t~r~ to pregneno~ne under t~s con~fion was reposed to b¢ 30 min-~37°C, and Kido a al. also ~ported that Tween20 ~ capa~e of activating the choles~rol fid~ch~n cleavage actifity for choles~rol as substrat~ and that the optim~ concentration of Tween20 was around 0.21 mM (0.023%) and Vm~ under t~s con~tion was 28m in -~ [1~. LateL Hanuko~u et a~ reposed that at 0.3% Tween2~ the reconstituted enzyme system showed Vm~ between 20-30rain -~ for chol~~ r ~ as subs~ate [11]. They ~so noted that the V~, was con~dera~y dependent on the ~nic ~ n g t h of the buffer. On the other hand, Lamb~h et a£ [1] showed that in an a~ifiO~ phosphotipid vesicle reconstituted cytochrome P-450sec system (at 37°C),

Tab~ Z Enzymatic a~ti~tics of r~constitut¢d bovine and porcine ¢ytochrom¢ P-450scc systems at ~1% Twcon20 Substra~P System Bo~ne system Porcine system B~nk inoubation b

Cholesterol

2~R],22[R ]-Dihydroxycholesterol

22[R]-Hydroxychole~¢rol

20[S]-Hydroxyeh~terol

~4 ~ ~5 %8 ~ 1~ <~ 1

172 ± 2~0 165 ± ISA <~1

24.3 ± 1~ 2~0 ± ~8 <0,1

l&7 ± ~5 1~7 ± 3~ <~1

'Data arc expressed in nmol pr~gncnolon~min/nmol P-450scc as the mean ± SD for 5 t~als, bwithout the r~consfituted enzyme system.

730

~ZUHIKO Iw~ASm et ~

each of the three hydroxylafions required for the c h o ~ e r o l side-chain cleavage reaction occur at approximately the same ra~, ff the number of hydrox~ation steps required for pregnenolone formation ~om each sub~ra~ ~ taken into account ~he c~cuiated Vm~ v~ues were ~Smin -~ for chok~erol, 15rain-m for 22[R]-hydroxychoks~rol and 31rain -~ for 20[R],2~R~dihydroxychok~ero0 [1]. Tuckey and Stevenson [1~ ~so showed that, at 37°C, the ~la~ve turnover rates for 20[R],2~R]-dihydroxycholes~rol (217 min-~),2~R~hydroxycholesterol ( 102 rain- ~ and cholester~ ~ 5 rain- ~ under saturating conditions were ~most 1:~5:~33 for bovine l u ~ cytochrome P450scc in phospholipid v e ~ e ~ indicating that each of the three hydrox~afions occurs at approximately the same ra~. It must b¢ noted, howeve~ the turnover ra~s for each substrate were all G to %fold higher than those reposed by Lambeth et al. [1]. Although the turnover rate for choks~rol (at satura~ng conditions) in our ~udy was considerab~ lower than those pre~ou~y reposed ~xcept for that reposed by Lambeth et aL [1], which was very ~ose to our v~ue), ~ was not due to the parti~ denatura~on of the reconstituted system, ~nce the same system ~so exhibited a very high turnover rate for 20[R],22[R~dihydroxychole~er~, which was dose to the value reported by Tuckey and S~venson [1~. In the present ~udy we have examined the effect of Tween20 for the turnover rates of cytochrome P-450scc with the various hydroxychoks~rols sys~matically. Our present data established that, fi~ti% the turnover ra~ for 20[R],2~R~dihydrnxychoks~rol was far greater than those for choks~rol, 22[R~hydroxycholes~rol or 2~S]-hydrnxych~es~rol. Furthermore, the turnover ra~ for 22[R~hydroxycholesterol was at kast 3-fold higher than that for choles~rnL even comparing them at optimum con~fion~ Thus, we could not observe any e~dence to suppo~ that the three hydroxylafions required for the s ~ c h a i n ckavage reaction occur at approximarry the same r a ~ as proposed by Lambeth et aL [1] and Tuckey and Stevenson [1~. Secondly, the p l a u ~ e first intermedia~ in the ~d~ch~n ~eavage reactio~ 22[~hydroxychole~erol, has an apprec~b~ higher turnover ra~ than that of 20[S]-hydroxycho~s~rol. These findings were valid for all the concentrations of Tween20 examined (from 0 to

~5%~

Lambeth [1~ an& ~te~ Kowluru et aL [1~ have shown th~ ch~esterol ~de~h~n cleavage ~acfions ~ phosph~ipid vesicl~ c o n t ~ n g both c h o ~ e r ~ and ¢ytochrome P-450scc are very sfimulatory upon addition of spedfic pho~ pholipids. These stimulatory effects of the phosphofi~ds are, howeve~ not observed for hydroxycholes~rol derivatives [1].T~s phenomenon may have some relation to the gim~atory effect of Tween20, wh~h ~ specific o~y for cho~s~rol as ob~rved ~ t~s ~ud~ The fir~ hydroxylafion s~p to yie~ 22[R}hydroxych~es~r~ ~ apparently the s~west process ~ the overfll reaction of the ~de~h~n cleavage and th~ proce~ ~ dependent on the Tween20 concentration. It is known that d~ergents and phospholipids do not ~gnificant~ affect the ~ n ~ n g of adrenodox~ to the cytochrom~ suggesting that the ~ec~on transfer rate ~om admnodo~n is ~so ~dependent of the Tween20 concen~ation[15, 1~. Fu~he~ mo~, ~ the ch~esmrol s ~ c h ~ n ckavage reactio~ the supposing ~e~rnn t r a n s i t rate ~om ad~nodox~ ~ 2-3 s-~ when the V ~ of the s ~ c h ~ n cleavage ~ 20-30 rain-~ [2]. The hydrox~ation step to yield 22[R]-hydroxych~r~ compris~ two processe~ an access of cholesterol to cytochrnme P-450scc and 2~R} hydrox~afion of choles~rol bound to the cytochrom~ It ~ unlikdy that Tween20 enhances the rate of 22[R]-hydroxy~fion reaction without affecting the subsequent hydrox~ation steps. Thus, R ~ in~ffed that Tween20 fa~fi~ s the access of c h o ~ e r ~ to cytochrnme P -450scc. Our present ~s~ts am con~s~nt with the widely-accepted concept that the ra~ of chol~ter~ transfer to the cytochrome P-450scc active fi~ fimits the overall ram of the side-chain cleavage ~actiom A~nowledgemen~--T~s w o ~ was supported by Os~ma Hospital and Odno Hospital. We ~ank Dr ~ Oshima, Dr K. Odn~ Mr % Naka~wa and Ms C $ ~ m i ~ of ~ e R e s ~ h Eq~pment C ~ t e r for their t ~ h n ~ a ~ B ~ n ~ .

REFERENCES I. Lambeth J. D., Ki~hen ~ ~ , Farooq~ A. A., Tuckey R. and Kam~ H.: Cytochrome P~50scc~ubetrate ~racfions. ~u~ of ~ n ~ n g and cambric activity u~ng hydroxycholesteroL ~ BioL Chem. ~g7 (1982) 1876-1884. ~ ~ o ~ e C. R.: C~ochrome P~50scc enzyme ~ sterol Eosynthesis and me~bolism. In Cytochrome P450 Struc~re, Mechan~m, and Biochemistry ('Edited by ~ R. Orfiz de Montellano). Henum Pres~ New York

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Eff~ & Tw~

on c

3, Tsuba~ M., Hiwata~hi A. and le~kawa Y.: Effect of chol~t~ol and adxenodo~n ~ n d ~ g on the heme mokff of cytochrome P450scc: a ~ s o n a n ~ Raman study. Biochem~try. 25 (1986) 3568-3569. ~ Tsuba~ M., Tomi~ $., Tsuneoka Y. and Ic~kawa Y.: Characterization of c h o l e s t ~ and adrenodoxin ~nd~ g on ~ e heine mokff of cytochrome P~50scc: a resonance Raman ~ud~ Biochim. Biophys. A c ~ 870 (1986) 564-57~ ~ Hiwatashi A., khikawa Y., Maruya N., Yamano T, and Aki K.: Ptopcrti~ of c~s~line reduced ~co~ne amide adenine d~ucleotide phospha~-ad~nudo~n r e d u c ~ ~om bo~ne adrenoco~ic~ mitochond~a. B~chemist~ 15 (197~ 3082-3090. ~ Hiwatashi A., Sakihama N., S~n M. and Ichikawa Y.: Heterogeneity of adrenocortic~ fe~edo~n, FEBS L e ~ 209 0986) 311-31~ % Morisa~ M., Sato ~ and Ikekawa N.: Stu~es on steroid. XLV. Synth~is of the ~ u r stereo ~omers of 20,22-dihydroxychol~rol. Chem. Pharm. Bu~ Tokyo 25 (197~ 2576-2583. & Morisaki M., S ~ M. and Ikekawa N.: Side~hain cleavage of cho~s~r~ by gas chroma~graphy-mass spec~ometry ~ a ~lec~d ~ n mortaring mode. Meth~. Enzym. 111 (198~ 352-364. Z Ta~kawa O., Gomi %, Suhara K., lmga~ L , Takemofi ~ and K ~ a o f i M : Prope~ies of an adren~ cytochrome P450scc for the ~de-cha~ cleavage ofchol~ W r ~ . Archs Biochem. Biophy~ 1~0 (197~ 300--30~

SBMB

3~E

~

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1~ Kido ~ , Arakawa M. and Kimura T.: AdmnM cortex m i t o c h o n d ~ cytochrome P~50scc specific to chol~mrol side~h~n cleavage ~acfio~ L BioL Chem. 254 (1979) 8377-8385. 1 I. Hanuko~u I., Privane C. T. and Jefcoa~ C. R.: Mec~ a~sms of ~ c a~ivation of admn~ mitochondrial cytochrome P~50scc and P4501 lfl. S BioL Chem. 256 (1981) 4329-4335. IZ Tuckey R. C. and S~venson E M.: Prope~i~ of bo~ne lute~ cytochrome P~50scc incorporated ~ a r f i f i ~ phospholipid vesicles. ~ ~ Biochem. 16 (198~ 497-50~ 13. Lambeth ~ D.: C~ochrome P~50scc. Cardiodi~n as an eff~tor of activi~ of a mitochond~al c~ochrome P-450scc. & BioL Chemo 256 (1981) 4757-4762. l ~ Kowluru R. A., George R. and Jefcoate C. R.: P~y phosph~no~fide activation of c h ~ t ~ ~de chain ~eavage with purified cytochrome P~5Oscc. ~ BioL Chem, 258 (198~ 8053-8059. 15. Seybe~ D. W., Lancaster ~ R. Jr., Lambeth J. D, and Kamin H.: Participation of the membrane ~ ~ e ~de c h i n c~avage of choles~r~. Reconstitufion of cytochrome P~50scc ~ t o phospholipid v e ~ c ~ ~ BioL Chem. 254 (197~ 12088-1209~ 16. HanukoOu L, S~tberge V., Bumpus ~ A., Dus K. M. and Jefcoate C. R.: Ad~nM mitochondri~ cytochrome P~50scc. Cholesterol and adrenodoxin infractions ~ eq~fibrium and during t u m o r s . ~ BioL Chem. 256

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