Studies on the mechanism of estrogen biosynthesis, V. stereochemical comparison of aromatization in placenta! and microbiological systems

BIOCHIMICA ET BIOPHYSICA hCTA

770

STUDIES

ON THE MECHANISM

STEREOCHEMICAL AND

COMPARISON

MICROBIOLOGICAL

HARRY

J. BRQDIE,

Worcester

Foundatim

(Received

February

OF ESTROGEN

GENUS

BIOSYNTHESIS.

OF AROMATIZATION

V.

IN PLACENTAL

SYSTEMS*

POSSANZA**

fofovExpeGmntal

Biology,

AND JOHN Shvewsbury,

D. TOWNSLEY Mass.

(U.S.A.)

zSth, 1968)

SUMMARY I. To provide definitive evidence as to whether rg-hydroxyandrost+ene-3,17dione (I) may aromatize by similar mechanisms in human placental and in microbiological systems, the stereochemistry of hydrogen removal at C-I in the conversion of I to estrogen was investigated. 2. Incubations of r9-hydroxy-[r-3H (83°/O-p)]androst-4-ene-3,17-dione with a placental microsomal preparation and a NADPH-generating system gave estrone in which 84% of the tritium was lost, showing that the $-hydrogen was eliminated. When the NADPH-generating system was replaced by an artificial electron acceptor active in converting q-hydroxyandrostenedione to estrone in microorganisms, no conversion to estrone was noted. 3. Incubations of the same steroid substrate with respiring cultures of Psetidomonas se. (ATCC 13262) and Nocardia ?festrictus showed that 22 and 28% of the tritium, respectively, was lost in the transformation to estrone, thus implicating removal of the m-hydrogen in the transformation. A similar result for N. restvictus was obtained with a 6000 xg supernate preparation and the artificial electron acceptor, phenazine methosulfate. 4. The results show that the transformation of I to estrone in the two types of systems occurs by different stereochemical mechanisms, and that probably different electronic factors are involved also.

INTRODUCTION

It is generally accepted that q-hydroxylation is the first step in the conversion of androstenedione (androst-4-ene-3,r7-dione) to estrogens in human placental and Abbreviations: Androstenedione, androst-4-ene-3,17-dione; Ig-hydroxyandrostenedione, droxyandrost-4-ene-3,17-dione. * Paper Iv iS TOWNSLEY AND BRODIE (Ig67)l. ** Postdoctoral trainee in the Training Program for Steroid Biochemistry (1964-1966). address: Ayerst Laboratories, Montreal, Canada. Biochim.

Biophys.

Acta,

152 (1968)

770-777

rg-hyPresent

STEREOCHEMXP.‘RY OF ESTROGEN HOSYNTHESIS

771

ovarian tissue. For a recent review, see ref. 2. The intermediate rg-hydroxyandro~~enediune(r9-hy~oxyandrost-4-ene-3~~7-d~o~e~ is a good precursor for arorn~~~za~on in both placental %4and some microbiological systemssj6 and there has been speculation about whether similar mechanisms are involved (see ref. 2, p. 365). This would be of interest to the comparative enzymologist and, as a practical corollary, microbiological preparations would be convenient for studying the general mechanism of estrogen biosynthesis It appeared that a definitive answer might be obtained from a consideration of the stereochemistry of aromatization in the two types of systems. ~~ndrostened~onehas been shown to aromatize with placental tissue with loss of the @-hydrogen ?*srand if rg-hydroxyandrostenedione is on the pathway, it too should lose the same hydrogen. On the other hand, if aromatization of rg-hydroxyandrostencdione takes place by C-r,z-dehydrogenation with concomitant expulsion of the C-20 hydrox~~eth~71 group, indications are that the ICE-hydrogenmay be lost, since dehydrogenation of and-r&en&one ~$8to give androsta-~,4~~ene-3,r7-~one proceeds by that path with preparations of Bacillus s$h~a&czas*,an organism which does not, however, metabolize rg-hydroxyandrostenedioueXO. To resolve this point, rg-hydroxyandrostenediane, stereo-selectively tritiated in the C-r;Bposition, was converted to estrone with preparations of human placental microsomes, Pseudmmm sp. and ~~~~~~d~~ ~est&tzs. A summary of portions of this work has appearedIf. METHODS

Materials rg-Hydroxy[r-3H (83%-~)!androste~edioae was prepared from [I-~H (83%~@)]an~osten~one as previously describedIe and its purity was established by thin-layer and paper chromato~aphy and by reverse isotope dilution and crystallization. NADP+, glucose &phosphate, glucose&phosphate dehydrogenase, phenazine methosulfate (Sigma Chemical Co.) and dichlorophenolindophenol (Hartman-Leddon Co., Philadelphia., Pa., U.S.A.) were used as received. Brinkmarr PX;,,, silica gel was used for thin-layer chromatography and Whatman No. r grade was used for paper systems. Solvents were distilled or were of “analytical” quality. Incubatioins (a) Placental microsomas. Microsomes prepared and washed three times in butfer accordmg to the procedure of RYAN~~ xxxe incubated as previously describedg. An amount obta.ined from 20 g wet weight of tissue was suspended in 3 mi of 0.05 M phosphate buffer (pH 7-z) and added to a 5o--xoo-ml erlenmeyer flask containing the steroid. A preincubatedt” NADPH-gene~~t~~g system consisting of 7.5 pmoles of NADP+, 140 pmoles of glucose 6-phosphate and I unit of @cased-phosphate, dehydrogenase was added and, after incubating the mixture in air for I h at 37”, it was extracted 5 times with ethyl acetate. fb) r’se~~~~~~~~ s$. (ATCC GJZ&Z).Three 4oo-ml cultures of this organism were grown for 24 h according to the procedure of DoEWX AND%JIR”, A solution of j0 mg

* The ra-hydrogen is also lost on C-I,z-dehydrogenation of the san1e substrate with a cell-free preparation from Bacillus cycloo;uydaxs (ATCC 12673). N. Sam AND W. J. BRODIE, unpublished observation.

772

H. J. BRODTE, G. POSSANZA,

J. D. TOWKSLET

of Ig-hydroxy[r-3~

(837O-p)] J an d ros-t enedione of specific activity 4735 disint, jmin per pmole, in 3 ml of ethanol-propylene glycol (I : I, v/v), was added to each culture and incubations

were continued

for an additional

36 h. After the addition

of ethyl

acetate and filtration the filtrate and residue were extracted with ethyl acetate and the combined extract was washed with water and then analyzed. restvictzls. Two incubations

(c) Nocavdia

were carried out with this organism,

one with the respiring cells and the other with a 6000 xg supernatant sonication.

Both preparations

were obtained

obtained

by

by culturing the organism from a slant

kindly supplied by Dr. C. J. SIH according to the procedures of Sra and co-workers”$15. In the whole-cell experiment, 68 mg of Ig-hydroxy activity

4220 disint./min

[r-FI

per !rmole were incubated

(83o,b-[j) jandrostenedione,

specific

for 24 h with a culture which had

previously grown for 24 h in the presence of progesterone. After filtration through cheesecloth, the supernatant and residue were extracted as described in Section b. After drying the extract

with Na,SO,,

graphic analysis. The 6000 xg supernatant a

24-h

culture in

20

it was evaporated

was obiained

by sonicating the precipitated

ml of 0.03 molar phosphate

were incubated photometer

utilized

0.15 ml of ethanol

at 30’ in a 3-ml cell placed in a Perkin-Elmer

set at 600 rnp (ref. 15). To 5 mi of the supernatant

2.3 mg of rg-hydroxy

[I-~H (83qd-P)]androstenedione,

min per pmole,

2

and

mg of phenazine

methosulfate

of dichlorophenol-

20 pg

per min as an initial rate when 0.1 ml of enzyme,

dione, 60 pg of dichlorophenolindopheno!,

cells from

buffer (pH 7) and then centrifuging

in a Servall Centrifuge for IO min. The supernatant indophenol

to dryness for chromato-

200 ,ug

and 202

of androsteneml of buffer

2.25

Recording

Spectro-

were added separatel>

specific activity

53 200 disint. /

each dissolved

in D.I ml of

ethanol. After incubation for 16 h at 3o”, the mixture was extracted with chloroform and the dried extract was treated as described in the RESULTS SECTION. Pzwijkation and qzhantijcatio9z Chromatographic procedures estimation

of mass for specific

light absorption

or direct weighing

describeda. Estrogen estimation to the method of BROwNIG.

including

activity

methods

determination

for visualization by fhrorometry,

and scintillation-counting

procedures

and elution, ultraviolet have been

using the Kober reaction was carried cut according

RESULTS

Placental incubations (a) Steveochemistvy ex$eviunent. To determine which hydrogen at C-I is eliminated during aromatization, IOO pg of q-hydroxy[r-YH (83°/O-/3)]androstenedione, specific activity 3.13. IO” disint./min per pmole, were incubated in each of two room1 erlenmeyer flasks containing 6 ml of the microsomal suspension and the NADPHgenerating system. The ethyl acetate extract contained 36yb of the original radioactivity indicating loss of tritium during incubation, since recovery from such incubations is usually 80-90%. The residue from evaporation of solvent was analyzed by thin-layer chromatography in benzene-ethyl acetate (8~2, v/v) and the areas corand substrate were eluted. There was only a trace responding to estrone, estradiol-q9 of substrate and this was not treated further. The estrogens were separately rechro-

STEREOCHEMISTRY OF ESTROGEN BIOSYNTHESIS

773

matographed in ether-hexane (I : I, v/v), eluted and partitioned between ether and IM NaOH. The base-soluble fractions after neutralization and extraction were rechromatographed on paper, estradiol-I7b in the BUSHB, System17 (ligroin (6o_go”)benzene-methanol-water (6.67 : 3.33 : 4 : I, v/v), and estrone in a modified B, System (4 : I : 4 : I, by vol.). The estrogen areas were eluted with redistilled absolute ethanol and the eluates were filtered through fine scintered-glass funnels. The yield of estrone at this point was 14 ,ug and estradiol-I7@, 27 ,ug, as judged by the amount of radioactivity present and subsequent specific activity determinations. The specific activity of the estrone as determined by fluorometryls and liquid scintillation counting was 4.92’105 disint./min per pmole representing a loss of 84% of the tritium originally present in the substrate. Another sample was estimated by the Kober procedure16 and the value obtained was 4.6.10~ disint./min per pmole representing a loss of 85% of the tritium. The procedure for purification prior to analysis and the analytical methods have been shown to provide accurate values for specific activity of estrone isolated from placental preparations8. When estradioLI7P was analyzed by fluorometry, a value of 3.7.10~ disint./min per pmole was obtained (88% loss of tritium). (b) Co-factor expevimed. Duplicate incubations of 50 ,ug of Ig-hydroxyandrostenedione were carried out in 3-ml suspensions of microsomes as detailed above. The NADPH-generating system was added to the first set, 750 ,ug (2.5 pmole) of phenazine methosulfate to the second, and no co-factors were added to the third set. After incubation for I h at 37” in air, each set was combined and extracted with ether. After evaporation to dryness, the residues were taken up in 20 ml of ethanol-water (4:I,v/v) and washed twice with IO-ml portions of ligroin. The residues from the alcoholic solution were then analyzed by thin-layer chromatography in benzene-ethyl acetate (4:1, v/v), The estrone and estradioLI7p areas were eluted and chromatographed in the Bush B, system together with varying amounts of estrone and estradioL17fi. The chromatograms were dipped in Turnbull’s blue reagentI and visual examination of the color intensities produced indicated that the incubations with NADPH-generating system yielded slightly more than 20 pg of estrone together with a trace of estradiol-171, whereas no estrone or estradiol-r7/3 was obtained from incubation without co-factor or with phenazine methosulfate. The results with the NADPH-generating system are in agreement with those obtained by MORATOand co- workerss. Microbiological

experiments

The extract from incubations with the respiring cultures of Pseudomonas sp. was chromatographed on two silica gel-layered (I mm) plates in benzene-acetone (9: I, v/v) and the areas corresponding in RF to estrone and rg-hydroxyandrostenedione were eluted. Crystallization of the estrone material from ethanol gave 13.5 mg. Two further crystallizations followed by thin-layer chromatography in the system cyclohexane-ethyl acetate (7 : 3, v/v) and recrystallization again gave material having specific activities of 3834, 3986 and 3977 disint./min per pmole, respectively. The infrared absorption spectrum was identical to that of authentic estrone. The purified estrone was acetylated with pyridine-acetic anhydride (I : I, v/v) at room temperature for 5 h. The estrone acetate formed was purified by thin-layer chromatography in benzene-ethyl acetate (93 : 7, v/v) and crystallized twice from benzene-hexane. The specific activities were 3679 and 3667 disint./min per pmole, respectively, showing Biochim.

Bio?hys.

Acta,

152 (1968)

770-777

N. J. BRODIE, G. POSSANZ.4, J. D. TOWNSLEY

774 a decrease recovered

in specific activity

from starting

material

of 22%.

from the r-mm thick silica gel plate contained

absorption

spectrum

crystallizations

identical

to that

from ethyl acetate

The starting

material

25 mg and had an infrared

of rg-hydroxyandrostenedione.

and ethyl acetate-benzene,

After

tv,ro

the specific activities

of the crystals were 4729 and 4830, and of the residues, 5121 and 4858 disint./min per pmole. The value is not significantly different from the original specific activity of 4735 showing that no isotopic The extract

effect was operating

from the incubation

only 25% of the original radioactivity with ether did not appreciably

in the transformation.

with respiring and additional

improve

recovery.

cells of N. rest&k extractions

contained

of the aqueous layer

The residue from the extract

was

analyzed by thin-layer chromatography on r-mm thick layers (two 20 cm x 20 cm plates) in benzene-ethyl acetate (4: I, v/v) and the estrone zone was eluted. No evidence

for starting

material

was obtained.

red spectrum three

times

of which was identical and analysis

The estrone

material

was rechromato-

(9 : I, v/v) to yield 6 mg of crystals,

graphed in the system benzene-acetone

to that of estrone.

for specific

activity

Recrystallization

by scintillation

the infra-

from ethanol

counting

and direct

weighing gave values of 3360, 3380 and 3294 disint./min per pmole. The estrone then was acetylated with equal volumes of acetic anhydride in pyridine for 16 h and, after evaporation

of reagents,

in benzene-ethyl

acetate

twice from benzene-hesane.

the residue was analyzed by thin-layer

(93 : 7, v/v). The estrone acetate The specific activities

chromatography

was eluted and crystallized

from the two crystallizations

3010 and 3060 disint. /min per bhmole, showing a loss of specific activity material of 28%. The extract contained

74%

lyzed by thin-layer Elution

from the incubation

of the original

chromatography

of the estrone

with the cell-free

radioactivity.

system

frcm AT. ~estricti-ts

The residue from the extract

in the benzene-ethyl

and Ig-hydroxyandrostenedione

acetate

were

from starting

was ana-

(4: I, v/v) system,

(origin) zones indicated

that

the ratio of these two materials was 6 : I. A portion of the estrone zone was chromatographed in the modified B, system on washed paper and, after elution of the estrone material

and determination

of specific activity

counting,

a value of 4230 disint./min

analyzed

in a gg-transfer

by fluorometry

per FLmole was obtained.

countercurrent

apparatus

and liquid scintillation A second sample was

in the system

methanol-weter-

carbon tetrachloride (7 : 3 : IO, v/v/v). Assay of tubes for radioactivity revealed a symmetrical distribution pattern of K value 1.27, theor. 1.25 (ref. zo), corresponding to estrone. Determination of the specific activities in tubes 50, 56 (peak) and 62 by fluorometry and scintillation counting gave values of 35 r5o,35 800 and 37 800 disint. ] min per ,umole, respectively. A portion of the combined estrone from the countercurrent distribution was acetylated with 0.2 ml each of pyridine and acetic anhydride at room temperature overnight. After evaporation of reagents, the residue was chromatographed in a modified Bush A system (heptane-methanol-water, 5 : 4: I, v/v/vi and the area corresponding to estrone acetate (RF = 0.85) was eluted and the specific activity was estimated by fluorometry. A value of 35350 disint./min per ,umole was obtained. Using this figure and the one obtained from estimation of the peak tube in the counter current distribution, a value of 32% loss of specific activity was obtained in the transformation to estrone. The r9-hydroxyandrostenedione starting material recovered was rechromatographed on a thin-layer silica gel plate in benzene-acetone (7:3, v/v) and the ultraBiochim. Biophys. Acta, 152 (1968) 770-777

STEREOCHEMISTRY

OF ESTROGEN

BIOSYNTHESIS

775

violet-absorbing material corresponding to standard rg-hydroxyandrostenedione was eluted and the specific activity was estimated by ultraviolet absorption and liquid scintillation counting. A value of 93600 disint./min per ,umole was obtained representing an increase in tritium specific activity over starting material of 71%. DISCUSSION

Aromatization of rg-hydroxy[r-JH (83%$)]androstenedione with placental microsomes caused a decrease in specific activity in the estrone product of 84-85%, whereas with respiring cells of Pseudomonas se. a ZZ~/~decrease was observed and with respiring cells of N. restrictus, or with the soluble enzyme system from the same organism, a 28-320/O decrease in estrone specific activity was obtained as compared to substrate. This indicates that aromatization of the Ig-hydroxy substrate with the placental system is effected with loss of the @-hydrogen and with the microbiological preparations the I&-hydrogen is mainly involved. We can conclude, therefore, that the aromatization in these two types of systems occurs by different stereochemical mechanisms. The loss of the @-hydrogen in the aromatization of rg-hydroxyandrostenedione is additional evidence that this substrate is on the pathway from androstenedione to estrogen, since predominately C-I beta-labeled androstenedione loses an equivalent amount of activity on aromatization and the C-I alpha-labeled androstenedione retains all its activity in the same transformation ‘s8. The other evidence for this is based on the ease of aromatization of Ig-hydroxyandrostenedione as compared to the unhydroxylated substrate3 and on kinetic data14. The nature of the oxidative attack in ring A is still obscure. It is tempting to suggest that this desaturation may involve a r/3-hydroxylation followed by dehydration since, in crude systems, NADPH and oxygen have been required as co-factors for all precursors tested and I/Lhydroxyestr-4-ene-3,17-dione is formed in good yield from incubations of estr-4-ene-3,17-dione 21.The inability of phenazine methosulfate to aromatize rg-hydroxyandrostenedione reported here and the inactivity of menadione and dichlorophenolindophenol as electron acceptors with estr-4-ene-3,r7-dione8 suggest that the aromatase does not have a dehydrogenase component, at least of the usual type. However, dehydration of r/3-hydroxy-Ig-norsteroids to give estrogen occurs to a negligible extent, if at all, and there is evidence that NADPH and oxygen may be required for some dehydrogenations 22. A detailed analysis of this situation has been presented8, some aspects of which are similar to results obtained recently from studies on C-5 desaturation during cholesterol biosynthesisZ3. The finding that the C-I tritium specific activity decreased only 22% in the aromatization of rg-hydroxy [IJH (83 %$)]androstenedione with PseudoPnonas sp. and 28-320/O with N. rest&m is evidence that the conversion in these systems occurs via a dehydrogenation mechanism for reasons noted in the introductory paragraph. SIH AND RAHIM~~also concluded this from the observation that the transformation with the soluble system from N. restrictus occurs anaerobically with artificial electron acceptors. It is logical to suppose, as has been noted often (see ref. 2, p. 366), that after C-1,2-dehydrogenation to give, at least formally, rg-hydroxyandrosta-r,4-diene3,r7-dione, elimination of the ro-hydroxymethyl group may occur spontaneously to give estrone. Although the enzymatic dehydrogenations with Pseudomonas sp. and

II. j. BKOIIIE, 6. POSSANZA,j. 0. TOWNSLBY

776 N. ~estrictz~s proceed Bacillus

cycle-oxydms,

stereochemistry

by the same stereochemistry at C-n as Bacillus allowing us to generalize with more assurance

of dehydrogenations

in microorganisms,

there

s@uerkxs

concerning

and

the

are significant

dif-

ferences which should be noted. Using 6. sphaeyiczds and SE-androstane-3,17-dione

or

androstenedione as substrates, the Is;-hydrogen is exchanged when the substrates are in contact with the respiring organism?a. r\‘o loss of the 17~~~0 alpha label could be demonstrated in the recovered substrate in the incubation of 1qhydroxyandrostenedione with respiring cells of Pse&oncorzas s$. In addition, the aromatization of Lghydroxyandrostenedione

by a respiring culture or

tus led to significantly nzonns sp. (22%)

higher loss of tritium,

or when androstenedione

dione) with the same tritium and 21%

loss, respectivelyr3).

increase in specific activity

distribution

a

cell-free preparation

of N. yestric-

28-3~~/~, than that found with Psez&o-

or Ig-ncrandrostenedione was dehydrogenated

(estr-4-ene-3,17-

withB.

sphuel~icz~s(17

The reason for this is not known although

in the recovered

starting

material

the large

from the cell-free experi-

ment suggests that an isotope effect is operating. To cause the results obtained, it would have to include a secondary one of rather high magnitude involving the IBposition. detailed

An evaluation kinetic

of this interesting

possibility

mnst

await the results

of a

investigation.

ACKNOWLEDGEMENTS We are grateful

to the National

Institutes

of Health,

U.S. Public

Health

Ser-

vice, for financial support to the following scientists

of this work (AM-6891; Training Grant CRTU-5001) and for their help in this investigation: Drs. A. BOWERS and

R. I. DORFXAN of Syntex,

Inc. for a gift of Ig-hydroxyandrostenedione,

D. S. LAPNE

for the counter current distribntion separation, N. SETO for culturing the microorganisms and C. J. SIH for donating a slant of Al. 1festrictsLs. We wish to thank the staffs of City, Hahnemann, Memorial and St. Vincent Hospitals, Worcester for providing placentae. The skilled technical assistance of Miss PHYLLIS A. WARG is appreciated. REFEREXCES I J. D. TOW-XSLEY AND H. J. BRODIE, Riochiw. Biophys. 1cta, 144 (1967) 410. 2 P. TALALAY, An%. Rev. Bioclw~., 34 (1965) 347. 3 T. MORATO,M. HAYANO, R. I.DORFMAN AND L. R. AXESROD, Biorhem.Bioph~s.Res.Co~~~~~~~~., 6 (1961) 334. 4 C. GUAL, T. MORATO,

M. HAYANo, ;vI. GUT AND R. I. DORFMAN, Endocviwology, 71 (1962)920. DODSON AXD R.D. MUIR, J. A?n. C?wm. Sot., 83 (1961) 4627. C. J. SIH AND R. E. BENNETT. Biochim. Biophys. dcta, 56 (1962) j84. T. MORATO, K. RAAB, H. J. RRODIE, M. HAYANO AND R. I. DORFNPAN, J. ,-1~.. Chew. Sot.,8~ (1962) 3764. J. D. TOWNSLEY ANS H. J. B~oorr:,Biockmis@, 7 (1968) 33. H. J. RINGOLD. M. HAYANO AND V. STEFANOVIC, J. Biol. Chew., 238 (1963) 1960. M. HAYANO, J. E.LONGCHAMPT, W. KELLY, C. GUAL AND R. I. DORFMAN, 1st Inter%.CO??@,. Endocvinol., Copelzlmgm, 1960, VIIIa, No. 351, 196% p. 699. J. D. TOWXSLEY, 6. POSSANZA AND H. J. BRODIE, FedwatiojL PYOC., 25 (1966)~82. H. J. BRODIE, Tetruhedvoqz, 23 (1967) 535. K. J. RYAN, J. Biol.Chew., 234 (1959) 268. R.B. WILCOX AND L. L. ENGEL, Steroids,Sup@,, I (1965)49. C. J. SIH AND A. M. RAHIx,J. Phnvm. Sci., 52 (1963) 1075.

5 R.M. 6 7

8 9 IO

II 12 13 14

15

16 Ezdocrinol., 8 (1952) 196. 17 1. BUSH, Biochem. J., 50 (1952) 370. 18 R. TN. BATES AND H. COHEN, Endocrinology, Biochim.

Bioph_vs. Acta,

152 (1968)

770-7 7 7

47 (rggo)

16b.

STEREOCHEMISTRY

OF ESTROGEN

BIOSYNTHESIS

777

19 G. M. BARTON, R. S. EVANS AND J. A. F. GARDNER, Nature, 170 (1952) 249. 20 T. F. GALLAGHER, S. KRAYCHY, J. FISHMAN, J. B.BROWN AND G. F.MARRIAN, 233 (1958)

J.Biol.Chem.,

1093.

J. D. TOWNSLEY AND H. J. BRODIE, Biochem. J., IOI (1966) 25C. 22 M. I. GURR AND K. BLOCH, Biochem J,, 99 (1966) 16C. 23 S. M. DEWHURST AND M. AKHTAR, Biochem. J., 105 (1967) 1187. 24 V. STEFANOVIC, M. HAYANO AND R. I. DORFMAN, Biochim.Biophys. 21

Biochim. Biofihys.

Acta, Acta,

71 (1963) 152 (1968)

429. 770-777