Studies of corticosterone binding and metabolism in rat thymocytes

557

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

26849

STUDIES OF CORTICOSTERONE BINDING AND METABOLISM IN RAT THYMOCYTES J O A N M. A U G U S T Y N * AND W I L L A K. B R U N K H O R S T " *

The Worcester Foundation for Experimental Biology, Shrewsbury, Mass. o~545 (U.S.A.) (Received N o v e m b e r 29th, 1971)

SUMMARY

I. I n vivo and in vitro experiments established that the radioactive compound bound to chromatin was corticosterone, thus supporting the hypothesis that the "specific" receptor for glucocorticoids in thymocytes is a component(s) of chromatin. 2. Identification of metabolites of corticosterone present in the cytoplasmic fraction, in in vitro experiments, showed that only the product oi oxidation at C-II, Ad-pregnene-2i-ol-3,ii,2o-trione, occurs to any significant extent. 3. In in vivo experiments, significant amounts oi the total radioactivity present in cytoplasm and nuclei occur as the following metabolites of corticosterone: A 4pregnene-2I-ol-3,II,2o-trione, Ad-pregnene-Ilfl,2o,2I-triol-3-one, 5fl-pregnane-3~¢,II/5, 2ofl,2I-tetrol, and 5fl-pregnane-3~.,II~,2I-triol-2o-one. It is suggested that the latter three metabolites do not originate in the thymus but reach it via the circulation. 4. Incubation of rat thymocytes in Eagle's minimal essential medium containing concentrations of corticosterone from I nM to 3 #M suggested the presence of "specific" binding sites for corticosterone, in both cytoplasm and nucleus, which were saturated at about IOO nM.

INTRODUCTION

The proposal that the primary action of hormones is mediated by interaction with a specific receptor molecule in cells of target tissues has been substantiated at the molecular level for several of the steroid hormones. Studies of intact hepatoma tissue culture cells indicate that receptors for glucocorticoids occur in both the nucleus and cytoplasm 1. Recent reports describe the properties of the cytoplasmic receptor in the hepatoma cells ~ and in normal rat liver s. Munck and Brinck-Johnsen 4 have presented evidence, obtained by measurement of steroid binding by whole cells, that specific receptors for cortisol are present in rat thymocytes. Subsequently, Wira and Munck s stated that these receptors are located mainly in the nucleus. We have reported previously on binding of corticosterone by fractions isolated from chromatin of rat thymocytes 6. This report presents evidence for cytoplasmic component(s) which * Present address: Veterans Administration Hospital, D e p a r t m e n t of Pathology, Albany, N.Y. 122o8, U.S.A. * * Present address: Searle Reference Laboratory, 2775 H o m e Road, Powell, Ohio 43o64, U.S.A.

Biochim. Biophys. Aeta, 264 (1972) 557-565

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J . M . AUGUSTYN, W. K. BRUNKHORST

specifically bind corticosterone and establishes that the steroid which is bound to chromatin is unmetabolized corticosterone. MATERIALS

rI,2-3H] Corticosterone (d4-pregnene-II/3, 2i- diol-3, 2o-dione) was obtained front New England Nuclear Corporation. Non-radioactive eorticosterone and I I dehydrocorticosterone (z~4-pregnene-2I-ol-3,II,2o-trione) were purchased from Calbiochem. 5fl-Pregnane-3~,Ilfi,2I-triol-2o-one and 5fl-pregnane-3~,Ilfi,2ofl,2I-tetrol were supplied by Ikapharm (Israel). We are indebted to Dr Marcel Gut of the Worcester Foundation for synthesizing z]4-pregnene-xlfl,2o,2I-triol-3-one. Corticosterone-2Iacetate was purchased from Sigma Chemical Co. and I I-dehydrocorticosterone acetate was purchased from Steraloids, Inc. Acetates of remaining standards were prepared under standard eonditionsL The purity of all steroids was verified by thin-layer chroInatography prior to their use as standards or carriers. METHODS

Preparatio~ of tissue fractions Male Sprague-Dawley rats 35-45 days old, were adrenalectolnized and maintained on Purina rat chow and 0. 9 % saline for 4 days. For in vivo experiments ea~ h rat was injected intraperitoneally with I ml saline containing 33 t tCi [I,2-3HJcorticosterone (44-52 Ci/mmole) and sufficient cold corticosterone to give a final concentration of I ¢tg/ml. At various time intervals after injection of hormone, animals were decapitated, thymus glands were removed, and thylnocytes were teased into 4 ml of a solution of ice-cold (0.25 M sucrose-3 mM MgCl~)-Eagle's minimal essential medium (3:I, v/v). Thymocytes were filtered through iniracloth (Chicopee Mills, New York, N.Y.) and cytoplasmic and nuclear fractions were prepared as indicated in Fig. I. Purified chrolnatin was prepared according to the method of Swaneck et al. s and solubilized in o.oi M Tris buffer, pH 8.0, by means of a Polytron (Type PTIo Kinematica GlnbIt, Luzern, Switzerland). For i~.zvitro experiments, filtered thymocyte suspensions, prepared as described for in vivo experiments, were diluted to a final concentration of 3" lO6-5 . Io6 cells per Inl with minimal essential medium. 5oo-ml aliquots were incubated with ~I,2-~Hieorti costerone (8 nM) under an atmosphere of O2-CO 2 (95:5, v/v) ior I5 or 9° Inin at 37 °C in a shaking water bath. After incubation, thylnocytes were chilled at 0- 4 °C for 15 rain and centrifuged at 900 × g for Io Inin. The resulting pellets were resuspended in a volume of (0.25 M sucrose-3 mM MgC12)-ininilnal essential medium (3 :I, v/v) equal to one-fifth the incubation volume. Nuclear, cytoplasmic and chromatin fractions were prepared as described in Fig. i.

Extractiot~ of steroids and chromatography Tissue fractions were made I3 % with respect to ethanol. In in vivo experiments 3-5 mg of free steroid standards were added to serve as carriers. In in vitro experiments 20 #g quantities oI carriers were added. The larger amounts were added in in vivo studies in anticipation of making acetate derivatives. Each fraction x, as extracted four times with 2 vol. of ethyl acetate. Extracts were taken to dryness Biochim. Biophys. Acta, 264 (1972) 557-565

559

CORTICOSTERONE BINDING AND METABOLISM Labeledthymoeytes(in 0.25 Msucrose-3mMMgCl2-minimalessentialmedium) Chilledto 0 °C to 4 °C, all procedures were carried out at 0 to 4°C. Centrifuge 900 ×g, 10 min

]

I

Supernatant

Pellet, resuspendin 0.25 Msucrose-3mMMgCI2 (finalconch= 10-106-15 • 106 cells/ml),homogenize (10 strokes Potter-Elvehjem teflon homogenizer) at 1300 rev./min. Filter through flannel. Homogenize filtrate 6 additional strokes I Centrifuge 900 x g , 10 rain

I

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I

Supematant= cytoplasm

I Centrifuge900 X g, 10 min

L

Washed nuclei, extract 3 times

I

Supematant

in 0.01 MTfis-3mMMgCl2, pH 7.1 (50. 106-100 • 10a cells/ml).Filterthird Trisnuclear suspension through flannel I I Centrifuge900 ×g, 10 rain

[

I

Pellet,fractionate to purified chromatinaccording to the method of Swaneck et al.8

I

Supernatants = 1st, 2nd, 3rd Tris washes

Fig. I. Procedure for fractionation of thymus tissue. u n d e r reduced pressure and applied to glass plates coated with silica gel G. Chromatograms were developed at room t e m p e r a t u r e in c h l o r o f o r m - m e t h a n o l - w a t e r (I88:24:2, b y vol.). Standards were located with ultraviolet absorbance or with phosphomolybdic acid spray. Sections of silica gel, corresponding to each standard, were scraped from the plates, transferred to scinter funnels a n d eluted with the developing solvent followed b y ethyl a c e t a t e - m e t h a n o l (3 :i, v/v). I n i n vitro experiments eluates were dried a n d counted i n toto. I n i n vivo experiments eluates from areas corresponding i n m o b i l i t y to s t a n d a r d s were dried, redissolved in a small volume of ethyl acetate a n d an aliquot removed for counting. The r e m a i n d e r of t h e sample was aeetylated u n d e r s t a n d a r d conditionsL Acetates were rechromatographed Oil silica gel using a solvent s y s t e m of c h l o r o f o r m - m e t h a n o l - w a t e r (242:7-5 :~, b y vol.). Identifications were made using s t a n d a r d steroid acetates a n d elutions were carried out as described for free steroids. Concentration curve

T h y m o c y t e suspensions (2. IO~ cells per ml), prepared as described for i n vitro experiments, were i n c u b a t e d for ~5 rain with [1,2 3Hlcorticosterone (44 Ci/mmole) Biochim. Biophys. Acta, 264 (1972) 557-565

560

J. M. AUGUSTYN, W. K. BRUNKHORST

with or without added non-radioactive steroid. The cells were chilled to o- 4 °C and centrifuged at 900 × g for IO rain. After washing the cells in minimal essential medium, nuclear and cytoplasmic fractions were prepared as described in Fig. I.

Analytical procedures Protein concentrations were determined by the method of Lowry et alY. DNA was determined by the method of Webb and Levy 1°. Purity of nuclear fractions was evaluated enzymatically by assaying for cytochrome oxidase activity n and histologically by viewing unstained preparations under the phase microscope and preparations stained with toluidine blue and fast green under the light microscope. Purified chromatin was examined by electron microscopy and its ultraviolet absorption spectrum was determined.

Determination of radioactivity Aliquots of cell fractions were solubilized in NCS (Nuclear Chicago Solubilizer) before IO ml of a toluene-based scintillation fluid were added. Eluates from thinlayer chromatograms were collected directly into scintillation vials. After evaporation of the solvent in a vacuum oven, scintillation fluid was added. The radioactivity of all samples was determined ill a liquid scintillation counter (Nuclear Chicago, Mark I). Correction for quenching was made using the Channel ratio method ~2. RESULTS

Purity of nuclei Cytochrome oxidase activity has been used traditionally as a measure of mitochondrial activity and hence of cytoplasmic contamination. Only lO% of the cytochrome oxidase activity present in whole cells was detected in our nuclear prepara tions. Examination of nuclei under a phase microscope and under the light microscope also established that maximum contamination of nuclei with whole cells or cytoplasmic tags was lO%. Extraction of nuclei 3 times with o.oi M Tris-3 mM MgCle, p H 7.1, removed essentially all cytoplasmic tags. In some experiments nuclei in 0.25 M sucrose-3 mM MgCI~ were purified by the convential procedure of centrifugation through a sucrose gradient composed of equal volumes of 1. 9 M sucrose-3 mM MgC12 and 1.6 M sucrose-3 mM MgC12. Metabolite studies on these preparations gave results comparable to those obtained from nuclei prepared as described in Fig. i. Examination of nuclei prepared by either procedure established that purity was comparable to that described by Allfrey et at. ~8.

Purity of chromatilz No cytochrome oxidase activity was measured in chromatin preparations. Electron microscopy also verified the preparations were pure except for occasional membrane profiles. The A26onm/A2sonm ratio was I. 7 ~ o.I, the A~6onm/A31onm ratio was 0.038 i 0.006 and the protein/DNA ratio was i . i ~ 0.4. These values satisfy the criteria of purity as discussed by Bonner et al. 14.

Biochirn. Biophys. Acta, 264

(I972) 557 565

I

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WITH CORTICOSTI~RONE

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36

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17

65

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I n vitro

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I n vivo

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5

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23

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I n vivo

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Cytoplasmic extracts(%)

from the origin to the position of corticcsterone.

I n vivo

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i I-Dehydrocorticosterone

Corticosterone

45 rain

In vivo

I n vitro

r5 rain

Nuclear extracts ( % )

have been

6

8

23

63

I n vitro

A d r e n a l e c t o m i z e d m a l e r a t s w e r e s a c r i f i c e d 15, 45 o r 9 0 r a i n a f t e r i n t r a p e r i t o n e a l i n j e c t i o n w i t h I m l o . 9 % s a l i n e c o n t a i n i n g 33 ~ C i [ i , 2 - a H ] c o r t i c o s t e r 0 n e ( 4 4 - 5 2 C i / m m o l e ) a n d s u f f i c i e n t c o l d c o r t i c o s t e r o n e t o g i v e a f i n a l c o n c e n t r a t i o n o f I # g / r o t . I n in vitro e x p e r i m e n t s , f i l t e r e d t h y m o c y t e suspgn'sions were incubated with [i,2-aH]corticosterone (8 nM) u n d e r a n a t m o s p h e r e o f O ~ - C O 2 ( 9 5 : 5 , v / v ) f o r 15 o r 9 0 r a i n a t 37 °C. N u c l e a r a n d cytoplasmic fractions were prepared as described (Methods). 2o-#g quantities of cold corticosterone and I I-dehydrocorticosterone were added as carriers. Extraction and chromatography were carried out as indicated for free steroids in Methods.

D I S T R I B U T I O N OF R A D I O A C T I V I T Y IN N U C L ~ ' I A N D C Y T O P L A S M OF RAT TI-IYMOCYTES F O L L O W I N G T R E A T M E N T

TABLE

H

562

j.M. AUGUSTYN, W. K. BRUNKHORST

I n vivo vs in vitro metabolism of corticosterone Only minor metabolic transformations occur during in vitro incubations (Table I). In nuclei, even after 9 ° rain, 80 % of corticosterone was unmetabolized. 5 % of the steroid was converted to II-dehydrocorticosterone; the remaining radioactivity was distributed over the chromatogram with the concentration in a single area never exceeding 2-3 %. Cytoplasm prepared from thymocytes incubated in w;tro shows that the major transformation product of corticosterone was ii-dehydrocorticosterone. 15 rain after injection of the hormone in vivo, unmetabolized corticosterone represented 39 % of the total recovered radioactivity in the nuclei and by 45 min, the value decreased to I6 %. Significant amounts of radioactivity, which increased with time, were detected in the section of the chromatogram between the origin and corticosterone. Very little radioactivity was detected in the corresponding regions Gf the chromatogram from in vitro experiments suggesting that in in vivo experiments these metabolites originate in other tissues, e.g. the liver, and reach the thymus via the circulation. In nuclei, ii-dehydrocorticosterone was never an important metabolite. Cytoplasmic fractions from tbymocytes labelled in vivo also showed major transformations to compounds more polar than corticosterone. In addition, significant amounts of ii-dehydrocoticosterone were present, which decreased with time after injection relative to the more polar compounds. Ide~ztificatio~ of metabolites Cell fractions were prepared from labelled thymocytes obtained from rats injected 15 rain previously with [I,2-3HJcorticosterone and were extracted with ethyl acetate. The extracts were subjected to chromatography on silica gel G using the system for free steroids described in Methods. Fig. 2 illustrates results from one such experiment. Some significant observations are the following: (I) Most of the metabolites of eorticosterone present in nuclei are removed by extracting with Tris buffer. The only steroid present in chromatin in significant amounts is unmetabolized corticosterone. (2) The major metabolites of corticosterone in cytoplasm and the Tris extract are ii-dehydrocorticosterone, 5fl-pregnane-3~,Ilfl,2ofl,2I-tetrol and d4-pregnene ii~,2o,2i-triol-3-one. Only small amounts of 5fl-pregnane-3~,II/~,2i-triol-2o-one occur, accounting for less than 5 % of the total radioactive compounds. (3) Only two areas of radioactivity remain unidentified. One appears in the Tris extract and is more polar than 5fl-pregnane-3~,Ilfi,2ofl,2I-tetrol. The second occurs in the cytoplasmic fraction and migrates between da-pregnene-Ilfl,2o,2I-triol-3-one and corticosterone. Only low activities are detected in comparable areas of other chromatograms. The identity of the metabolites produced in in vivo experiments was confirmed by acetylating the compounds recovered from the chromatograms and subjecting the acetates to thin layer chromatography. With three exceptions, to be discussed, all areas of radioactivity corresponded to aeetylated standards and recoveries for the acetates were 8O-lOO%. Chromatograms of the acetates of II-dehydrocorticosterone isolated from the Tris extract and from chromatin both contained a radioactive area equivalent to Biochim. Biophys. Mcla, 264 (1972) 557-565

563

CORTICOSTERONE BINDING_AND METABOLISM CYTOPLASM IN VIVO CYTOPLASM CI~ IN VITRO

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Fig. 2. D i s t r i b u t i o n of r a d i o a c t i v i t y in c y t o p l a s m , Tris e x t r a c t or p u r e c h r o m a t i n p r e p a r e d f r o m in vivo or in vitro i n c u b a t i o n s of r a t t h y m u s . A, 5fl-pregnane-3~,Ilfl,2ofl,2I-tetrol; B, A4-preg n e n e - i 1/5,2o,2 i-triol-3-one ; C, corticosterone ; D, i I - d e h y d r o c o r t i c o s t e r o n e a n d E, 5fl-pregnane3~, I Ifl, 21-triol-2o-one.

25-30% of the total, which had a mobility slower than II-dehydrocorticosterone acetate. The unacetylated steroid and ilfi-hydroxyandrostenedione have nobilities corresponding to this area. No attempt was made to establish whether these were the compounds present. It has been reported, however, that mouse thymus metabolizes cortisol to ii/%hydroxyandrostenedionO% The chromatogram of the acetate derivative of A*-pregnene-Ilfl,2o,2I-triol-3one from cytoplasm contained two areas of radioactivity between the acetate and the solvent front. Each represented about 20 % of the total radioactivity. The identity of these compounds is not known.

Effect of steroid concentration on amount of bound corticosterone To ascertain the effect of steroid concentration on the uptake of corticosterone by thymocytes, cell suspensions were incubated for 15 rain in minimal essential medium with the final concentrations of corticosterone ranging from I nM to 3 /~M (2-18o #Ci [I,2-3H]corticosterone). The amount of steroid bound b y whole cells and cytoplasmic and nuclear fractions was determined. The data from a representative experiment are plotted in Fig. 3. All three curves deviate from linearity at low hormone concentrations, however, above about IOO riM, corticosterone uptake is apparently linear, suggesting binding by "non-specific" sites which are not saturated even at these high concentrations. The non-linear portion of the curves indicates binding by components which become saturated at about ioo nM. Biochim. Biophys. Acta, 264 (1972) 557-565

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J. M. AUGUSTYN, W. K. BRUNKHORST

DISCUSSION

There are several reports in the literature on i.n vitro studies of metabolism of glucocorticoids by thymocytes. ]3urton 1~ found that dehydrogenation at C-II and, to a lesser extent, removal of the side chain occurred upon incubation of {4-14C] cortisol with mouse thymus. Rat thymus showed no demonstrable metabolism. In both species, unchanged cortisol was identified as the major or only component bound. Mahesh and Ulrich ~ identified metabolites of cortisol in different rat tissue preparations. A mince of thymus tissue converted trace amounts of cortisol to the 2odihydro derivative. The remainder was recovered unchanged. More recently, Munck and Brinck-Johnsen ~ studied the binding of cortisol by cell suspensions of rat thyrnocytes. They established that 80 % of the specifically bound steroid was cortisol. We present data in Fig. 2 which establishes that when rat thylnocytes are incubated in vitro with the naturally occurring hormone, corticosterone, 85-9o % of the steroid bound is unmetabolized corticosterone. 3'

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Fig. 3. Corticosterone bound by fractious of rat thymocytes. Corticosterone bound by cytoplasm (C), whole cells (WC), nuclei (N) in pmoles/mg protein (ordinate) is plotted (logarithmically) against the concentration of corticosterone during incubation (abscissa).

A detailed analysis of the metabolism of glucocorticoids following in viva administration of hormone has not been reported previously for thymus. Fig. 2 shows that 70-85 % of the radioactivity bound by chromatin isolated from thymocytes exposed in viva to hormone is ill the form of corticosterone. This chromatin was derived from nuclei which contain a high percentage of metabolites. We interpret this finding as evidence that the final "specific" receptor for glucocortieoids in thymus is in chromatin. Thymus tissue does not contain the glucocorticoid metabolizing enzymes present in liver, but it does contain Ilfl-hydroxydehydrogenase. Thus, the conversion of corticosterone to II/5-dehydrocorticosterone occurs in the thymus. The compounds, 5fl-pregnane-3c¢,ii/5,2o/3,2I-tetrol and 5/3-pregnane-3:qlIfl,2I-triol-2o-one have been identified by Lowry et al. 17 and Erickson and Gustafsson is as liver metabolites of corticosterone. Z14-Pregnene-II/J,2o,2I-triol-3-one has not been reported by others as a metabolite of thymus ceils and was not observed in high concentrations in our in vitro experiments. Its presence in high concentration in in viva experiments suggests it Biochim. Biophys. Acta, 264 (1972) 557-565

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565

may be a blood borne metabolite of extra-hepatic origin. These conclusions concerning the site of production of the metabolites are substantiated by our observation that extracts of serum derived from corticosterone-injected rats contain the same major metabolites. The data in Fig. 3 indicate that "specific" receptors in nuclei are saturated at approx. IOO nM concentrations of corticosterone. This is illustrated by the non-linear uptake at low concentrations of hormone. The linear uptake at high concentrations represents binding at "non-specific" sites which are not saturated even at the highest concentrations studied. Saturation of the "specific" sites occurs at physiologicM concentrations of corticosterone. Even though our metabolic transformation studies indicate the final receptor is in chromatin, some component(s) of cytoplasm specifically binds corticosterone. This result agrees with that obtained by Baxter et al. 19 in a study of cortisol binding by steroid-sensitive mouse lymphoma cells where saturation of specific sites in nuclei and cytosol occurs at about 30o nM eortisol. Baxter artd Tompkins ~ have studied specific cytoplasmic receptors for glucocortieoids in cultured rat hepatoma cells. Sekeris and his co-workers 3 have reported on specific binding sites for cortisol in normal rat liver cytosol and the transfer of ESHI cortisol from these sites to the nucleus. If specific glucocorticoid-binding sites occur in both the cytoplasm and nuclei of thymocytes, their it is quite possible that the sequence of events for the binding of these hormortes by their receptors will be similar to that which has been elucidated for other steroid hormones. ACKNOWLEDGEMENTS

We are indebted to Dr Peter Nickerson, Department of Pathology, S.U.N.Y. at Buffalo for carrying out electron micrograph studies. This work was supported by National Science Foundation Grant-GB-2517I, U.S. Public Health Service Grant RR-o5528 and U.S. Public Health Service Grant 5TolAMo5564 National Institutes Arthritis and Metabolic Diseases. REFERENCES I J. D. Baxter and G. M. Tompkins, Proc. Natl. Acad. Sci. U.S., 65 (197 o) 709. 2 J. D. Baxter and G. M. Tompkins, Pfoc. Natl. Acad. Sci. U.S., 68 (1971) 932. 3 M. E. Beato, W. Brandle, D. Biesewig and C. E. Sekeris, Biochim. Biophys. Acta, 208 (197 o) 125. 4 A. Munck and T. Brinck-Johnsen, J. Biol. Chem., 243 (1968) 5556. 5 C. Wira and A. Munck, J. Biol. Chem., 245 (I97 o) 3436. 6 W. K. Brunkhorst, Biochem. Biophys. Res. Commun., 35 (1969) 88o. 7 A. Zaffaroni and R. B. Burton, J. Biol. Chem., 193 (1951) 749. 8 G. E. Swaneck, L. L. H. Chu and I. S. Edelman, J. Biol. Chem., 245 (197o) 5382. 9 0 . H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem., 193 (1951) 265. IO J. M. Webb and H. B. Levy, J. Biol. Chem., 213 (1955) lO7. II S. J, Cooperstein and A. Lazarow, J. Biol. Chem., 189 (1951) 665. i2 Y. Kobayashi and D. V. Maudsley, Methods of Biochemical Analysis, Vol. 17, Interscience, New York, 1969, p. 55. 13 V. G. Allfrey, V. C. Littau and A. E. Mirsky, J. Cell Biol., 21 (1964) 213. 14 J. Bonner, G. R. Chalkley, M. Dahmus, D. Fambrough, F. Fujimura, R. C. Huang, J. Huberman, R. Jensen, K. Marushige, H. Ohlenbush, B. Olivera and J. Widholm, in L. Gorssman and K. Moldave, Methods in Enzymology, Vol. 12, Part B, Academic Press, New York, 1968, p. 7. 15 A. F. Burton, Cancer Res., 24 (1964) 470. 16 V. R. Mahesh and F. Ulrich, J. Biol. Chem., 235 (196o) 356. 17 J. Lowry, T. Albepart and J. R. Pasqualini, Acta Endocrinol. 61 (1969) 48318 H. Erickson and J. A. Gustafsson, Eur. J. Biochem., 2o (1971) 231. 19 J. D. Baxter, A. W. Harris and G. M. Tompkins, Science, 171 (1971) 189.

Biochim. Biophys. Acta, 264 (1972) 557-565