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Interactions of hormones with thymus-cell fractions
BIOCHIMICA ET BIOPHYSICA ACTA
385
BBA 4224
INTERACTIONS
OF HORMONES
WITH
THYMUS-CELL
FRACTIONS
WILLA K. B R U N K H O R S T AND E U G E N E L. HESS
Worcester Foundation for Experimental Biology, Shrewsbury, Mass. (U.S.A .) (Received May 9th, 1963)
SUMMARY
The binding of cortisol, progesterone and testosterone to thymus-cell fractions was studied b y the equilibrium dialysis procedure. The hormones appear to follow the "polarity rule", progesterone binding to the greatest extent, cortisol the least. Ribosomal particles seem to have a higher capacity for binding hormone than the p H 5.1 precipitate or the material in the supernatant resulting from treatment of microsomal fraction with deoxycholate. The possibility is discussed that progesterone is binding to a single component in the ribosomal fraction while cortisol and testosterone bind to more than one component.
INTRODUCTION
Interaction of steroid hormones with reactive sites on certain cellular proteins has been considered as a possible mechanism by which hormones initiate their physiological effect. The distribution of glucocorticoids in plasma and various tissues after intravenous injection of radioactive steroids has been studied by several groups of investigators 1-3. Recently DE VENUTO, KELLEHER AND WESTPHAL4 reported on the interaction of cortisol and corticosterone with cell fractions from rat-liver and muscle after injection of the hormones into the living animal, or after addition to organ homogenates. Nuclear, mitochondrial and microsomal fractions from liver interacted with cortisol and corticosterone while no significant binding was observed with muscle. It was considered worthwhile to study the interaction of cortisol with cell fractions from a gland or organ which undergoes a distinctive response to the hormone. It is well known that the thymus of animals treated with glucocorticoids undergoes rapid involution. FIRSCHEIN and associates 5 found a greater percentage of an injected dose of cortisolin the thymus of stressed rats as compared with control rats. Detection of an interaction with a particular cellular component which is specific for cortisol might indicate the site of initial action of the hormone. A previous paper described the binding of cortisol by a fraction from thvmus which contains most of the nucleic acid in the cytoplasm of lymphocytes 6. The work reported here was undertaken to study the binding in vitro of cortisol by various cell fractions which are involved in protein synthesis. Demonstration of a specific Abbreviation: DOC, deoxycholate.
Biochim. Biophys. Acta, 82 (I964) 385-393
386
w . K . BRUNKHORST, E. L. HESS
cortisol effect on one of these fractions could provide a plausible explanation of the involution produced by the glucocorticoids. EXPERIMENTAL
Various fractions were prepared from calf thymus obtained from a commercial slaughter house. Glands were chilled as quickly as they were removed from the animal and subsequent fractionation was carried out in the cold room at 4-5 °. After the tissue was minced and put through an onion press, three volumes o.25 M sucroseo.oo33 M CaC12 per gram wet tissue were added and the suspension was stirred in a Waring Blender at approximately IOOO rev./min for 4 min. The homogenate was centrifuged at 14oo >~, g for 7 min and the resulting supernatant was centrifuged at 15ooo × g for 2o min. The 15ooo "~ g supernatant was shell frozen and stored at - - 2 o ° until used.
Preparation of microsomal fraction, ribosomes and fraction S 5.r P The 15 ooo x g supernatant was centrifuged at lO5 ooo x g in a Spinco Model L centrifuge for I h to yield a pellet referred to as microsomal fraction. An additional fraction was precipitated from the lO5 ooo g supernatant by adjusting the p H to 5.1 with o.i M acetic acid. Centrifugation at 15ooo ~: g for 15 min brought down the precipitate, designated fraction S 5.IP, which contained soluble RNA and activating enzymes essential for amino acid incorporation into protein. In some experiments microsomal pellets were re-suspended in 3 ml o.25 M sucrose and 0.3 ml of 3 % sodium deoxvcholate (pH 8) was added. After IO min the volume was adjusted to 12 ml with o.25 M sucrose and the suspension was centriiuged again at lO5 ooo ~" g for I h. The resulting pellet contains ribosomal material. The deoxycholate supernatant from this step was dialyzed and lyophilized. All fractions were resuspended in o.o5 M NaHCO3 except in those experiments in which effects of various buffer ions were investigated. In this case the fractions were resuspended in the Tris or phosphate buffer as described under RESULTS. Concentrations were determined by analyzing the solutions for protein using the method of LowRY et al. 7 and correcting for RNA and lipid content. The amount of RNA and lipid in the fractions had been established previously.
Equilibrium dialysis procedure An equilibrium dialysis procedure was employed to study the interaction of the thymus fractions with various hormones. Since a significant amount of cortisol was bound by unwashed casing, Visking casing (32/lOO in diameter) was washed three times in 50 % ethyl alcohol followed by three rinses with distilled water at 50-60 °. Dialysis bags prepared from the washed Visking were air dried and used in the equilibrium dialysis studies. A volume of 1.5 ml of the thymus fraction, which contained the hormone, was placed in the dialysis bag along with an 8 mm glass rod. The bag was inserted in a 22 - 175 mm glass stoppered test tube containing 6 ml of 0.05 M NaHCOa. The tubes were placed in a rack which held them at a 4 °0 angle and shaken gently in an automatic shaker for 3 h at room temperature. Control experiments have shown that equilibrium is attained in this length of time. Amounts of steroids used are given in the tables. The specific activity of the Bioehim. Biophys. Acta, 82 (1964) 385-393
HORMONE INTERACTIONWITH THYMUS FRACTIONS
387
~4-14Clcortisol was 42.4 t~C/mg, that of [4-14CJprogesterone was 17 ~C/ml and that of E4-14Cltestosterone was 22/~C/mg.
Plating procedure An accurate correction for self-absorption could not be made by the usual method of determining the weight of the sample on the plate and applying a correction factor for self-absorption. For unknown reasons plating of hormone in the presence of thymus fractions resulted in counts in excess of the amount of radioactivity added. The following procedure was used to obtain correct binding data. The solution within the dialysis casing was diluted I : I with the buffer. The dialysate was diluted I : I with a protein solution of the same composition as that within the dialysis casing, except that no radioactive steroids were included. Triplicate samples of lOO-4 aliquots from both solutions were plated. Plates were counted on a thin-window gas.flow counter and counts were expressed as counts/min/ml. Since both parts of the equilibrium dialysis system contained the same amount of protein when plated, similar counting behavior was insured. Although the counts recorded did not represent the absolute number of counts present the difference between sack and dialysate was valid and an accurate calculation of the per cent bound hormone could be made. In experiments in which the data were analyzed using a SCATCHARDtype plot 8 it was necessary to establish the concentration of cortisol both in the sack and in the dialysate. A correction for the counting error was made, therefore, in the following manner. Control tubes were included in each experiment. Hormone equivalent to the amount in experimental tubes was dissolved in 1.5 ml buffer and placed in the sack. Dialysis was against 6 ml of buffer. After equilibrium dialysis the contents of the sack and the dialysate were diluted I ' i with buffer and triplicate aliquots of IOO ~ were plated. The connts on the plates gave an accurate measure of the radioactivity and could be related directly to the concentration of cortisol. The total counts in the system should be the same for control and experimental tubes since identical amounts of hormone were added to both. Higher counts were always obtained with experimental tubes presumably due to the protein on the plates. The per cent difference between total counts in experimental and control tubes was applied as a correction factor to the plates containing protein. The correct counts/min/ml thus determined were converted to concentrations of hormone in the sack and dialysate. The total amount of hormone in the system as calculated from the corrected counts/min/ml was in agreement with the amount added to the tubes.
Calculations The interaction between hormone and thymus fractions was expressed as per cent steroid bound. steroid bound % steroid bound steroid total × ioo At equilibrium the concentration of unbound steroid in the dialysis bag was assumed to be equal to the steroid concentration in the dialysate. The concentration of bound steroid could thus be obtained as the difference between the total steroid in the dialysis sack and the unbound or free steroid.
Biochim. Biophys. Acta, 82 (1964) 385-393
38~
W. K. B R U N K H O R S T ,
E. L. HESS
Data were plotted according to the equation (ref. 8): r
= k ( n - r) C
where r is the number of moles of bound cortisol per mole of total protein, c is the concentration of free cortisol, n is the number of binding sites per molecule protein and k is the association constant. The relation permits the determination of n and k under the assumed condition that combining sites on the protein are equivalent and independent. RESULTS A N D DISCUSSION
The extent of interaction of thymus fractions with cortisol, as well as progesterone and testosterone, was determined. The results are summarized in Table I. The hormones appear to follow the "polarity rule" as proposed by EIK-NES et alY and discussed by WESTPHALL10 for such proteins as albumin. The binding of cortisol was essentially the same for all fractions when the concentration of protein is expressed on a weight per volume basis. Weight average molecular weights of 3" lO8 for ribosomes, I. lOs for microsomes, I- lO 5 for DOC supernatant and 25000 for S 5.1 P were assumed and the binding was expressed on the basis of molarity of the various fractions. TABLE INTERACTION
OF
HORMONES
WITH
I
VARIOUS
CELL
FRACTIONS
OF
THYMUS
V a l u e s g i v e n a r e a v e r a g e s of t w o e x p e r i m e n t s a n d a r e e x p r e s s e d as p e r c e n t h o r m o n e b o u n d . T h e c o n c e n t r a t i o n s of a l l f r a c t i o n s w e r e i % i n 0.0 5 M N a H C O 3. Cell fraction Hormone
Cortisol* Progesterone** Testosterone** *
M icrosomal
Ribosomal
5 5. t P
Deoxycholate supernatant
i5.2 84.o 54.3
16.2 88. I 48-9
15.5 65.o 33.8
14.9 76. i 4 t .4
* 2 # g c o r t i s o l i n t o t a l s y s t e m (7.5 ml). C o n c e n t r a t i o n w o u l d b e 7" io- 7 M a t e q u i l i b r i u m if n o binding occurred. 1.4 P g p r o g e s t e r o n e in t o t a l s y s t e m . C o n c e n t r a t i o n w o u l d b e 6. lO -7 M a t e q u i l i b r i u m if n o binding occurred. 1. 5 p g t e s t o s t e r o n e in t o t a l s y s t e m . C o n c e n t r a t i o n w o u l d b e 7" lO-V M a t e q u i l i b r i u m if n o binding occurred.
Microsomes at approx, io -5 M bound 15 % cortisol while ribosomes bound 23.5 % S 5.IP bound 2.9 % and DOC supernatant bound 2.3 %. Mole for mole the microsomal and ribosomal fractions appear to have a higher capacity for binding the hormone than S 5.IP or DOC supernatant. The increased binding observed with the ribosomes suggests that the particles are largely responsible for binding by the microsomal fraction. Based on the binding in vitro reported here, the DOC supernatant and 5.IP fractions must be present in molar concentrations 30-40 times that of the particles if they bind a significant amount of hormone in the cell. According to findings in this laboratory these fractions are at most three to four times the concentrations of the ribosomal fraction. It would appear, therefore, that the particles bind most of the hormone in the cytoplasmic portion of the cell. B i o c h i m . B i o p h y s . A c l a , 82 (1964) 385 393
389
HORMONE INTERACTION WITH THYMUS FRACTIONS TABLE II BINDING
OF
CORTISOL
PRESENCE
OF
BY
MICROSOMAL
FRACTION OR
PROGESTERONE
OF
THYMUS
IN
TESTOSTERONE
Values given are averages of two experiments and are expressed as per cent h o r m o n e bound. Microsome concentration was i % in o.o 5 M N a H C O 3. 2/~g cortisol were present in total system of 7.5 ml. A m o u n t s given for second h o r m o n e were amounts present in total system. A mount o~ second hormone
Cortisolonly
Cortisoland progesterone
Cortisoland testosterone
I3. 3 i4.i 14.5
I6.O 14. 5 13.6
I3.9
O. I I /~g/ml 0.22 /~g/ml 2.20 /zg/ml
A
120
35 9C
25
X
X
~lu 6C 3C
%
I
I
10
20
I 30
r X
15
40
50
I
vo 25
60
I
75
:"
125
,
,75
"
300
aso
r X 10.2
1 0 -~
C 35
qo
°b%~
25
X
Fig. i. Binding of progesterone b y t h y m u s fractions. A, nficrosomes; B, ribosomes; C, DOC s u p e r n a t a n t ; r is n u m b e r of moles of b o u n d cortisol per mo]e of total protein, c is concentration of free progesterone.
~Ju 15
410
8I0 12G r x 10+*
160
280
320
A l t h o u g h none of t h e cell fractions i n t e r a c t exclusively with cortisol, t h e b i n d i n g of the h o r m o n e a p p e a r s to occur at a specific site in the microsome fraction. As seen in t h e d a t a in T a b l e I I , a d d i t i o n of increasing a m o u n t s of u n l a b e l e d progesterone or t e s t o s t e r o n e has no effect on the a m o u n t of cortisol bound. Thus, neither progesterone nor t e s t o s t e r o n e c o m p e t e s with cortisol for the same site. Using t h e SCATCHARD e q u a t i o n 8 a t y p i c a l plot with a n e g a t i v e slope was o b t a i n e d for p r o g e s t e r o n e i n t e r a c t i n g w i t h the m i c r o s o m a l fraction a n d also with t h e two m a i n c o m p o n e n t s of t h e m i c r o s o m a l fraction, ribosomes a n d DOC s u p e r n a t a n t . These results are r e p r e s e n t e d in Fig. i . N values can be considered as only a p p r o x i m a t e since t h e fractions are r e l a t i v e l y crude a n d a v e r a g e molecular weights were a s s u m e d for t h e calculations. Considered in this light t h e N values o b t a i n e d from t h e plot B i o c h i m . B i o p h y s . A c t a , 82 (1964) 385-393
39 °
w.K. BRUNKHORST, E. L. HESS
are approximately o.6 for microsome and 3.5 for ribosome fractions and o.o3 for DOC snpernatant. Curves with positive slopes were obtained for binding of testosterone and cortisol to all fractions studied. Similar results were obtained for fraction S 5.IP binding of progesterone. The positive slope suggests that: (a) the association constant changes with varying hormone concentration or (b) the number of sites available for binding is changing or (c) a mixture of several components having different binding affinities for the hormones are present in the fractions. The third possibility is most probable. The influence of other buffer systems on the interaction of cortisol with microsomal, S 5.IP and ribosomal fractions was investigated using potassium phosphate buffer, 0.05 M (pH 7.5), potassium phosphate buffer, 0.05 M (pH 7.6), containing 0.002 M MgC12 and 0.05 M Tris (pH 7.5). The extent of binding was the same as when the fractions were suspended in 0.05 M NaHCO 3, the buffer used in most of the experiments reported here. Phosphate, Tris or bicarbonate ions had no effect on the interaction, nor did the pH of the solution appear critical since the buffers varied from pH 7.5 for phosphate and Tris to 8.6 for 0.05 M NaHCO3. Binding of cortisol to transcortin has been reported to vary inversely with an increase in temperature. Interaction of cortisol with serum albumin, on the other hand, is not strongly temperature dependent 11. No thermal effect was found with thymus fractions. Cortisol was bound to the same extent by microsomal and pH 5 fractions at 4 ° and at room temperature. Effects of varying conditions under which microsomes were prepared and under which equilibrium dialysis was performed were investigated. Conditions were chosen which are known to affect the size distribution of ribosomes and their biological activity. I. Frozen instead of fresh thymus was used for preparation of the microsome fraction. It is known that biological activity of the microsomal fraction is lost when the tissue is frozen 12. Interaction with cortisol was not affected, however, by this treatment. It was also found that microsomes could be prepared from fresh or frozen tissue, suspended in 0.05 M NaHCO 3 and frozen for as long as one month without altering their ability to interact with cortisol. 2. The sedimentation properties of microsomal preparations resuspended in water and phosphate, Tris and NaHCOs buffers were examined. As seen in Fig. 2 sedimentation properties depend upon the buffer system. The maior component of microsomes in phosphate buffer has a sedimentation coefficient of 67 S, which, when corrected to infinite dilution, should correspond to the 74 S particles reported by HESS et al. 1~. Smaller amounts of 54 and 39 S components were present. The preparation in NaHCO3 also contained a component equivalent to 74 S particles, however, there were greater amounts of slower sedimenting material. Neither Tris nor water solutions contained 74 S particles. The preparation in water contained the largest amount of the slow sedimentating components. In spite of these differences all the preparations bound cortisol to the same extent. 3- It is well established that the concentration of Mg ions affects the size distribution and the extent of aggregation of ribosome particles 18,14. As seen in Fig. 2 the effect applies to the microsome fraction as well. Microsomes were prepared from fresh thymus and resuspended in 0.05 M Tris (pH 7.5), which contained varying Biochim. Biophys. Acta, 82 (1964) 385-393
HORMONE INTERACTION WITH THYMUS FRACTIONS
391
concentrations of Mg ions. The interaction of the solutions with cortisol was measured. As seen in Table I I I , Mg 2+ had no effect on the extent of binding. 4. The effect of aging on the capacity of microsomes to interact with cortisol was also investigated. A fraction resuspended in potassium phosphate buffer (pH 7.5), containing 0.002 M MgCI~ was dialyzed against the same buffer for 60 and 96 h at 4 ° without changing the dialysate. The amount of cortisol bound b y 0. 4 % solution of protein was 7.8 % after 60 h and 3.5 % at 96 h dialysis as compared with a value
6
B 4 6
4 I
3
J
Fig. 2. U l t r a c e n t r i f u g e p a t t e r n s from calf t h y m u s l y m p h o c y t e c y t o p l a s m . A. S o l v e n t s y s t e m p o t a s s i u m p h o s p h a t e buffer (pH 7.6) c o n t a i n i n g o.oo2 M MgC12, 0.05 M NaC1. T o t a l ionic s t r e n g t h o.I. C o n c e n t r a t i o n a p p r o x i m a t e l y i %. P h o t o g r a p h e d 735 sec a f t e r r o t o r a t t a i n e d s pe e d of 3345o r e v . / m i n . P h a s e p l a t e a n g l e 55 °. S e d i m e n t a t i o n coefficients: 3 = 39 S, 5 = 54 S, 6 = 67 S. ]3. S o l v e n t s y s t e m 0.o 5 M N a H C O a. C o n c e n t r a t i o n a p p r o x . 1.2 %. P h o t o g r a p h e d 595 sec a f t e r r o t o r a t t a i n e d speed of 33 450 r e v . / m i n . P h a s e p l a t e a n g l e 55 °- S e d i m e n t a t i o n coefficients : 2 = 32 S, 4 = 47 S, 6 = 63 S. C. S o l v e n t s y s t e m 0.05 M Tris (pH 7-5). C o n c e n t r a t i o n a p p r o x . 1.2 %. P h o t o g r a p h e d 520 sec a f t e r r o t o r a t t a i n e d speed of 33 45 ° r e v . / m i n . P h a s e p l a t e a ngl e 55 °. S e d i m e n t a t i o n coefficients: 2 = 32 S, 4 = 46 S, 5 = 57 S. D. S o l v e n t s y s t e m H 2 0 . C o n c e n t r a t i o n a p p r o x i m a t e l y 1.3 %. P h o t o g r a p h e d 52o sec a f t e r r o t o r a t t a i n e d speed of 33 45 ° r e v . / m i n . P h a s e p l a t e a n g l e 65 °. S e d i m e n t a t i o n coefficients: 2 = 28 S, 3 = 39 S, 4 = 48 S. All e x p e r i m e n t s were c o n d u c t e d a t 2o °.
Biochim. Biophys. dora, 82 (i964) 385-393
392
W. K. B R U N K H O R S T , E. L. HESS
of 9.2 % for a freshly prepared solution. No significant loss occurred, therefore, until after 60 h dialysis. The results discussed above offer evidence that the size distribution of the ribosome is not critical for binding of hormone. The findings are similar to the observations of ULRICH with respect to mitochondria 15. ULRICH reported that the uptake of corticosteroids is quite independent of the status of the mitochondria. The results also indicate that the binding of hormones by these thymus fractions is a general type reaction between steroids and proteins since the interaction of a particular fraction with a specific hormone could not be demonstrated. TABLE
II1
EFFECT OF VARYING M g ION CONCENTRATIONS ON THE INTERACTION OF MICROSOMES WITH CORTISOL V a l u e s a r e e x p r e s s e d a s p e r c e n t h o r m o n e b o u n d , C o n c e n t r a t i o n of m i c r o s o m e s w a s i % in 0.0,5 M N a H C O a , 2 /lg c o r t i s o l w a s p r e s e n t in t o t a l s y s t e m of 7.5 m l .
Mg ion concentration
% CortisoI Pound
o
to.6
0.000 5 M
[1.6
o.ooi
M
i2. 3
0.005
M
11.1
The difference in binding behavior of the hormones as indicated by the SCATCHAR1) plots appears significant. The negative slopes observed for progesterone binding to the microsomal fraction suggests specificity of this hormone for a single component. Conversely, the positive slope for cortisol indicates several components in the complex microsomal fraction are binding. Specificity of the site or sites for cortisol is indicated by the lack of competition by progesterone and testosterone. Preliminary evidence has been obtained which supports the hypothesis that the positive slope is indicative of binding by more than one component in the system. Separation of fraction S 5.1 P into several components was achieved by chromatography on DEAF-cellulose. Interaction of cortisol with each component was measured. Ii1 all cases negative slopes resulted when binding data were plotted. A second explanation, suggested above, for the positive slope is a change in number of binding sites. A hormone-induced alteration in the tertiary or quaternary structure of the macromolecule could produce such a change. Detection of this type of cortisol induced change was attempted by determining sedimentation coefficients of microsomal solutions with and without hormone present. No difference in sedimentation behavior was observed, however, between the two preparations. Further work will be directed toward isolating purified components from the fractions and studying their interactions with the various hormones. ACKNOWLEDGEMENTS
The capable technical assistance of Mrs. M. RUTH is gratefully acknowledged. The authors are indebted to the Endocrinology Study Section, National Institutes of B i o c h i m . B i o p h v s . A c l a , 82 (1964) 385 393
HORMONE INTERACTION WITH THYMUS FRACTIONS
393
Health for supplying the cortisol through Tracerlab Inc., Boston, Mass., and to Dr. M. GUT of the Worcester Foundation for the progesterone and testosterone. The work was supported by research grants from the American Cancer Society and the National Science Foundation. REFERENCES 1 C. J. MIGEON, A. A. SANDB]~RG, H. A. DECKER, D. F. SMITH, A. C. PAUL AND L. T. SAMUELS, J. Clin. Endocrinol. Metab., 16 (1956) 1137. 2 H . L . B R A D L O W , K . DOBRINER AND T. •. GALLAGER, Endocrinology, 54 (1954) 343. 3 p. BELLAMY, J. G. PHILLIPS, I. C. JONES AND R. A. LEONARD, Biochem. J., 85 (1962) 537. 4 F. DE VENUTO, P. C. KELLEHER ANn lJ. WESTPHAL, Biochim. Biophys. Acta, 63 (1962) 434. 5 H. E. FIRSCHEIN, F. DE VENUTO, W. M. FITCH, E. M. PEARCE AND U. ~VESTPHAL, Endocrinology, 60 (1957) 347. W. K. BRUNKHORST AND E. L. HESS, Biochem. Biophys. Res. Commen., 5 (1961) 238. 7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. BioL Chem., 193 (1951) 265. 8 G. SCATCHARD AND E. S. BLACK, J. Phys. and Colloid Chem., 53 (1949) 88. 9 K . EIK-NEs, J. A. SCHELLMAN, R. LUMRY AND L. SAMUELS, f . Biol. Chem., 2o6 (1954) 411. 10 U. WESTPHAL, in C. A. VIELEC, Mechanism of Action of Steroid Hormones, P e r g a m o n Press, N e w York, 1961, p. 42. 11 W. R. SLAUNWHITE AND A. A. SANDBERG, J. Clin. Invest., 38 (1959) 384 • 12 A. HERRANEN AND E. L. HESS, personal communication. 13 E. L. H E s s AND S. LAGG, Biochemistry, 2 (1963) 726. la M. G. HAMILTON AND M. L. PETERMAN, J. Biol. Chem., 234 (1959) 1441. 15 F. ULRICH, Am..[. Physiol., 196 (1959) 572.
Biochim. Biophys. Acta, 82 (I964) 385-393
385
BBA 4224
INTERACTIONS
OF HORMONES
WITH
THYMUS-CELL
FRACTIONS
WILLA K. B R U N K H O R S T AND E U G E N E L. HESS
Worcester Foundation for Experimental Biology, Shrewsbury, Mass. (U.S.A .) (Received May 9th, 1963)
SUMMARY
The binding of cortisol, progesterone and testosterone to thymus-cell fractions was studied b y the equilibrium dialysis procedure. The hormones appear to follow the "polarity rule", progesterone binding to the greatest extent, cortisol the least. Ribosomal particles seem to have a higher capacity for binding hormone than the p H 5.1 precipitate or the material in the supernatant resulting from treatment of microsomal fraction with deoxycholate. The possibility is discussed that progesterone is binding to a single component in the ribosomal fraction while cortisol and testosterone bind to more than one component.
INTRODUCTION
Interaction of steroid hormones with reactive sites on certain cellular proteins has been considered as a possible mechanism by which hormones initiate their physiological effect. The distribution of glucocorticoids in plasma and various tissues after intravenous injection of radioactive steroids has been studied by several groups of investigators 1-3. Recently DE VENUTO, KELLEHER AND WESTPHAL4 reported on the interaction of cortisol and corticosterone with cell fractions from rat-liver and muscle after injection of the hormones into the living animal, or after addition to organ homogenates. Nuclear, mitochondrial and microsomal fractions from liver interacted with cortisol and corticosterone while no significant binding was observed with muscle. It was considered worthwhile to study the interaction of cortisol with cell fractions from a gland or organ which undergoes a distinctive response to the hormone. It is well known that the thymus of animals treated with glucocorticoids undergoes rapid involution. FIRSCHEIN and associates 5 found a greater percentage of an injected dose of cortisolin the thymus of stressed rats as compared with control rats. Detection of an interaction with a particular cellular component which is specific for cortisol might indicate the site of initial action of the hormone. A previous paper described the binding of cortisol by a fraction from thvmus which contains most of the nucleic acid in the cytoplasm of lymphocytes 6. The work reported here was undertaken to study the binding in vitro of cortisol by various cell fractions which are involved in protein synthesis. Demonstration of a specific Abbreviation: DOC, deoxycholate.
Biochim. Biophys. Acta, 82 (I964) 385-393
386
w . K . BRUNKHORST, E. L. HESS
cortisol effect on one of these fractions could provide a plausible explanation of the involution produced by the glucocorticoids. EXPERIMENTAL
Various fractions were prepared from calf thymus obtained from a commercial slaughter house. Glands were chilled as quickly as they were removed from the animal and subsequent fractionation was carried out in the cold room at 4-5 °. After the tissue was minced and put through an onion press, three volumes o.25 M sucroseo.oo33 M CaC12 per gram wet tissue were added and the suspension was stirred in a Waring Blender at approximately IOOO rev./min for 4 min. The homogenate was centrifuged at 14oo >~, g for 7 min and the resulting supernatant was centrifuged at 15ooo × g for 2o min. The 15ooo "~ g supernatant was shell frozen and stored at - - 2 o ° until used.
Preparation of microsomal fraction, ribosomes and fraction S 5.r P The 15 ooo x g supernatant was centrifuged at lO5 ooo x g in a Spinco Model L centrifuge for I h to yield a pellet referred to as microsomal fraction. An additional fraction was precipitated from the lO5 ooo g supernatant by adjusting the p H to 5.1 with o.i M acetic acid. Centrifugation at 15ooo ~: g for 15 min brought down the precipitate, designated fraction S 5.IP, which contained soluble RNA and activating enzymes essential for amino acid incorporation into protein. In some experiments microsomal pellets were re-suspended in 3 ml o.25 M sucrose and 0.3 ml of 3 % sodium deoxvcholate (pH 8) was added. After IO min the volume was adjusted to 12 ml with o.25 M sucrose and the suspension was centriiuged again at lO5 ooo ~" g for I h. The resulting pellet contains ribosomal material. The deoxycholate supernatant from this step was dialyzed and lyophilized. All fractions were resuspended in o.o5 M NaHCO3 except in those experiments in which effects of various buffer ions were investigated. In this case the fractions were resuspended in the Tris or phosphate buffer as described under RESULTS. Concentrations were determined by analyzing the solutions for protein using the method of LowRY et al. 7 and correcting for RNA and lipid content. The amount of RNA and lipid in the fractions had been established previously.
Equilibrium dialysis procedure An equilibrium dialysis procedure was employed to study the interaction of the thymus fractions with various hormones. Since a significant amount of cortisol was bound by unwashed casing, Visking casing (32/lOO in diameter) was washed three times in 50 % ethyl alcohol followed by three rinses with distilled water at 50-60 °. Dialysis bags prepared from the washed Visking were air dried and used in the equilibrium dialysis studies. A volume of 1.5 ml of the thymus fraction, which contained the hormone, was placed in the dialysis bag along with an 8 mm glass rod. The bag was inserted in a 22 - 175 mm glass stoppered test tube containing 6 ml of 0.05 M NaHCOa. The tubes were placed in a rack which held them at a 4 °0 angle and shaken gently in an automatic shaker for 3 h at room temperature. Control experiments have shown that equilibrium is attained in this length of time. Amounts of steroids used are given in the tables. The specific activity of the Bioehim. Biophys. Acta, 82 (1964) 385-393
HORMONE INTERACTIONWITH THYMUS FRACTIONS
387
~4-14Clcortisol was 42.4 t~C/mg, that of [4-14CJprogesterone was 17 ~C/ml and that of E4-14Cltestosterone was 22/~C/mg.
Plating procedure An accurate correction for self-absorption could not be made by the usual method of determining the weight of the sample on the plate and applying a correction factor for self-absorption. For unknown reasons plating of hormone in the presence of thymus fractions resulted in counts in excess of the amount of radioactivity added. The following procedure was used to obtain correct binding data. The solution within the dialysis casing was diluted I : I with the buffer. The dialysate was diluted I : I with a protein solution of the same composition as that within the dialysis casing, except that no radioactive steroids were included. Triplicate samples of lOO-4 aliquots from both solutions were plated. Plates were counted on a thin-window gas.flow counter and counts were expressed as counts/min/ml. Since both parts of the equilibrium dialysis system contained the same amount of protein when plated, similar counting behavior was insured. Although the counts recorded did not represent the absolute number of counts present the difference between sack and dialysate was valid and an accurate calculation of the per cent bound hormone could be made. In experiments in which the data were analyzed using a SCATCHARDtype plot 8 it was necessary to establish the concentration of cortisol both in the sack and in the dialysate. A correction for the counting error was made, therefore, in the following manner. Control tubes were included in each experiment. Hormone equivalent to the amount in experimental tubes was dissolved in 1.5 ml buffer and placed in the sack. Dialysis was against 6 ml of buffer. After equilibrium dialysis the contents of the sack and the dialysate were diluted I ' i with buffer and triplicate aliquots of IOO ~ were plated. The connts on the plates gave an accurate measure of the radioactivity and could be related directly to the concentration of cortisol. The total counts in the system should be the same for control and experimental tubes since identical amounts of hormone were added to both. Higher counts were always obtained with experimental tubes presumably due to the protein on the plates. The per cent difference between total counts in experimental and control tubes was applied as a correction factor to the plates containing protein. The correct counts/min/ml thus determined were converted to concentrations of hormone in the sack and dialysate. The total amount of hormone in the system as calculated from the corrected counts/min/ml was in agreement with the amount added to the tubes.
Calculations The interaction between hormone and thymus fractions was expressed as per cent steroid bound. steroid bound % steroid bound steroid total × ioo At equilibrium the concentration of unbound steroid in the dialysis bag was assumed to be equal to the steroid concentration in the dialysate. The concentration of bound steroid could thus be obtained as the difference between the total steroid in the dialysis sack and the unbound or free steroid.
Biochim. Biophys. Acta, 82 (1964) 385-393
38~
W. K. B R U N K H O R S T ,
E. L. HESS
Data were plotted according to the equation (ref. 8): r
= k ( n - r) C
where r is the number of moles of bound cortisol per mole of total protein, c is the concentration of free cortisol, n is the number of binding sites per molecule protein and k is the association constant. The relation permits the determination of n and k under the assumed condition that combining sites on the protein are equivalent and independent. RESULTS A N D DISCUSSION
The extent of interaction of thymus fractions with cortisol, as well as progesterone and testosterone, was determined. The results are summarized in Table I. The hormones appear to follow the "polarity rule" as proposed by EIK-NES et alY and discussed by WESTPHALL10 for such proteins as albumin. The binding of cortisol was essentially the same for all fractions when the concentration of protein is expressed on a weight per volume basis. Weight average molecular weights of 3" lO8 for ribosomes, I. lOs for microsomes, I- lO 5 for DOC supernatant and 25000 for S 5.1 P were assumed and the binding was expressed on the basis of molarity of the various fractions. TABLE INTERACTION
OF
HORMONES
WITH
I
VARIOUS
CELL
FRACTIONS
OF
THYMUS
V a l u e s g i v e n a r e a v e r a g e s of t w o e x p e r i m e n t s a n d a r e e x p r e s s e d as p e r c e n t h o r m o n e b o u n d . T h e c o n c e n t r a t i o n s of a l l f r a c t i o n s w e r e i % i n 0.0 5 M N a H C O 3. Cell fraction Hormone
Cortisol* Progesterone** Testosterone** *
M icrosomal
Ribosomal
5 5. t P
Deoxycholate supernatant
i5.2 84.o 54.3
16.2 88. I 48-9
15.5 65.o 33.8
14.9 76. i 4 t .4
* 2 # g c o r t i s o l i n t o t a l s y s t e m (7.5 ml). C o n c e n t r a t i o n w o u l d b e 7" io- 7 M a t e q u i l i b r i u m if n o binding occurred. 1.4 P g p r o g e s t e r o n e in t o t a l s y s t e m . C o n c e n t r a t i o n w o u l d b e 6. lO -7 M a t e q u i l i b r i u m if n o binding occurred. 1. 5 p g t e s t o s t e r o n e in t o t a l s y s t e m . C o n c e n t r a t i o n w o u l d b e 7" lO-V M a t e q u i l i b r i u m if n o binding occurred.
Microsomes at approx, io -5 M bound 15 % cortisol while ribosomes bound 23.5 % S 5.IP bound 2.9 % and DOC supernatant bound 2.3 %. Mole for mole the microsomal and ribosomal fractions appear to have a higher capacity for binding the hormone than S 5.IP or DOC supernatant. The increased binding observed with the ribosomes suggests that the particles are largely responsible for binding by the microsomal fraction. Based on the binding in vitro reported here, the DOC supernatant and 5.IP fractions must be present in molar concentrations 30-40 times that of the particles if they bind a significant amount of hormone in the cell. According to findings in this laboratory these fractions are at most three to four times the concentrations of the ribosomal fraction. It would appear, therefore, that the particles bind most of the hormone in the cytoplasmic portion of the cell. B i o c h i m . B i o p h y s . A c l a , 82 (1964) 385 393
389
HORMONE INTERACTION WITH THYMUS FRACTIONS TABLE II BINDING
OF
CORTISOL
PRESENCE
OF
BY
MICROSOMAL
FRACTION OR
PROGESTERONE
OF
THYMUS
IN
TESTOSTERONE
Values given are averages of two experiments and are expressed as per cent h o r m o n e bound. Microsome concentration was i % in o.o 5 M N a H C O 3. 2/~g cortisol were present in total system of 7.5 ml. A m o u n t s given for second h o r m o n e were amounts present in total system. A mount o~ second hormone
Cortisolonly
Cortisoland progesterone
Cortisoland testosterone
I3. 3 i4.i 14.5
I6.O 14. 5 13.6
I3.9
O. I I /~g/ml 0.22 /~g/ml 2.20 /zg/ml
A
120
35 9C
25
X
X
~lu 6C 3C
%
I
I
10
20
I 30
r X
15
40
50
I
vo 25
60
I
75
:"
125
,
,75
"
300
aso
r X 10.2
1 0 -~
C 35
qo
°b%~
25
X
Fig. i. Binding of progesterone b y t h y m u s fractions. A, nficrosomes; B, ribosomes; C, DOC s u p e r n a t a n t ; r is n u m b e r of moles of b o u n d cortisol per mo]e of total protein, c is concentration of free progesterone.
~Ju 15
410
8I0 12G r x 10+*
160
280
320
A l t h o u g h none of t h e cell fractions i n t e r a c t exclusively with cortisol, t h e b i n d i n g of the h o r m o n e a p p e a r s to occur at a specific site in the microsome fraction. As seen in t h e d a t a in T a b l e I I , a d d i t i o n of increasing a m o u n t s of u n l a b e l e d progesterone or t e s t o s t e r o n e has no effect on the a m o u n t of cortisol bound. Thus, neither progesterone nor t e s t o s t e r o n e c o m p e t e s with cortisol for the same site. Using t h e SCATCHARD e q u a t i o n 8 a t y p i c a l plot with a n e g a t i v e slope was o b t a i n e d for p r o g e s t e r o n e i n t e r a c t i n g w i t h the m i c r o s o m a l fraction a n d also with t h e two m a i n c o m p o n e n t s of t h e m i c r o s o m a l fraction, ribosomes a n d DOC s u p e r n a t a n t . These results are r e p r e s e n t e d in Fig. i . N values can be considered as only a p p r o x i m a t e since t h e fractions are r e l a t i v e l y crude a n d a v e r a g e molecular weights were a s s u m e d for t h e calculations. Considered in this light t h e N values o b t a i n e d from t h e plot B i o c h i m . B i o p h y s . A c t a , 82 (1964) 385-393
39 °
w.K. BRUNKHORST, E. L. HESS
are approximately o.6 for microsome and 3.5 for ribosome fractions and o.o3 for DOC snpernatant. Curves with positive slopes were obtained for binding of testosterone and cortisol to all fractions studied. Similar results were obtained for fraction S 5.IP binding of progesterone. The positive slope suggests that: (a) the association constant changes with varying hormone concentration or (b) the number of sites available for binding is changing or (c) a mixture of several components having different binding affinities for the hormones are present in the fractions. The third possibility is most probable. The influence of other buffer systems on the interaction of cortisol with microsomal, S 5.IP and ribosomal fractions was investigated using potassium phosphate buffer, 0.05 M (pH 7.5), potassium phosphate buffer, 0.05 M (pH 7.6), containing 0.002 M MgC12 and 0.05 M Tris (pH 7.5). The extent of binding was the same as when the fractions were suspended in 0.05 M NaHCO 3, the buffer used in most of the experiments reported here. Phosphate, Tris or bicarbonate ions had no effect on the interaction, nor did the pH of the solution appear critical since the buffers varied from pH 7.5 for phosphate and Tris to 8.6 for 0.05 M NaHCO3. Binding of cortisol to transcortin has been reported to vary inversely with an increase in temperature. Interaction of cortisol with serum albumin, on the other hand, is not strongly temperature dependent 11. No thermal effect was found with thymus fractions. Cortisol was bound to the same extent by microsomal and pH 5 fractions at 4 ° and at room temperature. Effects of varying conditions under which microsomes were prepared and under which equilibrium dialysis was performed were investigated. Conditions were chosen which are known to affect the size distribution of ribosomes and their biological activity. I. Frozen instead of fresh thymus was used for preparation of the microsome fraction. It is known that biological activity of the microsomal fraction is lost when the tissue is frozen 12. Interaction with cortisol was not affected, however, by this treatment. It was also found that microsomes could be prepared from fresh or frozen tissue, suspended in 0.05 M NaHCO 3 and frozen for as long as one month without altering their ability to interact with cortisol. 2. The sedimentation properties of microsomal preparations resuspended in water and phosphate, Tris and NaHCOs buffers were examined. As seen in Fig. 2 sedimentation properties depend upon the buffer system. The maior component of microsomes in phosphate buffer has a sedimentation coefficient of 67 S, which, when corrected to infinite dilution, should correspond to the 74 S particles reported by HESS et al. 1~. Smaller amounts of 54 and 39 S components were present. The preparation in NaHCO3 also contained a component equivalent to 74 S particles, however, there were greater amounts of slower sedimenting material. Neither Tris nor water solutions contained 74 S particles. The preparation in water contained the largest amount of the slow sedimentating components. In spite of these differences all the preparations bound cortisol to the same extent. 3- It is well established that the concentration of Mg ions affects the size distribution and the extent of aggregation of ribosome particles 18,14. As seen in Fig. 2 the effect applies to the microsome fraction as well. Microsomes were prepared from fresh thymus and resuspended in 0.05 M Tris (pH 7.5), which contained varying Biochim. Biophys. Acta, 82 (1964) 385-393
HORMONE INTERACTION WITH THYMUS FRACTIONS
391
concentrations of Mg ions. The interaction of the solutions with cortisol was measured. As seen in Table I I I , Mg 2+ had no effect on the extent of binding. 4. The effect of aging on the capacity of microsomes to interact with cortisol was also investigated. A fraction resuspended in potassium phosphate buffer (pH 7.5), containing 0.002 M MgCI~ was dialyzed against the same buffer for 60 and 96 h at 4 ° without changing the dialysate. The amount of cortisol bound b y 0. 4 % solution of protein was 7.8 % after 60 h and 3.5 % at 96 h dialysis as compared with a value
6
B 4 6
4 I
3
J
Fig. 2. U l t r a c e n t r i f u g e p a t t e r n s from calf t h y m u s l y m p h o c y t e c y t o p l a s m . A. S o l v e n t s y s t e m p o t a s s i u m p h o s p h a t e buffer (pH 7.6) c o n t a i n i n g o.oo2 M MgC12, 0.05 M NaC1. T o t a l ionic s t r e n g t h o.I. C o n c e n t r a t i o n a p p r o x i m a t e l y i %. P h o t o g r a p h e d 735 sec a f t e r r o t o r a t t a i n e d s pe e d of 3345o r e v . / m i n . P h a s e p l a t e a n g l e 55 °. S e d i m e n t a t i o n coefficients: 3 = 39 S, 5 = 54 S, 6 = 67 S. ]3. S o l v e n t s y s t e m 0.o 5 M N a H C O a. C o n c e n t r a t i o n a p p r o x . 1.2 %. P h o t o g r a p h e d 595 sec a f t e r r o t o r a t t a i n e d speed of 33 450 r e v . / m i n . P h a s e p l a t e a n g l e 55 °- S e d i m e n t a t i o n coefficients : 2 = 32 S, 4 = 47 S, 6 = 63 S. C. S o l v e n t s y s t e m 0.05 M Tris (pH 7-5). C o n c e n t r a t i o n a p p r o x . 1.2 %. P h o t o g r a p h e d 520 sec a f t e r r o t o r a t t a i n e d speed of 33 45 ° r e v . / m i n . P h a s e p l a t e a ngl e 55 °. S e d i m e n t a t i o n coefficients: 2 = 32 S, 4 = 46 S, 5 = 57 S. D. S o l v e n t s y s t e m H 2 0 . C o n c e n t r a t i o n a p p r o x i m a t e l y 1.3 %. P h o t o g r a p h e d 52o sec a f t e r r o t o r a t t a i n e d speed of 33 45 ° r e v . / m i n . P h a s e p l a t e a n g l e 65 °. S e d i m e n t a t i o n coefficients: 2 = 28 S, 3 = 39 S, 4 = 48 S. All e x p e r i m e n t s were c o n d u c t e d a t 2o °.
Biochim. Biophys. dora, 82 (i964) 385-393
392
W. K. B R U N K H O R S T , E. L. HESS
of 9.2 % for a freshly prepared solution. No significant loss occurred, therefore, until after 60 h dialysis. The results discussed above offer evidence that the size distribution of the ribosome is not critical for binding of hormone. The findings are similar to the observations of ULRICH with respect to mitochondria 15. ULRICH reported that the uptake of corticosteroids is quite independent of the status of the mitochondria. The results also indicate that the binding of hormones by these thymus fractions is a general type reaction between steroids and proteins since the interaction of a particular fraction with a specific hormone could not be demonstrated. TABLE
II1
EFFECT OF VARYING M g ION CONCENTRATIONS ON THE INTERACTION OF MICROSOMES WITH CORTISOL V a l u e s a r e e x p r e s s e d a s p e r c e n t h o r m o n e b o u n d , C o n c e n t r a t i o n of m i c r o s o m e s w a s i % in 0.0,5 M N a H C O a , 2 /lg c o r t i s o l w a s p r e s e n t in t o t a l s y s t e m of 7.5 m l .
Mg ion concentration
% CortisoI Pound
o
to.6
0.000 5 M
[1.6
o.ooi
M
i2. 3
0.005
M
11.1
The difference in binding behavior of the hormones as indicated by the SCATCHAR1) plots appears significant. The negative slopes observed for progesterone binding to the microsomal fraction suggests specificity of this hormone for a single component. Conversely, the positive slope for cortisol indicates several components in the complex microsomal fraction are binding. Specificity of the site or sites for cortisol is indicated by the lack of competition by progesterone and testosterone. Preliminary evidence has been obtained which supports the hypothesis that the positive slope is indicative of binding by more than one component in the system. Separation of fraction S 5.1 P into several components was achieved by chromatography on DEAF-cellulose. Interaction of cortisol with each component was measured. Ii1 all cases negative slopes resulted when binding data were plotted. A second explanation, suggested above, for the positive slope is a change in number of binding sites. A hormone-induced alteration in the tertiary or quaternary structure of the macromolecule could produce such a change. Detection of this type of cortisol induced change was attempted by determining sedimentation coefficients of microsomal solutions with and without hormone present. No difference in sedimentation behavior was observed, however, between the two preparations. Further work will be directed toward isolating purified components from the fractions and studying their interactions with the various hormones. ACKNOWLEDGEMENTS
The capable technical assistance of Mrs. M. RUTH is gratefully acknowledged. The authors are indebted to the Endocrinology Study Section, National Institutes of B i o c h i m . B i o p h v s . A c l a , 82 (1964) 385 393
HORMONE INTERACTION WITH THYMUS FRACTIONS
393
Health for supplying the cortisol through Tracerlab Inc., Boston, Mass., and to Dr. M. GUT of the Worcester Foundation for the progesterone and testosterone. The work was supported by research grants from the American Cancer Society and the National Science Foundation. REFERENCES 1 C. J. MIGEON, A. A. SANDB]~RG, H. A. DECKER, D. F. SMITH, A. C. PAUL AND L. T. SAMUELS, J. Clin. Endocrinol. Metab., 16 (1956) 1137. 2 H . L . B R A D L O W , K . DOBRINER AND T. •. GALLAGER, Endocrinology, 54 (1954) 343. 3 p. BELLAMY, J. G. PHILLIPS, I. C. JONES AND R. A. LEONARD, Biochem. J., 85 (1962) 537. 4 F. DE VENUTO, P. C. KELLEHER ANn lJ. WESTPHAL, Biochim. Biophys. Acta, 63 (1962) 434. 5 H. E. FIRSCHEIN, F. DE VENUTO, W. M. FITCH, E. M. PEARCE AND U. ~VESTPHAL, Endocrinology, 60 (1957) 347. W. K. BRUNKHORST AND E. L. HESS, Biochem. Biophys. Res. Commen., 5 (1961) 238. 7 0 . H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. BioL Chem., 193 (1951) 265. 8 G. SCATCHARD AND E. S. BLACK, J. Phys. and Colloid Chem., 53 (1949) 88. 9 K . EIK-NEs, J. A. SCHELLMAN, R. LUMRY AND L. SAMUELS, f . Biol. Chem., 2o6 (1954) 411. 10 U. WESTPHAL, in C. A. VIELEC, Mechanism of Action of Steroid Hormones, P e r g a m o n Press, N e w York, 1961, p. 42. 11 W. R. SLAUNWHITE AND A. A. SANDBERG, J. Clin. Invest., 38 (1959) 384 • 12 A. HERRANEN AND E. L. HESS, personal communication. 13 E. L. H E s s AND S. LAGG, Biochemistry, 2 (1963) 726. la M. G. HAMILTON AND M. L. PETERMAN, J. Biol. Chem., 234 (1959) 1441. 15 F. ULRICH, Am..[. Physiol., 196 (1959) 572.
Biochim. Biophys. Acta, 82 (I964) 385-393