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Studies of the glucocorticoid-binding protein from thymocytes
414
BIOCHIMICAET BIOPHYSICAACTA
BBA 36099 STUDIES OF T H E G L U C O C O R T I C O I D - B I N D I N G P R O T E I N FROM THYMOCYTES UI. pH D E P E N D E N C E OF T H E B I N D I N G AND D E N S I T Y - G R A D I E N T C E N T R I F U G A T I O N OF T H E P R O T E I N
BJORN P. SCHAUMBURG Institute of Experimental Endocrinology, University of Copenhagen, Narre All~ 71, 2Ioo (Denmark)
(Received 9 December, 1971)
SUMMARY
I. The binding of eorticosterone to the glucocorticoid-binding protein from thymocytes was maximal at p H 7-8 and decreased rapidly below p H 72. Glucocorticoid-binding protein was precipitated at pH 5-6 together with most of the protein in crude extracts from thymocytes. 3- After density gradient centrifugation of glucocorticoid-binding protein at a low salt concentration (o.I M KC1) 2 peaks of binding activity were found. The sedimentation constant of the major peak was IO.O ~: 0.5 S and that of the minor peak 4.5 :[: 0.2 S. At higher salt concentrations (o.25-1.o M KC1) only the 4.5-S peak was observed. 4. The complex with corticosterone and with triamcinolone acetonide showed the same sedimentation behaviour, although a partial dissociation of the corticosterone complex occurred during the run. 5. Binding of triamcinolone acetonide in intact cells at 37 °C followed by extraction with a buffer containing 0.6 M KC1 caused the appearance of a 3.5-S form of bound hormone, the sedimentation constant of which was uninfluenced by the KC1 concentration in the density gradient.
INTRODUCTION Glucocorticoids have a direct effect on the metabolism of lymphocytes i n vitro1, 2 when applied in physiological concentrations. Furthermore, the glucocorticoids are bound to a well-defined "receptor" protein in the cells, and the properties of the binding (specificity, kinetics and affinity) support the contention that this binding is the initial event in the action of the hormonesa-t The role of the glucocorticoid-binding protein from thymocytes in the hormonal mechanism of action is furthermore supported by the finding that some steroids are bound to glucocorticoidbinding protein without causing any metabolic effect and that these steroids at the same time inhibit the effects of cortisol 1,~. The glucocorticoid-binding protein is very unstable in crude extracts from Biochim. Biophys. Acta, 263 (1972) 414-423
GLUCOCORTICOID-BIND.ING PROTEIN FROM THYMOCYTES
415
thymocytes 5, but is stabilized by glycerol at low temperatures. Under these conditions the kinetics of binding and dissociation is simple and has been investigated for several steroids 6. In the experiments described below the effect of changing the pH was studied as well as the sedimentation behaviour of glucocorticoid-binding protein at low and high salt concentrations taking advantage of the extremely slow dissociation of bound triamcinolone acetonide from glucocorticoid-binding protein. A preliminary report of these experiments has been presented 7. METHODS
The preparation of thymocyte suspensions from adrenalectomized rats and the sonication of the cells followed by ultracentrifugation was performed as previously describedS, 6. The binding of tritiated steroids was measured by adsorption of unbound steroid by dextran-coated coalS, 6 and counting of the unadsorbed radioactivity in a liquid scintillation counter. The concentration of steroid-binding sites in the preparation was approximately i nM. Assuming a molecular weight of 50 ooo per binding site (corresponding roughly to the molecular weight of a protein with a sedimentation constant of 4.5 S) the steroid-binding protein could be calculated to account for o.oi% of the protein in the extract, containing 3-5 mg protein per ml.
Gradient centrifugation Density gradients (20-30% glycerol) were prepared from buffers containing 20 and 35% glycerol using three perpex pumps (LKB, Sweden). The preparation of glucocorticoid-binding protein was mixed with solutions of marker proteins and the concentration of glycerol adjusted to approx. 15°/o. 0.3 ml was layered on the top of the gradients. The centrifugation was performed either at 35 ooo rev./min using a SW 39 L rotor in a Spinco L5o preparative ultracentrifuge at approx. 4 °C or at 50 ooo rev./min using a SW 5o.1 rotor in a Spinco L2-65B preparative ultracentrifuge at --2 - --5 °C. After the run the bottom of the tubes were pierced in a self-constructed elution device and the content of the tubes displaced using a perpex pump. 30-35 fractions were normally collected using a fraction collector. In some of the experiments the glucocorticoid-binding protein preparation was incubated with radioactive hormone before the gradient centrifugation (prelabeled glucocorticoid-binding protein), while in other experiments the glucocorticoid-binding protein was unlabelled, in which cases the content of glucocorticoid-binding protein in the fractions was determined by incubation of aliquots with tritiated corticosterone followed by coal adsorption. The position of marker proteins was determined by measurements of the absorbance in a spectrophotometer after dilution of 5o-#1 aliquots with 125/A water. The buffers used for the preparation of the gradients contained the concentration of KC1 indicated in the separate experiments and IO mM potassium phosphate, 5 mM/~-mercaptoethanol and 0.5 mM EDTA (pH 7-4) before the mixing with glycerol. The sedimentation constants (s°20,w) of the marker proteins 8-10 were catalase 11.3 S, ovalbumin 3.55 S, bovine serum albumin 4.6 S,/~-amylase 8. 9 S and hemoglobin 4.1 S. Biochim. Biophys. Acta, 263 (1972) 414-423
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B. P. SCHAUMBURG
RESULTS
pH dependence of the binding The binding of [I,2-3H~]corticosterone to glucocorticoid-binding protein was determined by incubation of a IOO ooo × g supernatant from sonicated thymocytes in the presence of i nM of the steroid (40% glycerol, --5 °C). After 2 days of incubation the binding was measured. Maximal binding was found between p H 7 and 8, while the binding decreased sharply below p H 7 and more gradually above p H 8 (Fig. i).
SO0-
400-
300-
O
O
200o
u
0 pH
Fig. i. p H dependence of the binding of corticosterone to glucocorticoid-binding protein. A IOO ooo × g s u p e r n a t a n t was p r e p a r e d from sonicated r a t t h y m o c y t e s . Aliquots of the supern a t a n t were incubated in 4 o~o glycerol at --5 °C after titration to different p H values. The binding of tritiated corticosterone was measured after 48 h incubation with i nM [I,2-3H2]corticosterone ( © - - © ) or with i nM [I,2-3H~]corticosteroneplus I/~M non-radioactive corticosterone ( Q - - O ) . The specific binding of [I,2-SH~]corticosterone to glucocorticoid-binding protein was obtained as the difference between the two curves ((~---(}).
The kinetics of dissociation of bound corticosterone have previously been investigatedS, 6 at p H values from 6.5-1o and the half-life of the complex found to be approx. 800 min between p H 6. 5 and 8.5, while it decreased to 30-60 min at p H IO.O. From these findings it can be concluded that the period of incubation should be more than sufficient to reach equilibrium of the binding in the whole p H interval, and that the decrease of the binding on both sides of p H 7-8 must be explained either by a decreased number of binding sites or by a decreased affinity of the existing sites. No decrease of the number of binding sites occurred, however, between p H 6 Biochim. Biophys./tcta, 263 (1972) 414-423
6LUCOCORTICOID-BINDING PROTEIN FROM THYMOCYTES
417
and 9, as shown by an experiment in which aliquots of glucocorticoid-binding protein were incubated for 24 h at different pH values, the pH adjusted to 7.4 and thereupon the binding of [I,2-~H21corticosterone measured after 2 days at pH 7.4. Above pH 9 and below pH 6, however, the binding activity appeared to be slowly and irreversibly destroyed. The pH dependence of the binding was thus due to the decreased affinity of glucocorticoid-binding protein for the steroid. As the steroid concentration (I nM) was well below the dissociation constant of the binding of [I,2-3H2]corticosterone (3.5 nM) 6 the binding should be inversely proportional to the dissociation constant, and Kd thus increased rapidly below pH 7 and more slowly above pH 8.
Precipitation at low pH Aliquots of a IOO ooo × g supernatant of sonicated thymocytes in 40% glycerol were titrated to different pH values in an ice bath and incubated for IOO min at --5 °C, centrifuged and the supernatants adjusted to pH 7-4, while the precipitates were dissolved in buffer, also at pH 7.4. The content of glucocorticoid-binding protein was determined by incubation with i nM Ii,2-3H2~corticosterone for 2 days followed by coal adsorption.
1o0 illll / i o[_AL£tLL 4.0
4.4
4.7 5.0 5.3 5.6 pFI of precipitation
6.0
74
Fig. 2. P r e c i p i t a t i o n of glucocorticoid-binding p r o t e i n in a IOO ooo x g s u p e r n a t a n t f r o m r a t t h y m o c y t e s t o g e t h e r w i t h t h e b u l k of p r o t e i n a r o u n d p H 5. T h e specific b i n d i n g of t h e s u p e r n a t a n t s a n d p r e c i p i t a t e s w a s c a l c u l a t e d from i n c u b a t i o n s a t p H 7.4 w i t h i n M [I,2-3H2]corticosterone a n d w i t h t h e s a m e c o n c e n t r a t i o n of r a d i o a c t i v e h o r m o n e plus I/zM n o n - r a d i o a c t i v e corticosterone. F o r t h e s u p e r n a t a n t s t h e specific b i n d i n g is g i v e n as c p m per ioo/~1 after correction for t h e slight dilution d u e to t h e t i t r a t i o n s (open bars), while t h e b i n d i n g to t h e redissolved prec i p i t a t e s are g i v e n as c p m p e r IOO/zl of p r e c i p i t a t e d glucocorticoid-binding p r o t e i n p r e p a r a t i o n (black bars).
Between pH 6.0 and 9.0 virtually all the binding activity was found in the supernatants, but below pH 6.0 (Fig. 2) the binding activity occurred mainly in the redissolved precipitates. At pH 5-6, however, most of the protein in the preparation was precipitated. No binding activity at all could be shown after incubation at pH 4.0 or below. Biochim. Biophys. Acta, 263 (1972) 414-423
418
B. P. SCHAUMBURG
I n o t h e r e x p e r i m e n t s it was shown t h a t the i n a c t i v a t i o n at p H 4 occurred within a few minutes. The r e c o v e r y of binding a c t i v i t y in the p r e c i p i t a t e s o b t a i n e d between p H 5 a n d 6 was at the m o s t a p p r o x . 6O~o in the described e x p e r i m e n t , b u t when t h e acidification was p e r f o r m e d at a lower t e m p e r a t u r e ( - - 1 5 °C) a r e c o v e r y of n e a r l y lOO% could be obtained, b u t w i t h o u t appreciable purification.
Gradient centrifugation Sedimentation of glueocorticoid-binding protein in the absence of steroids T h e glucocorticoid-binding p r o t e i n s e d i m e n t e d as a m a j o r p e a k just b e h i n d catalase a n d a m i n o r p e a k j u s t a h e a d of hemoglobin at a KC1 concentration of IOO mM (Fig. 3). B y i n c u b a t i o n with r a d i o a c t i v e corticosterone plus an excess of unlabelled corticosterone it was controlled t h a t t h e p e a k s were due to high-affinity binding of corticosterone. T h e unspecific, low-affinity b i n d i n g was small a n d n e a r l y the same in all fractions e x c e p t in the u p p e r one or two fractions, where a slight increase of the unspecific b i n d i n g was noticed.
C~T~I~SE
H E M O G L O B IN
60:1 O
2 cx
'F,
.$ 20 ~ 01
~ -
-
i 10
ToP 210
T 30
P-rac t i o n N o .
Fig. 3. Gradient eentrifugation (2o-3O~o glycerol) of a IOO ooo × g supernatant from rat thymocytes. Marker proteins are catalase (11.3 S) and hemoglobin (4.1 S). The binding activity towards corticosterone was measured after the centrifugation by incubation with [i,2-aH~]corticosterone. At low salt concentration two peaks with sedimentation constants IO S and 4.5 S are observed, while at higher salt concentrations only the 4.5-S peak is seen. A t higher ionic s t r e n g t h (o.25, o.4, o.6 a n d I.O M KCI) only a single p e a k of binding a c t i v i t y was observed in the same position as the m i n o r p e a k in t h e presence of 0.I M KC1. No shift of t h e position of this p e a k was observed from 0.25 M KC1 to 1.0 M KC1 (except a slight d i s p l a c e m e n t of b o t h b i n d i n g a c t i v i t y a n d m a r k e r proteins due to tile higher d e n s i t y of the g r a d i e n t a t high KC1 concentrations) a n d t h e t r a n s f o r m a t i o n of glucocorticoid-binding p r o t e i n from the h e a v y form to the light form a p p e a r e d to be c o m p l e t e d a t a c o n c e n t r a t i o n of 0.25 M KC1. T h e s e d i m e n t a t i o n c o n s t a n t s could be e s t i m a t e d from the distance of sedimentation, t h e k n o w n s e d i m e n t a t i o n c o n s t a n t s of t h e m a r k e r proteins a n d the v a r i a t i o n of the density, viscosity a n d centrifugal force t h r o u g h the gradient. Values of I0.0 :J:
Biochim. Biophys. Acta, 263 (i972) 414-423
419
GLUCOCORTICOID-BINDING PROTEIN FROM THYMOCYTES
o.5 S and 4-5 zk o.2 S were obtained for the two peaks of glucocorticoid-binding protein.
Sedimentation of the glucocorticoid-binding protein-[z,2-3H2]corticosterone complex The complex was formed by incubation of a glucocorticoid-binding protein preparation with I nM [I,2-3H2]corticosterone at --5 °C in 40% glycerol. The incubate was in some cases adsorbed b y dextran-coated coal before the gradient centrifugation while in other cases the adsorption was performed after the centrifugation. However, the two methods gave identical values for the sedimentation constants.
60
CAT~,~/ASE
a
40-
g
HEMOGLOBIN
,k
o.1 MKCt
J'
_
TOP
OI
1
,
10
,
Fraction No.
20
'
30
Fig. 4. G r a d i e n t c e n t r i f u g a t i o n (20-30 % glycerol) of a i o o ooo X g s u p e r n a t a n t f r o m r a t t h y m o c y t e s a f t e r p r e v i o u s i n c u b a t i o n w i t h 2 n M [i,2-aH~]eorticosterone in 4 o % glycerol at - - 5 °C. T h e f r a c t i o n s were directly c o u n t e d .
At low ionic strength (Fig. 4) a peak of radioactivity with a sedimentation constant of IO S was observed, and there was a slight indication of a binding in the region of hemoglobin (approx. 4 S). At higher ionic strength only one broad peak was observed sedimenting just ahead of hemoglobin. This 4-5-S peak could also in this case be shown to be due to high-affinity binding as no such peak was observed when glucocorticoid-binding protein was preincubated with tritiated corticosterone plus an excess of unlabelled corticosterone. However, the peaks of bound tritiated corticosterone were not always reproducible and sometimes no binding at all could be shown. The contrast to the reproducibility of the experiments with free glucocorticoid-binding protein suggests that the cause is the dissociation of the corticosterone-glucocorticoid-binding protein complex during the centrifugation.
Sedimentation of the glucocorticoid-binding protein-[z,2,4-SH~]triamcinolone acetonide complex Gradient centrifugations of the [I,2,4-~H3]triamcinolone acetonide complex with glucocorticoid-binding protein gave much better results than the experiments with the corticosterone complex, in accordance with the much slower dissociation rate of the triamcinolone acetonide complex. At low salt concentration only one peak of bound radioactivity was found with a sedimentation constant of approx. IO S, Biochim. Biophys. $cta, 263 (1972) 414-423
420
B.P.
SCHAUMBURG
while at higher salt concentrations (from 0.25 M KC1) the peak was changed to 4.5 ~: 0.2 S (Figs. 5 and 6).
200-
o o
u
>, 100>
0.1M KCI
o
rn ¸
O' Bottom
10
Fraction No.
20
30
Fig. 5, G r a d i e n t c e n t r i f u g a t i o n (20-30% glycerol) of a Ioo ooo X g s u p e r n a t a n t from r a t t h y m o c y t e s a f t e r p r e v i o u s i n c u b a t i o n w i t h 2 nM [i,2,4-~H3~triamcinolone acetonide in 40% glycerol a t - - 5 °C. T h e fractions were directly counted.
emogtobin
~
300,
-30
q
Q25MKCt
o
o 20o-
~"
q
-20
~ lOO-
-lO
,-n o
o
10
o
20
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o
Fraction No.
Fig. 6. See legend for Fig. 5. M a r k e r p r o t e i n s were m e a s u r e d a t 41o n m ( O
O).
Sedimentation of the glucocorticoid-binding protein-Ex,2,4-SHs]triamcinolone acetonide complex from cells incubated at 37 °C A thymocyte suspension was incubated for 3o rain at 37 °C in the presence of IO nM [i,2,4-aH~]triamcinolone acetonide in a modified Krebs-Ringer-phosphate buffer s . Thereupon the cells were rapidly cooled b y the addition of ice-cold buffer and washed twice. The cells were resuspended in normal SH-buffer (o.z M KC1)5 or in SH-buffer with 0.6 M KC1, sonicated and ultracentrifuged (zoo ooo X g, 4 ° min, 4 °C). The Biochim. Biophys. Acta, 263 (1972) 414-423
GLUCOCORTICOID-BINDING PROTEIN FROM THYMOCYTES
421
content of bound and unbound radioactivity was then determined in the supernatants. The content of unbound radioactivity was nearly the same in the two supernatants, while the amount of bound radioactivity in the supernatant at low ionic strength was only 40% of that obtained at high ionic strength. It thus appeared that a part of the bound triamcinolone acetonide was released from the cells as free hormone during the sonication, probably following denaturation of glucocorticoid-binding protein. This denaturation was largely independent of the ionic strength. However, the extraction of the intact glucocorticoid-binding protein-steroid complex occulted most efficiently at high ionic strength. No peak of bound triamcinolone acetonide could be obselved after gradient centrifugation of the o.I M KC1 extract in a o.I M KC1 gradient, while a single peak was observed after centrifugation of the o.6 M KC1 extract in a o.6 M KC1 gradient (Fig. 7)- This peak sedimented more slowly than hemoglobin and bovine serum albumin, and had a sedimentation constant of 3-5 S. Serurn/l~
75-
0.6M KC|
50-
..~ 25-
m
01 10
20
30
Fraction No.
Fig. 7. G r a d i e n t centrifugation of a IOO ooo x g s u p e r n a t a n t obtained b y sonication in o.6 M KC1 and centrifugation of t h y m o c y t e s previously incubated w i t h io nM [I,2,4-3Ha]triamcinolone acetonide at 37 °C and w a s h e d at 4 °C. A single peak of b o u n d radioactivity is observed, h a v i n g a s e d i m e n t a t i o n c o n s t a n t of a p p r o x . 3.5 S irrespective of t h e salt concentration of the density gradient. No peaks were observed, w h e n the cells were incubated w i t h [I,2,4-aH3]triamcinolone acetonide in the presence of i t,lV[ of the unlabelled h o r m o n e .
The sedimentation constant of the bound triamcinolone acetonide was unchanged when the 0.6 M KC1 extract was centrifuged through a gradient with o.i M KC1.
Sedimentation of transcortin from rat plasma 50 times diluted rat plasma was subjected to gradient centrifugation. The sedimentation constant of transcortin was identical at high and low salt concentrations (0. 4 and o.I M KC1) as determined from the binding activity of the fractions towards EI,2-aH~]corticosterone after the run. The sedimentation constant was determined to 3.9 4- 0.2 S in agreement with the results of Chader and Westpha111. Biochim. Biophys. Acta, 263 (1972) 414-423
422
B.P. SCHAUMBURG
DISCUSSION
The pH dependence of the binding of corticosterone to glucocorticoid-binding protein is nearly identical with that described for the binding of glucocorticoids to transcortin 1~ and for the binding of oestradiol to the oestradiol "receptor" from uterus 13. The variation of the extent of binding appears to be due to a variation of the dissociation constant, and as the dissociation constant is equal to the ratio between the rate constants of dissociation and association for a simple binding reaction and furthermore, the rate constant for the dissociation 6 is unchanged between pH 6.5 and 8.5, the decreased affinity below p H 7 and above p H 8 must be due to a decreased rate of binding. The rapid diminution of the affinity below p H 7.4 can be explained if at least one group with a p K value of approx. 6.5 is involved in the binding. In the case of another steroid-binding protein, the 2ofl-hydroxysteroid dehydrogenase the presence of a histidine group in the binding site is indicated by affinity labelling 14. I f this group was a part of the binding site, the invariance of the dissociation rate could also be explained if the bound steroid made the group inaccessible to the influence of the medium. However, the group(s) with a p K of 6.5 could also be situated outside the binding site, excerting an indirect influence on the binding. The sedimentation behaviour of glucocorticoid-binding protein is nearly identical with that described for m a n y other steroid "receptors"13,15,16 except for the tlansformatior, of glucocorticoid-binding protein in the nuclei to a form with a smaller sedimentation constant than that observed for the cytoplasmic receptor at high ionic strength. It is possible that the different transformation of glucocorticoid-binding protein and, for example, the oestradiol receptor in the uterus should be considered in relation to the roughly opposite effects of glucocorticoids in lymphocytes and oestradiol in uterus; glucocorticoids inhibiting metabolic activity, while oestradiol stimulates.
ACKNOWLEDGEMENTS
Mrs Hanne Hegelund is thanked for the performance of the greatest part of the density gradient experiments. The performance of adrenalectomies, coal adsorptions and drawings bv Miss Susanne Ostergaard is appreciated.
REFERENCES i M. H. M a k m a n , S. N a k a g a w a a n d A. W h i t e , Recent Prog. Horm. Res., 23 (1968) 195. 2 A. M u n c k a n d C. Wira, in Schering Workshop on Steroid Hormone Receptors, Berlin Jr97o, Adv. Biosci., Vol. 7, P e r g a m o n Press, Vieweg, B r a u n s c h w e i g , 1971, p. 3Ol. 3 B. P. S c h a u m b u r g a n d E. Bojesen, Biochim. Biophys. Acta, 17o (1968) 172. 4 A. M u n c k a n d T. B r i n c k - J o h n s o n , J. Biol. Chem., 243 (1968) 55565 B. P. S c h a u m b u r g , Biochim. Biophys..dcta, 214 (197 o) 520. 6 B. P. S c h a u m b u r g , Biochim. Biophys. Acta, 261 (1972) 219. 7 B. P. S c h a u m b u r g , in 3rd fnt. Congr. Horm. Steroids, Abstr. papers presented, Hamburg, I97 o, E x c e r p t a Medica F o u n d a t i o n , A m s t e r d a m , 197 o, p. 156.
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8 E. S. West and W. R. Todd, Textbook of Biochemistry, Macmillan Company, New York, 1961. 9 P- I. Brecher, J.-P. Chabaud, V. Colucci, E. R. DeSombre, J. W. Flesher, G. N. Gupta, D. J. Hurst, M. Ikeda, H. J. Jacobson, E. V. Jensen, P. W. Jungblut, T. Kawashima, K. A. Kyser, H.-G. Neumann, M. Numata, G. A. Puca, N. Saha, S. Smith and T. Suzuki, in Schering Workshop on Steroid Hormone Receptors, Berlin, z97o, Adv. Biosci., Vol. 7, Pergamon Press, Vieweg, Braunschweig, 1971, p. 75io E. M. Reimann, D. A. Walsh and E. G. Krebs, J. Biol. Chem., 246 (1971) 1986. II G. J. Chader and U. Westphal, Biochemistry, 7 (1968) 4272. 12 U. Westphal, in G. Litwack, Biochemical Actions of Hormones, Vol. I, Academic Press, New York, 197 o, p. 209. 13 1R. Hahnel, Steroids, 17 (1971) lO5. 14 M. Ganguly and J. C. Warren, J. Biol. Chem., 246 (1971) 3646. 15 E.-E. Baulieu, I. Jung, J. P. Blondeau and P. Robel, in Sehering Workshop on Steroid Hormone Receptors, Berlin, z97o, Adv. Biosei., Vol. 7, Pergamon Press, Vieweg, Braunschweig, 1971, p. 179. 16 B. W. O'Malley, M. R. Sherman, D. O. Tort, T, C. Spelsberg, W. T. Schrader and A. W. Steggles, in Schering Workshop on Steroid Hormone Receptors, Berlin, z97 o, Adv. Biosei., Vol. 7, Pergamon Press, Vieweg, Braunschweig, 1971, p. 213.
Bioehim. Biophys. Acta, 263 (1972) 414-423
BIOCHIMICAET BIOPHYSICAACTA
BBA 36099 STUDIES OF T H E G L U C O C O R T I C O I D - B I N D I N G P R O T E I N FROM THYMOCYTES UI. pH D E P E N D E N C E OF T H E B I N D I N G AND D E N S I T Y - G R A D I E N T C E N T R I F U G A T I O N OF T H E P R O T E I N
BJORN P. SCHAUMBURG Institute of Experimental Endocrinology, University of Copenhagen, Narre All~ 71, 2Ioo (Denmark)
(Received 9 December, 1971)
SUMMARY
I. The binding of eorticosterone to the glucocorticoid-binding protein from thymocytes was maximal at p H 7-8 and decreased rapidly below p H 72. Glucocorticoid-binding protein was precipitated at pH 5-6 together with most of the protein in crude extracts from thymocytes. 3- After density gradient centrifugation of glucocorticoid-binding protein at a low salt concentration (o.I M KC1) 2 peaks of binding activity were found. The sedimentation constant of the major peak was IO.O ~: 0.5 S and that of the minor peak 4.5 :[: 0.2 S. At higher salt concentrations (o.25-1.o M KC1) only the 4.5-S peak was observed. 4. The complex with corticosterone and with triamcinolone acetonide showed the same sedimentation behaviour, although a partial dissociation of the corticosterone complex occurred during the run. 5. Binding of triamcinolone acetonide in intact cells at 37 °C followed by extraction with a buffer containing 0.6 M KC1 caused the appearance of a 3.5-S form of bound hormone, the sedimentation constant of which was uninfluenced by the KC1 concentration in the density gradient.
INTRODUCTION Glucocorticoids have a direct effect on the metabolism of lymphocytes i n vitro1, 2 when applied in physiological concentrations. Furthermore, the glucocorticoids are bound to a well-defined "receptor" protein in the cells, and the properties of the binding (specificity, kinetics and affinity) support the contention that this binding is the initial event in the action of the hormonesa-t The role of the glucocorticoid-binding protein from thymocytes in the hormonal mechanism of action is furthermore supported by the finding that some steroids are bound to glucocorticoidbinding protein without causing any metabolic effect and that these steroids at the same time inhibit the effects of cortisol 1,~. The glucocorticoid-binding protein is very unstable in crude extracts from Biochim. Biophys. Acta, 263 (1972) 414-423
GLUCOCORTICOID-BIND.ING PROTEIN FROM THYMOCYTES
415
thymocytes 5, but is stabilized by glycerol at low temperatures. Under these conditions the kinetics of binding and dissociation is simple and has been investigated for several steroids 6. In the experiments described below the effect of changing the pH was studied as well as the sedimentation behaviour of glucocorticoid-binding protein at low and high salt concentrations taking advantage of the extremely slow dissociation of bound triamcinolone acetonide from glucocorticoid-binding protein. A preliminary report of these experiments has been presented 7. METHODS
The preparation of thymocyte suspensions from adrenalectomized rats and the sonication of the cells followed by ultracentrifugation was performed as previously describedS, 6. The binding of tritiated steroids was measured by adsorption of unbound steroid by dextran-coated coalS, 6 and counting of the unadsorbed radioactivity in a liquid scintillation counter. The concentration of steroid-binding sites in the preparation was approximately i nM. Assuming a molecular weight of 50 ooo per binding site (corresponding roughly to the molecular weight of a protein with a sedimentation constant of 4.5 S) the steroid-binding protein could be calculated to account for o.oi% of the protein in the extract, containing 3-5 mg protein per ml.
Gradient centrifugation Density gradients (20-30% glycerol) were prepared from buffers containing 20 and 35% glycerol using three perpex pumps (LKB, Sweden). The preparation of glucocorticoid-binding protein was mixed with solutions of marker proteins and the concentration of glycerol adjusted to approx. 15°/o. 0.3 ml was layered on the top of the gradients. The centrifugation was performed either at 35 ooo rev./min using a SW 39 L rotor in a Spinco L5o preparative ultracentrifuge at approx. 4 °C or at 50 ooo rev./min using a SW 5o.1 rotor in a Spinco L2-65B preparative ultracentrifuge at --2 - --5 °C. After the run the bottom of the tubes were pierced in a self-constructed elution device and the content of the tubes displaced using a perpex pump. 30-35 fractions were normally collected using a fraction collector. In some of the experiments the glucocorticoid-binding protein preparation was incubated with radioactive hormone before the gradient centrifugation (prelabeled glucocorticoid-binding protein), while in other experiments the glucocorticoid-binding protein was unlabelled, in which cases the content of glucocorticoid-binding protein in the fractions was determined by incubation of aliquots with tritiated corticosterone followed by coal adsorption. The position of marker proteins was determined by measurements of the absorbance in a spectrophotometer after dilution of 5o-#1 aliquots with 125/A water. The buffers used for the preparation of the gradients contained the concentration of KC1 indicated in the separate experiments and IO mM potassium phosphate, 5 mM/~-mercaptoethanol and 0.5 mM EDTA (pH 7-4) before the mixing with glycerol. The sedimentation constants (s°20,w) of the marker proteins 8-10 were catalase 11.3 S, ovalbumin 3.55 S, bovine serum albumin 4.6 S,/~-amylase 8. 9 S and hemoglobin 4.1 S. Biochim. Biophys. Acta, 263 (1972) 414-423
416
B. P. SCHAUMBURG
RESULTS
pH dependence of the binding The binding of [I,2-3H~]corticosterone to glucocorticoid-binding protein was determined by incubation of a IOO ooo × g supernatant from sonicated thymocytes in the presence of i nM of the steroid (40% glycerol, --5 °C). After 2 days of incubation the binding was measured. Maximal binding was found between p H 7 and 8, while the binding decreased sharply below p H 7 and more gradually above p H 8 (Fig. i).
SO0-
400-
300-
O
O
200o
u
0 pH
Fig. i. p H dependence of the binding of corticosterone to glucocorticoid-binding protein. A IOO ooo × g s u p e r n a t a n t was p r e p a r e d from sonicated r a t t h y m o c y t e s . Aliquots of the supern a t a n t were incubated in 4 o~o glycerol at --5 °C after titration to different p H values. The binding of tritiated corticosterone was measured after 48 h incubation with i nM [I,2-3H2]corticosterone ( © - - © ) or with i nM [I,2-3H~]corticosteroneplus I/~M non-radioactive corticosterone ( Q - - O ) . The specific binding of [I,2-SH~]corticosterone to glucocorticoid-binding protein was obtained as the difference between the two curves ((~---(}).
The kinetics of dissociation of bound corticosterone have previously been investigatedS, 6 at p H values from 6.5-1o and the half-life of the complex found to be approx. 800 min between p H 6. 5 and 8.5, while it decreased to 30-60 min at p H IO.O. From these findings it can be concluded that the period of incubation should be more than sufficient to reach equilibrium of the binding in the whole p H interval, and that the decrease of the binding on both sides of p H 7-8 must be explained either by a decreased number of binding sites or by a decreased affinity of the existing sites. No decrease of the number of binding sites occurred, however, between p H 6 Biochim. Biophys./tcta, 263 (1972) 414-423
6LUCOCORTICOID-BINDING PROTEIN FROM THYMOCYTES
417
and 9, as shown by an experiment in which aliquots of glucocorticoid-binding protein were incubated for 24 h at different pH values, the pH adjusted to 7.4 and thereupon the binding of [I,2-~H21corticosterone measured after 2 days at pH 7.4. Above pH 9 and below pH 6, however, the binding activity appeared to be slowly and irreversibly destroyed. The pH dependence of the binding was thus due to the decreased affinity of glucocorticoid-binding protein for the steroid. As the steroid concentration (I nM) was well below the dissociation constant of the binding of [I,2-3H2]corticosterone (3.5 nM) 6 the binding should be inversely proportional to the dissociation constant, and Kd thus increased rapidly below pH 7 and more slowly above pH 8.
Precipitation at low pH Aliquots of a IOO ooo × g supernatant of sonicated thymocytes in 40% glycerol were titrated to different pH values in an ice bath and incubated for IOO min at --5 °C, centrifuged and the supernatants adjusted to pH 7-4, while the precipitates were dissolved in buffer, also at pH 7.4. The content of glucocorticoid-binding protein was determined by incubation with i nM Ii,2-3H2~corticosterone for 2 days followed by coal adsorption.
1o0 illll / i o[_AL£tLL 4.0
4.4
4.7 5.0 5.3 5.6 pFI of precipitation
6.0
74
Fig. 2. P r e c i p i t a t i o n of glucocorticoid-binding p r o t e i n in a IOO ooo x g s u p e r n a t a n t f r o m r a t t h y m o c y t e s t o g e t h e r w i t h t h e b u l k of p r o t e i n a r o u n d p H 5. T h e specific b i n d i n g of t h e s u p e r n a t a n t s a n d p r e c i p i t a t e s w a s c a l c u l a t e d from i n c u b a t i o n s a t p H 7.4 w i t h i n M [I,2-3H2]corticosterone a n d w i t h t h e s a m e c o n c e n t r a t i o n of r a d i o a c t i v e h o r m o n e plus I/zM n o n - r a d i o a c t i v e corticosterone. F o r t h e s u p e r n a t a n t s t h e specific b i n d i n g is g i v e n as c p m per ioo/~1 after correction for t h e slight dilution d u e to t h e t i t r a t i o n s (open bars), while t h e b i n d i n g to t h e redissolved prec i p i t a t e s are g i v e n as c p m p e r IOO/zl of p r e c i p i t a t e d glucocorticoid-binding p r o t e i n p r e p a r a t i o n (black bars).
Between pH 6.0 and 9.0 virtually all the binding activity was found in the supernatants, but below pH 6.0 (Fig. 2) the binding activity occurred mainly in the redissolved precipitates. At pH 5-6, however, most of the protein in the preparation was precipitated. No binding activity at all could be shown after incubation at pH 4.0 or below. Biochim. Biophys. Acta, 263 (1972) 414-423
418
B. P. SCHAUMBURG
I n o t h e r e x p e r i m e n t s it was shown t h a t the i n a c t i v a t i o n at p H 4 occurred within a few minutes. The r e c o v e r y of binding a c t i v i t y in the p r e c i p i t a t e s o b t a i n e d between p H 5 a n d 6 was at the m o s t a p p r o x . 6O~o in the described e x p e r i m e n t , b u t when t h e acidification was p e r f o r m e d at a lower t e m p e r a t u r e ( - - 1 5 °C) a r e c o v e r y of n e a r l y lOO% could be obtained, b u t w i t h o u t appreciable purification.
Gradient centrifugation Sedimentation of glueocorticoid-binding protein in the absence of steroids T h e glucocorticoid-binding p r o t e i n s e d i m e n t e d as a m a j o r p e a k just b e h i n d catalase a n d a m i n o r p e a k j u s t a h e a d of hemoglobin at a KC1 concentration of IOO mM (Fig. 3). B y i n c u b a t i o n with r a d i o a c t i v e corticosterone plus an excess of unlabelled corticosterone it was controlled t h a t t h e p e a k s were due to high-affinity binding of corticosterone. T h e unspecific, low-affinity b i n d i n g was small a n d n e a r l y the same in all fractions e x c e p t in the u p p e r one or two fractions, where a slight increase of the unspecific b i n d i n g was noticed.
C~T~I~SE
H E M O G L O B IN
60:1 O
2 cx
'F,
.$ 20 ~ 01
~ -
-
i 10
ToP 210
T 30
P-rac t i o n N o .
Fig. 3. Gradient eentrifugation (2o-3O~o glycerol) of a IOO ooo × g supernatant from rat thymocytes. Marker proteins are catalase (11.3 S) and hemoglobin (4.1 S). The binding activity towards corticosterone was measured after the centrifugation by incubation with [i,2-aH~]corticosterone. At low salt concentration two peaks with sedimentation constants IO S and 4.5 S are observed, while at higher salt concentrations only the 4.5-S peak is seen. A t higher ionic s t r e n g t h (o.25, o.4, o.6 a n d I.O M KCI) only a single p e a k of binding a c t i v i t y was observed in the same position as the m i n o r p e a k in t h e presence of 0.I M KC1. No shift of t h e position of this p e a k was observed from 0.25 M KC1 to 1.0 M KC1 (except a slight d i s p l a c e m e n t of b o t h b i n d i n g a c t i v i t y a n d m a r k e r proteins due to tile higher d e n s i t y of the g r a d i e n t a t high KC1 concentrations) a n d t h e t r a n s f o r m a t i o n of glucocorticoid-binding p r o t e i n from the h e a v y form to the light form a p p e a r e d to be c o m p l e t e d a t a c o n c e n t r a t i o n of 0.25 M KC1. T h e s e d i m e n t a t i o n c o n s t a n t s could be e s t i m a t e d from the distance of sedimentation, t h e k n o w n s e d i m e n t a t i o n c o n s t a n t s of t h e m a r k e r proteins a n d the v a r i a t i o n of the density, viscosity a n d centrifugal force t h r o u g h the gradient. Values of I0.0 :J:
Biochim. Biophys. Acta, 263 (i972) 414-423
419
GLUCOCORTICOID-BINDING PROTEIN FROM THYMOCYTES
o.5 S and 4-5 zk o.2 S were obtained for the two peaks of glucocorticoid-binding protein.
Sedimentation of the glucocorticoid-binding protein-[z,2-3H2]corticosterone complex The complex was formed by incubation of a glucocorticoid-binding protein preparation with I nM [I,2-3H2]corticosterone at --5 °C in 40% glycerol. The incubate was in some cases adsorbed b y dextran-coated coal before the gradient centrifugation while in other cases the adsorption was performed after the centrifugation. However, the two methods gave identical values for the sedimentation constants.
60
CAT~,~/ASE
a
40-
g
HEMOGLOBIN
,k
o.1 MKCt
J'
_
TOP
OI
1
,
10
,
Fraction No.
20
'
30
Fig. 4. G r a d i e n t c e n t r i f u g a t i o n (20-30 % glycerol) of a i o o ooo X g s u p e r n a t a n t f r o m r a t t h y m o c y t e s a f t e r p r e v i o u s i n c u b a t i o n w i t h 2 n M [i,2-aH~]eorticosterone in 4 o % glycerol at - - 5 °C. T h e f r a c t i o n s were directly c o u n t e d .
At low ionic strength (Fig. 4) a peak of radioactivity with a sedimentation constant of IO S was observed, and there was a slight indication of a binding in the region of hemoglobin (approx. 4 S). At higher ionic strength only one broad peak was observed sedimenting just ahead of hemoglobin. This 4-5-S peak could also in this case be shown to be due to high-affinity binding as no such peak was observed when glucocorticoid-binding protein was preincubated with tritiated corticosterone plus an excess of unlabelled corticosterone. However, the peaks of bound tritiated corticosterone were not always reproducible and sometimes no binding at all could be shown. The contrast to the reproducibility of the experiments with free glucocorticoid-binding protein suggests that the cause is the dissociation of the corticosterone-glucocorticoid-binding protein complex during the centrifugation.
Sedimentation of the glucocorticoid-binding protein-[z,2,4-SH~]triamcinolone acetonide complex Gradient centrifugations of the [I,2,4-~H3]triamcinolone acetonide complex with glucocorticoid-binding protein gave much better results than the experiments with the corticosterone complex, in accordance with the much slower dissociation rate of the triamcinolone acetonide complex. At low salt concentration only one peak of bound radioactivity was found with a sedimentation constant of approx. IO S, Biochim. Biophys. $cta, 263 (1972) 414-423
420
B.P.
SCHAUMBURG
while at higher salt concentrations (from 0.25 M KC1) the peak was changed to 4.5 ~: 0.2 S (Figs. 5 and 6).
200-
o o
u
>, 100>
0.1M KCI
o
rn ¸
O' Bottom
10
Fraction No.
20
30
Fig. 5, G r a d i e n t c e n t r i f u g a t i o n (20-30% glycerol) of a Ioo ooo X g s u p e r n a t a n t from r a t t h y m o c y t e s a f t e r p r e v i o u s i n c u b a t i o n w i t h 2 nM [i,2,4-~H3~triamcinolone acetonide in 40% glycerol a t - - 5 °C. T h e fractions were directly counted.
emogtobin
~
300,
-30
q
Q25MKCt
o
o 20o-
~"
q
-20
~ lOO-
-lO
,-n o
o
10
o
20
3tO
o
Fraction No.
Fig. 6. See legend for Fig. 5. M a r k e r p r o t e i n s were m e a s u r e d a t 41o n m ( O
O).
Sedimentation of the glucocorticoid-binding protein-Ex,2,4-SHs]triamcinolone acetonide complex from cells incubated at 37 °C A thymocyte suspension was incubated for 3o rain at 37 °C in the presence of IO nM [i,2,4-aH~]triamcinolone acetonide in a modified Krebs-Ringer-phosphate buffer s . Thereupon the cells were rapidly cooled b y the addition of ice-cold buffer and washed twice. The cells were resuspended in normal SH-buffer (o.z M KC1)5 or in SH-buffer with 0.6 M KC1, sonicated and ultracentrifuged (zoo ooo X g, 4 ° min, 4 °C). The Biochim. Biophys. Acta, 263 (1972) 414-423
GLUCOCORTICOID-BINDING PROTEIN FROM THYMOCYTES
421
content of bound and unbound radioactivity was then determined in the supernatants. The content of unbound radioactivity was nearly the same in the two supernatants, while the amount of bound radioactivity in the supernatant at low ionic strength was only 40% of that obtained at high ionic strength. It thus appeared that a part of the bound triamcinolone acetonide was released from the cells as free hormone during the sonication, probably following denaturation of glucocorticoid-binding protein. This denaturation was largely independent of the ionic strength. However, the extraction of the intact glucocorticoid-binding protein-steroid complex occulted most efficiently at high ionic strength. No peak of bound triamcinolone acetonide could be obselved after gradient centrifugation of the o.I M KC1 extract in a o.I M KC1 gradient, while a single peak was observed after centrifugation of the o.6 M KC1 extract in a o.6 M KC1 gradient (Fig. 7)- This peak sedimented more slowly than hemoglobin and bovine serum albumin, and had a sedimentation constant of 3-5 S. Serurn/l~
75-
0.6M KC|
50-
..~ 25-
m
01 10
20
30
Fraction No.
Fig. 7. G r a d i e n t centrifugation of a IOO ooo x g s u p e r n a t a n t obtained b y sonication in o.6 M KC1 and centrifugation of t h y m o c y t e s previously incubated w i t h io nM [I,2,4-3Ha]triamcinolone acetonide at 37 °C and w a s h e d at 4 °C. A single peak of b o u n d radioactivity is observed, h a v i n g a s e d i m e n t a t i o n c o n s t a n t of a p p r o x . 3.5 S irrespective of t h e salt concentration of the density gradient. No peaks were observed, w h e n the cells were incubated w i t h [I,2,4-aH3]triamcinolone acetonide in the presence of i t,lV[ of the unlabelled h o r m o n e .
The sedimentation constant of the bound triamcinolone acetonide was unchanged when the 0.6 M KC1 extract was centrifuged through a gradient with o.i M KC1.
Sedimentation of transcortin from rat plasma 50 times diluted rat plasma was subjected to gradient centrifugation. The sedimentation constant of transcortin was identical at high and low salt concentrations (0. 4 and o.I M KC1) as determined from the binding activity of the fractions towards EI,2-aH~]corticosterone after the run. The sedimentation constant was determined to 3.9 4- 0.2 S in agreement with the results of Chader and Westpha111. Biochim. Biophys. Acta, 263 (1972) 414-423
422
B.P. SCHAUMBURG
DISCUSSION
The pH dependence of the binding of corticosterone to glucocorticoid-binding protein is nearly identical with that described for the binding of glucocorticoids to transcortin 1~ and for the binding of oestradiol to the oestradiol "receptor" from uterus 13. The variation of the extent of binding appears to be due to a variation of the dissociation constant, and as the dissociation constant is equal to the ratio between the rate constants of dissociation and association for a simple binding reaction and furthermore, the rate constant for the dissociation 6 is unchanged between pH 6.5 and 8.5, the decreased affinity below p H 7 and above p H 8 must be due to a decreased rate of binding. The rapid diminution of the affinity below p H 7.4 can be explained if at least one group with a p K value of approx. 6.5 is involved in the binding. In the case of another steroid-binding protein, the 2ofl-hydroxysteroid dehydrogenase the presence of a histidine group in the binding site is indicated by affinity labelling 14. I f this group was a part of the binding site, the invariance of the dissociation rate could also be explained if the bound steroid made the group inaccessible to the influence of the medium. However, the group(s) with a p K of 6.5 could also be situated outside the binding site, excerting an indirect influence on the binding. The sedimentation behaviour of glucocorticoid-binding protein is nearly identical with that described for m a n y other steroid "receptors"13,15,16 except for the tlansformatior, of glucocorticoid-binding protein in the nuclei to a form with a smaller sedimentation constant than that observed for the cytoplasmic receptor at high ionic strength. It is possible that the different transformation of glucocorticoid-binding protein and, for example, the oestradiol receptor in the uterus should be considered in relation to the roughly opposite effects of glucocorticoids in lymphocytes and oestradiol in uterus; glucocorticoids inhibiting metabolic activity, while oestradiol stimulates.
ACKNOWLEDGEMENTS
Mrs Hanne Hegelund is thanked for the performance of the greatest part of the density gradient experiments. The performance of adrenalectomies, coal adsorptions and drawings bv Miss Susanne Ostergaard is appreciated.
REFERENCES i M. H. M a k m a n , S. N a k a g a w a a n d A. W h i t e , Recent Prog. Horm. Res., 23 (1968) 195. 2 A. M u n c k a n d C. Wira, in Schering Workshop on Steroid Hormone Receptors, Berlin Jr97o, Adv. Biosci., Vol. 7, P e r g a m o n Press, Vieweg, B r a u n s c h w e i g , 1971, p. 3Ol. 3 B. P. S c h a u m b u r g a n d E. Bojesen, Biochim. Biophys. Acta, 17o (1968) 172. 4 A. M u n c k a n d T. B r i n c k - J o h n s o n , J. Biol. Chem., 243 (1968) 55565 B. P. S c h a u m b u r g , Biochim. Biophys..dcta, 214 (197 o) 520. 6 B. P. S c h a u m b u r g , Biochim. Biophys. Acta, 261 (1972) 219. 7 B. P. S c h a u m b u r g , in 3rd fnt. Congr. Horm. Steroids, Abstr. papers presented, Hamburg, I97 o, E x c e r p t a Medica F o u n d a t i o n , A m s t e r d a m , 197 o, p. 156.
Biochim. Biophys..dcta, z63 (1972) 414-423
GLUCOCORTICOID-BINDING PROTEIN FROM THYMOCYTES
423
8 E. S. West and W. R. Todd, Textbook of Biochemistry, Macmillan Company, New York, 1961. 9 P- I. Brecher, J.-P. Chabaud, V. Colucci, E. R. DeSombre, J. W. Flesher, G. N. Gupta, D. J. Hurst, M. Ikeda, H. J. Jacobson, E. V. Jensen, P. W. Jungblut, T. Kawashima, K. A. Kyser, H.-G. Neumann, M. Numata, G. A. Puca, N. Saha, S. Smith and T. Suzuki, in Schering Workshop on Steroid Hormone Receptors, Berlin, z97o, Adv. Biosci., Vol. 7, Pergamon Press, Vieweg, Braunschweig, 1971, p. 75io E. M. Reimann, D. A. Walsh and E. G. Krebs, J. Biol. Chem., 246 (1971) 1986. II G. J. Chader and U. Westphal, Biochemistry, 7 (1968) 4272. 12 U. Westphal, in G. Litwack, Biochemical Actions of Hormones, Vol. I, Academic Press, New York, 197 o, p. 209. 13 1R. Hahnel, Steroids, 17 (1971) lO5. 14 M. Ganguly and J. C. Warren, J. Biol. Chem., 246 (1971) 3646. 15 E.-E. Baulieu, I. Jung, J. P. Blondeau and P. Robel, in Sehering Workshop on Steroid Hormone Receptors, Berlin, z97o, Adv. Biosei., Vol. 7, Pergamon Press, Vieweg, Braunschweig, 1971, p. 179. 16 B. W. O'Malley, M. R. Sherman, D. O. Tort, T, C. Spelsberg, W. T. Schrader and A. W. Steggles, in Schering Workshop on Steroid Hormone Receptors, Berlin, z97 o, Adv. Biosei., Vol. 7, Pergamon Press, Vieweg, Braunschweig, 1971, p. 213.
Bioehim. Biophys. Acta, 263 (1972) 414-423