Molecular end Cellular Endocrinology,
0 Elsevier/North-Holland
I (1977) 49-66 Scientific Publishers, Ltd.
BINDING OF THE PARTIALLY PURIFIED GLUCOCORTICOID OF RAT LIVER TO CHROMATIN AND DNA *
RECEPTOR
Harald BUGANY and Miguel BEAT0 ** Institut fiir Physiologische
Chemie, D-3550 MarburgjLahn,
Deutschhausstr.
I-2, G.F.R.
Received 8 June 1976; accepted 2 September 1976
The binding of the glucocorticoid receptor of rat liver to chromatin and DNA has been studied with crude and partially purified preparations of cytosol receptor labelled with [3H]triamcinolone acetonide in vitro. The use of crude preparations of receptor and increasing protein concentrations leads to an apparent saturation of chromatin and DNA, suggesting a limited number of high affinity nuclear acceptor sites for the receptor. Appropriate controls indicate that the observed saturability of chromatin acceptor sites is due to the presence in crude receptor preparations of heat-stable protein factors which interfere with the binding of the receptor to the genome; whereas the apparent saturation of DNA is due to contamination with deoxyribonucleases. If the activated complex of receptor and triamcinolone acetonide (R-TA) is partially purified to a step where it is free from nucleases and inhibitors, its binding to both chromatin and DNA is linearly dependent on the concentration of free (R-TA) in the incubation medium. There is no absolute specificity with respect to the source of DNA or chromatin, although liver chromatin has considerably higher receptor binding capacity than chromatin from avian erythrocytes. The rate kinetics of association and dissociation for the binding of (R-TA) to DNA and chromatin are very similar, but DNA exhibits a lo-fold higher receptor binding capacity than chromatin. These data, in conjunction with the effect of poly-(D)-lysine and NaCl on the binding of (R-TA) to chromatin and DNA, suggest that most of the receptor molecules bound to chromatin in vitro interact with the ‘accessible’ DNA stretches. Although a small population of receptor molecules may bind specifically to target tissue genome, the detection of these specific sites against the background of unspecific binding is not possible with unfractionated chromatin or DNA preparations. Keywords:
protein-DNA
interaction; DNA-cellulose;
triamcinolone acetonide.
Glucorticoid hormones are known to act on their target organs through a multistep mechanism involving an interaction with specific receptor proteins in the cytosol, followed by the activation of the receptor-steroid complex and its migration to the cell nucleus. Within the nucleus the receptor-steroid complex binds to chromatin, and is supposed to modulate the transcription of specific genes (for * This work was supported by a grant of the Deutsche Forschungsgemeinschaft ** To whom correspondence should be sent.
49
(SFB 103-1,2).
H. Bugany, M. Beato
50
review see Beato and Doenecke, 1976). Various parts of this pathway have been reproduced in cell-free systems, in which it has been demonstrated that the activation of the receptor-glucocorticoid complex is dependent on temperature and ionic strength, and confers to the complex affinity for chromatin and DNA (Baxter et al., 1972; Beato et al., 1973; Hamana and Iwai, 1973; Higgins et al., 1973a; Kalimi et al., 1973; Milgrom et al., 1973). A preferential binding of the glucocorticoid receptor to the nuclei of the target organs has been reported (Hamana and Iwai, 1973; Higgins et al., 1973b; Kalimi et al., 1973), and Lippman and Thompson (1974) claimed that the nuclear acceptor sites can distinguish between glucocorticoid receptors from target and non-target tissues. On the other hand, some groups have described the existence of a limited number of high affinity nuclear acceptor sites for the glucocorticoid receptor (Baxter et al., 1972; Higgins et al., 1973a; Kalimi et al., 1973; Lippman and Thompson, 1974), whereas other laboratories could not observe a saturation of the nuclear sites with receptor-steroid complex (Rousseau et al., 1974; Milgrom and Atger, 1975; Climent et al., 1976; Simons et al., 1976). The binding of the activated receptor to purified DNA shows no saturation in the range of physiological receptor concentrations, and exhibits very little specificity in respect to the source of the DNA (Baxter et al., 1972; Beato et al., 1973; Milgrom et al., 1973; Rousseau et al., 1974, 1975; Simons et al., 1976). Therefore, the physiological significance of the interaction of the receptor-steroid complex with DNA is not clear and very little is known about the role of the chromosomal proteins in nuclear binding of the complex. In this paper we present a comparison of the binding of the receptor-glucocorticoid complex to chromatin and DNA.
MATERIALS
AND METHODS
[6,7-3H] Triamcinolone acetonide *, spec. act. 33.7 Ci/mmol was obtained from New England Nuclear Inc. The nonradioactive steroids, DNA from calf thymus, salmon sperm and E. coEi, were purchased from Sigma and Co. Crystallized bovine serum albumin (BSA) was obtained from the Behringwerke, Marburg. Pronase, grade B, pancreatic ribonuclease A, and Nonidate P40, were purchased from Serva, Heidelberg. Cellulose (Cellex 410) und Bio-Gel PlO were obtained from Bio-Rad Laboratories Inc, and phosphocellulose (Pll) was from Whatman Inc. Poly-(D)lysine hydroxybromide, molecular weight 140,000, was purchased from Sigma and Co. Male Wistar II rats weighing 120-140 g were used throughout. The animals were adrenalectomized 5-10 days before the beginning of the experiments.
* TA: triamcinolone acetonide: 9~fluoro-1 3,20dione cyclic 16,17-acetal with acetone.
lp,16a,17q2l-tetrahydroxy-pregnan-l,4diene-
Preparation and fractionation of liver cytosol The animals were killed by cervical dislocation and the liver was perfused in situ through the portal vein with 10 ml of cold TSS buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCla, 2 mM mercaptoethanol, and 50 mM Tris-HCI, pH 7.5). The livers were then removed, and the cytosol was prepared as previously described (Beat0 and Feigelson , 1972). For some experiments the cytosol was used directly after incubation with radioactive triamcinolone acetonide (TA) as indicated in the legend to the corresponding figures. In another series of experiments the cytosol was fractionated with ammonium sulphate (Beat0 and Feigelson, 1972). For these experiments cytosol was first incubated with 10 mM [3H]TA for 30 min at 22’C, before addition of saturated ammonium sulphate, pH 7.0, to 30% saturation. After slowly stirring for 30 min at 0°C the cytosol was centrifuged at 20,OOOg for 10 min and the pellet was resuspended in TSS buffer (0.1 vol. of the original cytosol). The ammonium sulphate was then removed by passing the sample through a Bio-Gel PlO column equilibrated with the appropriate binding buffer. Partial purification of the activated receptor-steroid complex, (R-TA) For another series of experiments a partially purified receptor-steroid complex, (R-TA), was used. The details of the purification procedure, and the characterization of the partially purified receptor have been published elsewhere (Climent et al., 1976; Climent and Beato, in preparation). In summary, the procedure is as follows: Cytosol is incubated with 50 nM [3HJ TA at 0°C for IO min in medium of low ionic strength (25 mM KCl), and passed through two large phosphocellulose columns. The unbound material is incubated at 22°C for 30 min in order to activate the (R-TA), and passed through a small phosphocellulose column, to which the activated (R-TA) is bound, and can be eluted by increasing the salt concentration. This very simple procedure usually yielded over 3000-fold purification of the (RTA), and the final preparation was free of deoxyribonuclease (DNase) activity (Climent et al., 1976). The concentration of (R-TA) in different preparations was determined by the charcoal technique (Beat0 and Feigelson, 1972). When partially purified receptor was used, the concentration of charcoal was reduced by lo-fold, and BSA (final concentration of 0.1%) was added to the samples in order to prevent adsorption of the receptor to the charcoal. Preparation ofrat liver and au&n erythrocyte chromatic The pellet obtained after centrifugation of the liver homogenate at 750g was resuspended in two volumes of TSS buffer and mixed with an equal volume of a I% solution of Nonidet P40 in TSS buffer. After 30 s the nuclei were centrifuged at XOOg for 5 min. The pellet was resuspended in 4 volumes of TSS buffer and centrifuged again to remove the detergent. Further purification of chromatin was as previously described (Beat0 et al., 1970). For the preparation of erythrocyte chromatin from duck or chicken a proce-
52
H. Bugany, M. Beato
dure similar to that used for rat liver was employed. The erythrocytes were purified by repeated washings in 0.15 M KC1 containing 3 mM MgCla, and disrupted by hypotonic lysis in buffer containing 25 mM Tris-HCl, pH 8.0. After completion of the cell lysis, 0.16 volumes of 7-fold concentrated TSS buffer were added and a crude nuclear pellet was obtained by centrifugation at 75Og for 10 min. The rest of the chromatin preparation was identical to that previously described for rat liver (Beat0 et al., 1970). Beparation of rat liver DNA and coupling of DNA to cellulose DNA was prepared from purified nuclei of rat liver according to the procedure of Marmur (1961), modified to include treatments with pronase and ribonuclease (RNase) (Beat0 et al., 1970). DNA of various sources was coupled to cellulose according to the procedure of Litman (1968). The DNA content of the cellulose was in the order of 3-7 mg/g cellulose. Assay of receptor binding to chromatin and DNA-cellulose Binding of (R-TA) to chromatin or DNA was measured in 550-d assays containing 100 fl of a suspension of chromatin, DNA-cellulose or cellulose alone, and different amounts of (R-TA). When specified, heat-denatured cytosol or cytosol which was not incubated with radioactive steroid was added to the assay. The incubation media contained 10% glycerol, 1 mM EDTA-Naa, 10 mM Tris-HCl, pH 8.0, and variable amounts of NaCl as indicated. The salt concentration was adjusted with a concentrated solution of NaCl. Incubation was performed in Eppendorf reaction tubes for 30 min at 22’C. Every 5 min the tubes were shaken to resuspend cellulose or chromatin. When partially purified (R-TA) was used, bovine serum albumin was added to a final concentration of 0.1%. At the end of the incubation, the tubes were centrifuged at 10,OOOg for 2 min and the pellets washed twice with cold incubation medium of the appropriate ionic strength. Radioactivity measurements were performed by resuspending the pellets in 7 ml of Bray’s solution (Bray, 1960). Determination of DNA content was performed in a parallel assay by the procedure of Burton (1956). When BSA was used, the corresponding blanks in the absence of the receptor and with similar amounts of [3H] TA were performed. The kinetics of association and dissociation for the reaction of the partially purified (R-TA) with chromatin and DNA were determined as described in the legend to fig. 7. Effect of NaCl concentration The influence of NaCl on the binding of (R-TA) to chromatin and DNA was investigated with partially purified receptor preparations. In one set of experiments (R-TA) was incubated with chromatin, DNA-cellulose or cellulose in buffer containing 25 mM NaCl for 30 min at 22°C and after centrifugation the pellets were washed twice in incubation buffer containing the appropriate NaCl concentration. For each wash, the resuspended samples were allowed to
~l~c~cortic~id receptor minding to the genome
53
stand in the ice-bath for 10 min before centrifugation. The final pellet was used for radioactivity determination. In another set of experiments the incubation of the receptor with chromatin or DNA-cellulose was carried out at different ionic strengths and the washing of the pellets was performed at the same NaCl concentration as was present during the incubation. determination of the receptor birzditzgcapacity of chromutin and DIVA For these experiments increasing amounts of a suspension of chromatin, DNAcellulose or cellulose alone were resuspended in 550 $ of incubation medium containing 0.15 M NaCl, the partially purified (R-TA) and 0.5 mg BSA. After incuba” tion at 22°C for 30 min the samples were centrifuged, the pellets washed twice and the radioactivity and DNA content determined as described above.
Rat liver chromatin was resuspended by sonication in 50 mM sodium phosphate buffer, pH 7.0, and centrifuged at 10,OOOg for 10 min. To the supernatant (4.90 ml containing 5 X 10V4 M nucleotides) 100 111aliquots of polylysine solution in 50 mM sodium phosphate buffer, pH 7.0, (2 X 10m3 M lysine) were added. After standing for 10 min at 0°C aliquots of 50 111were taken for incubation with (R-TA) and for the determination of the amounts of DNA in chromatin which was complexed with polylysine, as described below. This procedure was repeated 11 times after addition each time of 100 ,ul of pdylysine. The binding of the receptor to the polylysine-treated chromatin was measured by incubating 0.5 ml of (R-TA) with a 50 4 aliquot of polylysine-treated chromatin at 22°C for 1 h in 50 mM sodium phosphate buffer, pH 7.0. The samples were then centrifuged at 10,OOOg for 10 min and the pellets washed twice with 1.5 ml of incubation medium. The final pellets were resuspended by sonication in 200 4 of incubation medium and aliquots were taken for the determination of DNA and for radioactivity measurements. The determination of the amount of chromatin DNA which was precipitated by polylysine was performed by mixing a 50 &l aliquot of polylysine-treated chromatin with 1 ml incubation medium and centrifuging at 10,OOOg for 10 min. The DNA content in the supernatant was determined, and the difference between this value and the total DNA content of the chromatin gave the amount of DNA precipitated by polylysine.
RESULTS Binding of the receptor-steroid complex to chromatin The binding of (R-TA) to chromatin was first investigated using as receptor preparation either the crude cytosol labelled with [3H]TA or the ammonium sulphate fraction containing the receptor-triamcinolone complex. Under these con-
H. Bugany, M. Beato
54
ditions incubation of a given amount of chromatin with increasing concentrations of receptor-steroid complex leads to a saturation curve, comparable to that obtained with isolated rat liver nuclei (Kalimi et al., 1973). The extent of binding is very sensitive to the ionic conditions of the incubation medium (fig. 1). In medium containing 25 mM NaCl and no divalent cations a high binding capacity is observed and half saturation is reached at concentrations of (R-TA) in the order of lo-* M, as calculated by Scatchard analysis. Raising the concentration of NaCl to 0.15 M results in a considerable decrease in the amount of bound (R-TA), and half saturation is reached at lower concentrations of (R-TA). Addition of MgCla (3 mM) to a medium containing low concentration of NaCl(25
bl
.3mM
1
MgC12
2.0
LIVER.25mM
1.0
LIVER.lSOrnM
LIVER.lSOmM
J
Fig. 1. Effect of ions on the binding of crude (R-TA) to chromatin. A crude (R-TA) was prepared from liver cytosol incubated with [3H] TA (50 nM) by precipitation with 30% saturated ammonium sulphate, as indicated in Methods. Increasing amounts of this (R-TA) preparation were incubated at 20°C for 1 h with a constant amount of either rat liver (125 Mg DNA) or duck erythrocyte (405 pg DNA) chromatin in a final volume of 0.55 ml, containing different final concentrations of NaCl and MgClZ. The amount of chromatin-bound (R-TA) and the DNA content of the chromatin pellets were determined as described in Methods. (a) Incubations performed in the absence of divalent cations: at 25 mM NaCl with liver (0) and erythrocyte (0) chromatin; or at 150 mM NaCl, with liver (e) and erythrocyte chromatin (0); (b) incubation performed in the presence of 3 mM MgCls. All other conditions and symbols as in (a).
Glucocorticoid
receptor
binding to the genome
55
mM) also results in a reduction of the binding capacity of chromatin for (R-TA). However, when the concentration of NaCl in the incubation mixture is 0.15 M, MgCl, has little effect on either the affinity or the number of binding sites for the receptor in isolated chromatin (compare Fig. 1, a and b). Under these more physiological conditions (0.15 M NaCI) the number of molecules of (R-TA) bound per haploid genome, calculated by Scatchard analysis, is in the order of 4-7 X 103, but this number varies considerably with the amount of DNA used in the different experiments. When a small amount of DNA in chromatin is used and (R-TA) is added in large excess, the number of receptor molecules bound at saturation is low (range of 4 experiments: 600-2400 molecules per haploid genome). Raising the amount of chromatin-DNA leads to a higher number of bound molecules of (RTA) and the apparent affinity is lower. These latter results already indicate that some components of the cytosol preparation influence the binding of the receptor to the chromatin and prevent a precise quantitation of the binding parameters. Nevertheless, under these conditions a considerable tissue specificity is observed as demonstrated by a comparison of the amount of (R-TA) bound to rat liver versus erythrocyte chromatin (Fig. la). The form of the saturation curve, however, is very similar for both chromatins indicating that the binding process is not essentially different, but just that there are fewer acceptor sites in the erythrocyte chromatin. In order to explore the behaviour of the saturation curve under different incubation conditions, and following the suggestion of Chamness et al. (1974), a series of control experiments were performed, in which the concentration of cytosol proteins was maintained constant and the concentration of receptor-steroid complex was the only variable. This was achieved in one set of experiments by incubating a constant amount of cytosol with different concentrations of [3H] TA and determining concentration of (R-TA) by the charcoal procedure. Under these conditions the binding of receptor-steroid complex to chromatin was linearly dependent on the concentration of free (R-TA). Similar results were obtained when increasing amounts of cytosol labelled with [3H]TA were incubated with chromatin and the volume was maintained constant by the addition of non-labelled cytosol or cytosol heated at 50°C for IO min before the incubation (data not shown). This latter control eliminates the possibility, that the apparent saturation is due to free receptor molecules present in non-labelled cytosol, as they will be inactivated by the heating step (Koblinsky et al., 1972) A linear dependence on (R-TA) concentration was also observed when a receptor preparation enriched by fractionation with ammonium sulphate was used and the volume was kept constant by adding an ammonium sulphate fraction prepared from cytosol which was not incubated with [3H] TA (fig. 2). In all the experiments with added cytosol fractions the values obtained at low concentrations of (R-TA) are lower than those observed with a conventional assay (fig. 2) suggesting the existence in the cytosol preparation, and in the ammonium sulphate fraction of heat-stable inhibitors of the binding of the receptor to chromatin. A similar conclusion can be drawn from the experiments with partially purified (R-TA), in
H. Bugany, M. Beato
IR-TA
I
FREE’
nM
Fig. 2. Effect of cytosol. components on the binding of (R-TA) to rat liver chromatin. Constant amounts of rat liver chromatin (140 pg DNA) were incubated with increasing amounts of a crude preparation of (R-TA) obtained by precipitation of the cytosol with ammonium sulphate as indicated in Methods. Conditions of incubation were 22°C for 1 h in buffer containing 0.15 M NaCl. In one series of experiments the final voiume of 0.55 ml was maintained by addition of incubation buffer (=), whereas in another series of experiments the final volume and protein concentration were kept constant by the addition of a 30% ammonium sulphate fraction prepared from unlabelled cytosol (0). Also shown are the results obtained with a partially purified preparation of (R-TA) incubated with chromatin under similar conditions (0). The determination of chromatin-bound (R-TA) and DNA was performed as described in Methods.
which the amount of receptor bound to chromatin was also a linear function of the concentration of free (R-TA), and the values obtained were considerably higher than those observed with the crude cytosol preparations or the ~monium sulphate fraction (fig. 3). Under these conditions, the binding capacity of rat liver chromatin for the partially purified (R-TA) is 3.fold higher than that of chicken erythrocyte chromatin (fig. 3). biding of receptor-steroid complex to DIVA Incubation of calf thymus DNA-cellulose with increasing
amounts
of cytosol
Glucocorticoid
receptor binding to the genome
0.25
0.5 IR -TA IFREE n M
Fig. 3. Binding of partially purified (R-TA) to chromatin. Constant amounts of chromatin prepared either from rat liver (211 pg DNA) or from chicken erythrocytes (625 Hg DNA) were incubated in buffer containing 0.15 M NaCl, with increasing concentrations of partially purified (R-TA), prepared as described in Methods. The incubations were performed in a final volume of 0.55 ml for 1 h, at 22”C, and the amount of (R-TA) bound to rat liver (9) and erythrocyte (0) chromatin, as well as the DNA content were determined as described in Methods.
labelled with [3H] TA leads to an apparent saturation of the DNA binding capacity (fig. 4). However, determination of the DNA content in the pellet obtained after incubation shows that the crude cytosol contains considerable DNase activity. When the values of (R-TA) bound are expressed per mg DNA recovered in the pellet, a linear dependence on the concentration of free (R-TA) in the incubation medium is observed. It is interesting to note that the apparent saturation of DNA with crude cytosol receptor preparations is only detected in the presence of MgCla. This observation is in agreement with the finding that the DNase activity of the cytosol, as measured in the DNA-cellulose assay, is dependent on Mg*+. In the absence of MgCla, even using crude cytosol, there is no indication of saturation of the DNA-cellulose with (R-TA). Similar linear relationship between free and bound (R-TA) was observed when partially purified receptor preparations were
H. Bugany, M. Beato
Fig. 4. Binding of crude (R-TA) to calf thymus DNA. A constant amount of calf thymus DNA-cellulose (30 Irg DNA) or an equivalent amount of plain cellulose were incubated at 22°C for 1 h with increasing amounts of rat liver cytosol, labeled with [ %I] TA. The final volume was maintained at 0.55 ml by addition of buffer containing NaCl and MgCIz to give fii concentrations of 0.15 M and 3 mM, respectively. The amount of (R-TA) bound to DNA (a) was determined by subtracting from the DNA-cellulose values, the values obtained with cellulose alone. In parallel analysis the DNA content of the cellulose was determined after incubation with the same amount of crude (R-TA), and used to calculate the amount of (R-TA) bound per mg of DNA (s).
used in the binding assays, both in the absence and presence of Mg’+. There is little specificity in respect to the source of the DNA, as only quantitative differences are detected in the binding capacity of rat liver, calf thymus and salmon sperm (fig. 5). Similar binding was also observed with DNA prepared from E. coli (data not shown). Comparison between receptor binding to chromatin and DNA The comparison of the values in figs. 3 and 5 clearly shows that the capacity of DNA for binding (R-TA) is much higher than that of chromatin (Climent et al.,
Glucocorticoid
receptor
binding to the genome
1
RAT
:
59
LIVER
CALF
THYMUS
Q e \ 2aI
SALMON
50-
=. P 2 -_Q : E
25-
I
I
2.5
5.0
‘R-‘“IFREE , n
M
Fig. 5. Binding of partially purified (R-TA) to DNA of various sources. A constant amount (30 pg DNA) of DNA-cellulose, or an equivalent amount of plain cellulose, was incubated at 22°C for 1 h with increasing concentrations of partially purified (R-TA) in buffer containing 0.15 M NaCl. The amount of (R-TA) bound to DNA of rat liver (a), calf thymus (o), and salmon sperm (e), was calculated by subtracting the blank values obtained with plain cellulose. The DNA content was determined in parallel assays and shown to be constant, independent of the amount of (R-TA) added.
1976). A direct comparison of relative capacities of rat liver chromatin and calf thymus DNA-cellulose for receptor binding was performed by exposing a constant amount of partially purified (R-TA) to increasing concentration of either chromatin or DNA-cellulose (fig. 6). From the initial slopes of the saturation curves, it can be calculated that DNA has at least 10 times higher binding capacity than chromatin for the partially purified (R-TA). The results presented above indicate quantitative differences in the binding of (R-TA) to chromatin and DNA. In order to detect the possible existence of qualitative differences, the kinetics of association and dissociation, as well as the sensitivity to ionic strength of the binding of the receptor to chromatin and DNA were compared. As depicted in fig. 7a the time kinetic of association is very similar for receptor binding to chromatin and DNA, and reaches a plateau after 1 h of incubation at O’C. At 22°C the kinetics are also very similar, but the binding is much faster and cannot be measured precisely. The rate of dissociation of the receptor from chromatin and DNA at 22°C follows first order kinetics (fig. 7b). After subtraction of the blank values obtained
60
H. Bugany, M. Beat0
y
j 2,o
1.0
DNA.
mg
Fig. 6. Titration of DNA and chromatin with partially purified (R-TA). A constant amount of partially purified (R-TA) was incubated with increasing amounts of either rat liver chromatin, calf thymus DNA-cellulose or equivalent amounts of plain cellulose. Incubation was performed at 22°C for 30 min in a final volumeof 0.55 ml, containing 0.15 M NaCl. The amount of (R-TA) bound to either chromatin (0) or DNA (m) was determined after subtraction of the blank values obtained with plain cellulose (see Methods).
with pure cellulose, the half-times of the dissociation of (R-TA) from DNA and chromatin are practically indistinguishable (trip N 90 min). The sensitivity to salt extraction of the receptor-steroid complex bound to, DNA or chromatin is depicted in fig. 8. In these experiments the incubations were performed in 25 mM NaCl and the washing of the pellets at the indicated concentrations of NaCl. It is obvious that the course of both curves is very similar, with most of the extractable (R-TA) being removed at 0.3 M NaCl. However, even at 0.4 M NaCl around 10% of the originally bound (R-TA) remains associated with the chromatin pellet. This percentage of salt-res~tant binding was also found with chicken erythrocyte chromatin, indicating that it is not an exclusive property of target tissue chromatin. A similar influence of ionic strength was observed in experiments in which the incubations, and not only the washing steps, were performed at different concentrations of NaCl.
Polylysine was used to cover the so called free or accessible DNA stretches in chromatin, in order to decide whether these accessible DNA sequences are involved in the binding of (R-TA) ln fact, as the amount of polylysine bound to chromatin increases, the ability of the chromatin to bind the receptor decreases in parallel (fig. 91. As the number of lysin residues per nucleotide reaches 0.5, most of the
Uucocorticoid
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binding to the genome
61
1
;0
60
120 TIME.
I
120
1
180
min
Fig. 7. Binding of (R-TA) to DNA and chromatin: kinetics of association and dissociation. Le.& Kinetics of association. A constant amount of rat liver chromatin (374 E.cgDNA), calf thymus DNA-cellulose (232 pg DNA) or an equivalent amount of plain cellulose were incubated at 0°C with a constant concentration of partially purified (R-TA) in a final volume of 0.55 ml containing 0.15 M NaCl. The reaction was started by the addition of (R-TA) and stopped after different time intervals by dilution with 1 ml incubation buffer, immediately followed by centrifugation. The value obtained at time 0, that means, by centrifugation immediately after adding (R-TA), was subtracted from all experimental values. To calculate the values for calf thymus DNA, the blanks obtained with plain cellulose were subtracted from the DNA-cellulose values. The values are expressed as percentage of the (R-TA) bound to chromatin (0) and DNA (m) in the plateau region of the curve, after 180 min of incubation. Right, kinetics of dissociation. A constant amount of rat liver chromatin (82 pg DNA), calf thymus DNA-cellulose (66 pg DNA) or an equivalent amount of plain cellulose were incubated for 30 min at 22°C with a high concentration (5 X lop9 M) of partially purified (R-TA) in a final volume of 0.55 ml containing 0.15 M NaCl. After centrifugation and two washes with 1 ml of incubation buffer at O”C, the pellets were resuspended in 0.45 ml incubation buffer containing 0.15 M NaCl, and incubated at 22°C. At the indicated time intervals, the reaction was stopped by centrifugation, and the amount of radioactivity in the pellet was measured. The values are expressed as perce.ttage of the amount of (R-TA) bound at time 0, that means, centrifugation immediately after resuspension of the pellet. All the values are corrected for the inactivation of (R-TA) under the incubation conditions, which was determined in parallel assays. The value obtained after incubation for 12 h has been subtracted from all the experimental values-, as this was shown to represent the value for the new equilibrium reached after 9-10 h of incubation. Chromatin (0) and DNA (u) were calculated after subtracting the cellulose blank.
H. Bugany, hf. Beato
62
NOCI ~ONCEN TRATIION, M
Fig. 8. Effect of NaCl on the binding of (R-TA) to chromatin and DNA. A constant amount of rat liver chromatin (273 wg DNA), calf thymus DNA-cellulose (224 fig DNA) or an equivalent weight of plain cellulose were incubated at 22°C for 30 min with a constant amount of partially purified (R-TA) in a final volume of 0.55 ml containing 25 mM NaCl. After centrifugation the pellet was washed twice with 1 ml cold incubation buffer containing the indicated concentration of NaCl, and used for radioactivity determination. The values are expressed a’s percentage of the value obtained with 50 mM NaCl in the washing buffer. Chromatin (0) and DNA (m) were calculated after subtraction of the blanks obtained with plain cellulose.
DNA in chromatin has been covered with polylysine and only a small residual binding of (R-TA) to chromatin is detected, comparable to that observed at high ionic strength, in the absence of polylysine (fig. 8).
DISCUSSION The results presented above clearly demonstrate the importance of the degree of purification of the receptor preparations for binding studies to DNA or chromatin. The comparison of the results obtained with crude cytosol preparations and with partially purified (R-TA) suggests the existence in the cytosol of inhibitors of receptor binding to chromatin. The existence of such inhibitors is responsible for the apparent saturation of the nuclear and chromatin acceptor sites, which is ob-
Glucocorticoid
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I
I
0.2
0.L LYSIN/
1
0.6
63
I
0.8
1.0
NUCLEOTIDE
Fig. 9. Effect of poly-(D)-lysine on the binding of (R-TA) to chromatin. Aliquots of sonicated rat liver chromatin were incubated with increasing amounts of polylysine as described in Methods. The amount of DNA not precipitated by polylysine was determined by the procedure of Burton (1956) in the supernatant obtained after centrifugation of an’aliquot at 10,OOOgfor 10 min. This value was subtracted from the total DNA, to give the amount of DNA in the precipitated chromatin, which is expressed as a percentage of total DNA (m). Another aliquot of the polylysine-treated chromatin was used to determine the binding capacity for partially purified (R-TA) as described in Methods. The values are expressed as radioactivity bound per mg DNA in the pellet (*).
sulphate fractions are used as from control experiments in which the concentration of cytosol protein was maintained constant in the entire range of (R--TA) concentration tested. Similar results have been initially reported by Chamness et al. (1974) and more recently by Milgrom et al. (1975) and Simons et al. (1976). A physiological significance of these inhibitory factors in cytosol can, however, not be excluded. Using the partially purified receptor preparation, a linear relationship was observed between the concentration of free (R-TA) in the incubation medium and the amount of receptor bound to chromatin. Even at concentrations of receptorsteroid complex 3-4 times higher than those present in cytosol, no saturation of the chromatin acceptor sites could be detected. Under these conditions, chromatin prepared from avian erythrocytes binds considerably less .(R-TA) than liver chromatin. As we will discuss below, this finding probably reflects the more condensed structure of the physiologically inactive erythrocyte chromatin. served when crude cytosol preparations or ammonium a source of (R-TA). This interpretation is derived
64
H. Bugany, M. Beat0
The study of the interaction of (R-TA) with purified DNA is also markedly affected by the purity of the receptor preparation. The use of crude cytosol as the source of the receptor leads to an apparent saturation curve, which is dependent on the presence of Mg2+. This apparent saturability of the DNA with (R-TA) is, however, due to the presence of DNases in crude cytosol. If this activity is taken into consideration, a linear relationship is observed between the concentration of (RTA) in the incubation medium and the amount of receptor bound to DNA-cellulose. A comparison of the binding of partially purified (R-TA) to chromatin and DNA shows that calf thymus DNA has at least 10 times higher binding capacity than liver chromatin, and about 50 times higher binding capacity than erythrocyte chromatin. These figures are in good agreement with estimates of the amount of DNA in these two types of chromatins that can be transcribed by bacterial DNA-dependent RNA polymerases (Seligy and Miyagi, 1974). It is therefore conceivable that the binding of the (R-TA) to chromatin takes place by interaction with those sequences of DNA which are also accessible to other proteins and to RNA polymerases. This assumption is supported by the finding that around 90% of the receptor binding capacity of the chromatin can be inhibited by titration with polylysine, which is known to interact primar~y with the accessible DNA stretches (Clark and Felsenfeld, 1971). The similarities observed in the kinetic experiments, as well as in the sensitivity of the binding to the concentration of NaCl also suggests that the majority of the (R-TA) molecules bound to chromatin are interacting with the so called free DNA stretches. We cannot confirm the report by Simons et al. (1976), that the receptor dissociates more slowly from chromatin that it does from DNA. In our experiments the rates of dissociation of (R-TA) from chromatin and DNA were indistinguishable (fig. 7b). The results presented here indicate that there is a relatively unspecific binding of the receptor-steroid complex to the DNA in DNA-cellulose and in chromatin, but do not exclude the possible existence of a limited number of specific receptor binding sites in the genome of the target tissue. A similar difficulty in detecting specific binding to DNA has been observed in studies on the interaction of the cyclic AMP binding protein with the lac operon in E. coli. Only after a sequence of about 200 bases containing the binding site for the cyclic AMP binding protein was obtained with restriction endonucleases, could the binding of this protein be demonstrated to be sequence specific (Majors, 1975). In fact, a general affinity for DNA appears to be an important property of all regulatory proteins which recognize specific DNA sequences, including the lac repressor (von Hippel et al., 1974; Lin and Riggs, 1975). We Cannot exclude that the weak interaction of (R-TA) with DNA is physiologically significant. Under in vivo conditions, after injection of radioactive glucocorticoids, a considerable amount of the radioactivity can be removed in the nucleus bound to chromatin. Calculations of the number of receptor molecules per haploid genome give values in the order of 5-10 X lo3 indicating a weak inter-
Glucocorticoid receptor binding to the genome
65
action with a large number of acceptor sites within the nucleus, similar to that observed using crude cytosol preparation (fig. 1). In addition to the interaction with free DNA, we have to take into consideration the possibility that the (R-TA) recognizes other components of the chromatin, such as the chromosomal proteins. Reports from other steroid target tissues have shown an interaction of the corresponding receptors with basic and acidic proteins of the chromatin (O’Malley et al., 1972; Puca et al., 1975). Such an interaction could be responsible for the residual binding to chromatin observed at high salt concentrations or after titration of chromatin with polylysine. The clarification of these points, awaits the development of procedures able to distinguish between specific and non-specific binding of the receptor to DNA and chromatin.
REFERENCES Baxter, J.D., Rousseau, G.G., Benson, M.C., Garcea, R.L., Ito, J. and Tomkins, G.M. (1972) J. Biol. Chem. 69,1892-1896. Beato, M. and Doenecke, D. (1976) Metabolic effects and modes of action of glucocorticoids. In: General, Comparative and Clinical Endocrinology, Vol. HI, Eds.: I. Chester Jones and I.W. Henderson (Academic Press) in press. Beato, M. and Feigelson, P. (1972) J. Biol. Chem. 247, 7890-7896. Beato, M., Kalimi, M., Konstam, M. and Feigelson, P. (1973) Biochemistry 12,3372-3379. Beato, M,, Seifart, K.H. and Sekeris, C.E. (1970) Arch. Biochem. Biophys. 138,272-284. Bray, GA. (1960) Anal, Biochem. 1,279-285. Burton, K. (1956) Biochem. J. 62,315-321. Chamness, G.C., Jennings, A.W. and McGuire, W.L. (1974) Biochemistry 13,327-331. Clark, R.J. and Felsenfeld, G. (1971) Nature New Biol. 229, 101-106. Climent, F., Bugany, H. and Beato, M. (1976) FEBS Lett. 66, 317-321. Hamana, K. and Iwai, K. (1973) Gunma Symp. Endocrinol. 10,77-88. Higgins, S.J., Rousseau, G.G., Baxter, J.D. and Tomkins, G.M. (1973a) J. Biol. Chem. 248, 5866-5872. Higgins, S.J., Rousseau, G.G., Baxter, J.D. and Tomkins, G.M. (1973b) J. Biol. Chem. 58735879. Kalimi, M., Beato, M. and Feigelson, P. (1973) Biochemistry 12, 3365-3371. Koblinsky, M., Beato, M., Kalimi, M. and Feigelson, P. (1972) J. Biol. Chem. 247, 7897-7904. Lin, S. and Riggs, A.D. (1975) Cell 4,107-l 11. Lippman, M.E. and Thompson, E.B. (1974) J. Biol. Chem. 249,2483--2488. Litman, R.M. (1968) J. Biol. Chem. 243,6222-6231. Majors, J. (1975) Nature 256,672-674. Marmur, J. (1961) J. Mol. Biol. 3, 208-220. Milgrom, E. and Atger, M. (1975) J. Steroid Biochem. 6,487-492. Milgrom, E., Atger, M. and Baulieu, E.-E. (1973) Biochemistry 12,5198-5205. O’Malley, B.W., Spelsberg, Th.C., Schrader, W.T., Chytil, F. and Steggles, A.W. (1972) Nature 235,141-145. Puca, G.A., Nola, E., Hibner, U., Cicala, G. and Sica, V. (1975) J. Biol. Chem. 250, 64526459. Rousseau, G.G., Higgins, S.J., Baxter, J.D., Gelfant, D. and Tomkins, G.M. (1975) J. Biol. Chem. 250,6015-6021.
66
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Rousseau, G.G., Higgins, S.J., Baxter, J.D. and Tomkins, G.M. (1974) J. Steroid Biochem. 5, 935-939. Seligy, V.L. and Miyagi, M. (1974) Eur. J. Biochem. 46,259-269. Simons, S.S., Martinez, H.M., Garcea, R.L., Baxter, J.D. and Tomkins, G.M. (1976) J. Biol. Chem. 251,334-343. Von Hippel, P.H., Renin, A., Gross, CA. and Wang, A.M. (1974) Proc. Acad. Sci. U.S.A. 7 I, 4808-4812.
0 Elsevier/North-Holland
I (1977) 49-66 Scientific Publishers, Ltd.
BINDING OF THE PARTIALLY PURIFIED GLUCOCORTICOID OF RAT LIVER TO CHROMATIN AND DNA *
RECEPTOR
Harald BUGANY and Miguel BEAT0 ** Institut fiir Physiologische
Chemie, D-3550 MarburgjLahn,
Deutschhausstr.
I-2, G.F.R.
Received 8 June 1976; accepted 2 September 1976
The binding of the glucocorticoid receptor of rat liver to chromatin and DNA has been studied with crude and partially purified preparations of cytosol receptor labelled with [3H]triamcinolone acetonide in vitro. The use of crude preparations of receptor and increasing protein concentrations leads to an apparent saturation of chromatin and DNA, suggesting a limited number of high affinity nuclear acceptor sites for the receptor. Appropriate controls indicate that the observed saturability of chromatin acceptor sites is due to the presence in crude receptor preparations of heat-stable protein factors which interfere with the binding of the receptor to the genome; whereas the apparent saturation of DNA is due to contamination with deoxyribonucleases. If the activated complex of receptor and triamcinolone acetonide (R-TA) is partially purified to a step where it is free from nucleases and inhibitors, its binding to both chromatin and DNA is linearly dependent on the concentration of free (R-TA) in the incubation medium. There is no absolute specificity with respect to the source of DNA or chromatin, although liver chromatin has considerably higher receptor binding capacity than chromatin from avian erythrocytes. The rate kinetics of association and dissociation for the binding of (R-TA) to DNA and chromatin are very similar, but DNA exhibits a lo-fold higher receptor binding capacity than chromatin. These data, in conjunction with the effect of poly-(D)-lysine and NaCl on the binding of (R-TA) to chromatin and DNA, suggest that most of the receptor molecules bound to chromatin in vitro interact with the ‘accessible’ DNA stretches. Although a small population of receptor molecules may bind specifically to target tissue genome, the detection of these specific sites against the background of unspecific binding is not possible with unfractionated chromatin or DNA preparations. Keywords:
protein-DNA
interaction; DNA-cellulose;
triamcinolone acetonide.
Glucorticoid hormones are known to act on their target organs through a multistep mechanism involving an interaction with specific receptor proteins in the cytosol, followed by the activation of the receptor-steroid complex and its migration to the cell nucleus. Within the nucleus the receptor-steroid complex binds to chromatin, and is supposed to modulate the transcription of specific genes (for * This work was supported by a grant of the Deutsche Forschungsgemeinschaft ** To whom correspondence should be sent.
49
(SFB 103-1,2).
H. Bugany, M. Beato
50
review see Beato and Doenecke, 1976). Various parts of this pathway have been reproduced in cell-free systems, in which it has been demonstrated that the activation of the receptor-glucocorticoid complex is dependent on temperature and ionic strength, and confers to the complex affinity for chromatin and DNA (Baxter et al., 1972; Beato et al., 1973; Hamana and Iwai, 1973; Higgins et al., 1973a; Kalimi et al., 1973; Milgrom et al., 1973). A preferential binding of the glucocorticoid receptor to the nuclei of the target organs has been reported (Hamana and Iwai, 1973; Higgins et al., 1973b; Kalimi et al., 1973), and Lippman and Thompson (1974) claimed that the nuclear acceptor sites can distinguish between glucocorticoid receptors from target and non-target tissues. On the other hand, some groups have described the existence of a limited number of high affinity nuclear acceptor sites for the glucocorticoid receptor (Baxter et al., 1972; Higgins et al., 1973a; Kalimi et al., 1973; Lippman and Thompson, 1974), whereas other laboratories could not observe a saturation of the nuclear sites with receptor-steroid complex (Rousseau et al., 1974; Milgrom and Atger, 1975; Climent et al., 1976; Simons et al., 1976). The binding of the activated receptor to purified DNA shows no saturation in the range of physiological receptor concentrations, and exhibits very little specificity in respect to the source of the DNA (Baxter et al., 1972; Beato et al., 1973; Milgrom et al., 1973; Rousseau et al., 1974, 1975; Simons et al., 1976). Therefore, the physiological significance of the interaction of the receptor-steroid complex with DNA is not clear and very little is known about the role of the chromosomal proteins in nuclear binding of the complex. In this paper we present a comparison of the binding of the receptor-glucocorticoid complex to chromatin and DNA.
MATERIALS
AND METHODS
[6,7-3H] Triamcinolone acetonide *, spec. act. 33.7 Ci/mmol was obtained from New England Nuclear Inc. The nonradioactive steroids, DNA from calf thymus, salmon sperm and E. coEi, were purchased from Sigma and Co. Crystallized bovine serum albumin (BSA) was obtained from the Behringwerke, Marburg. Pronase, grade B, pancreatic ribonuclease A, and Nonidate P40, were purchased from Serva, Heidelberg. Cellulose (Cellex 410) und Bio-Gel PlO were obtained from Bio-Rad Laboratories Inc, and phosphocellulose (Pll) was from Whatman Inc. Poly-(D)lysine hydroxybromide, molecular weight 140,000, was purchased from Sigma and Co. Male Wistar II rats weighing 120-140 g were used throughout. The animals were adrenalectomized 5-10 days before the beginning of the experiments.
* TA: triamcinolone acetonide: 9~fluoro-1 3,20dione cyclic 16,17-acetal with acetone.
lp,16a,17q2l-tetrahydroxy-pregnan-l,4diene-
Preparation and fractionation of liver cytosol The animals were killed by cervical dislocation and the liver was perfused in situ through the portal vein with 10 ml of cold TSS buffer (0.25 M sucrose, 25 mM KCl, 5 mM MgCla, 2 mM mercaptoethanol, and 50 mM Tris-HCI, pH 7.5). The livers were then removed, and the cytosol was prepared as previously described (Beat0 and Feigelson , 1972). For some experiments the cytosol was used directly after incubation with radioactive triamcinolone acetonide (TA) as indicated in the legend to the corresponding figures. In another series of experiments the cytosol was fractionated with ammonium sulphate (Beat0 and Feigelson, 1972). For these experiments cytosol was first incubated with 10 mM [3H]TA for 30 min at 22’C, before addition of saturated ammonium sulphate, pH 7.0, to 30% saturation. After slowly stirring for 30 min at 0°C the cytosol was centrifuged at 20,OOOg for 10 min and the pellet was resuspended in TSS buffer (0.1 vol. of the original cytosol). The ammonium sulphate was then removed by passing the sample through a Bio-Gel PlO column equilibrated with the appropriate binding buffer. Partial purification of the activated receptor-steroid complex, (R-TA) For another series of experiments a partially purified receptor-steroid complex, (R-TA), was used. The details of the purification procedure, and the characterization of the partially purified receptor have been published elsewhere (Climent et al., 1976; Climent and Beato, in preparation). In summary, the procedure is as follows: Cytosol is incubated with 50 nM [3HJ TA at 0°C for IO min in medium of low ionic strength (25 mM KCl), and passed through two large phosphocellulose columns. The unbound material is incubated at 22°C for 30 min in order to activate the (R-TA), and passed through a small phosphocellulose column, to which the activated (R-TA) is bound, and can be eluted by increasing the salt concentration. This very simple procedure usually yielded over 3000-fold purification of the (RTA), and the final preparation was free of deoxyribonuclease (DNase) activity (Climent et al., 1976). The concentration of (R-TA) in different preparations was determined by the charcoal technique (Beat0 and Feigelson, 1972). When partially purified receptor was used, the concentration of charcoal was reduced by lo-fold, and BSA (final concentration of 0.1%) was added to the samples in order to prevent adsorption of the receptor to the charcoal. Preparation ofrat liver and au&n erythrocyte chromatic The pellet obtained after centrifugation of the liver homogenate at 750g was resuspended in two volumes of TSS buffer and mixed with an equal volume of a I% solution of Nonidet P40 in TSS buffer. After 30 s the nuclei were centrifuged at XOOg for 5 min. The pellet was resuspended in 4 volumes of TSS buffer and centrifuged again to remove the detergent. Further purification of chromatin was as previously described (Beat0 et al., 1970). For the preparation of erythrocyte chromatin from duck or chicken a proce-
52
H. Bugany, M. Beato
dure similar to that used for rat liver was employed. The erythrocytes were purified by repeated washings in 0.15 M KC1 containing 3 mM MgCla, and disrupted by hypotonic lysis in buffer containing 25 mM Tris-HCl, pH 8.0. After completion of the cell lysis, 0.16 volumes of 7-fold concentrated TSS buffer were added and a crude nuclear pellet was obtained by centrifugation at 75Og for 10 min. The rest of the chromatin preparation was identical to that previously described for rat liver (Beat0 et al., 1970). Beparation of rat liver DNA and coupling of DNA to cellulose DNA was prepared from purified nuclei of rat liver according to the procedure of Marmur (1961), modified to include treatments with pronase and ribonuclease (RNase) (Beat0 et al., 1970). DNA of various sources was coupled to cellulose according to the procedure of Litman (1968). The DNA content of the cellulose was in the order of 3-7 mg/g cellulose. Assay of receptor binding to chromatin and DNA-cellulose Binding of (R-TA) to chromatin or DNA was measured in 550-d assays containing 100 fl of a suspension of chromatin, DNA-cellulose or cellulose alone, and different amounts of (R-TA). When specified, heat-denatured cytosol or cytosol which was not incubated with radioactive steroid was added to the assay. The incubation media contained 10% glycerol, 1 mM EDTA-Naa, 10 mM Tris-HCl, pH 8.0, and variable amounts of NaCl as indicated. The salt concentration was adjusted with a concentrated solution of NaCl. Incubation was performed in Eppendorf reaction tubes for 30 min at 22’C. Every 5 min the tubes were shaken to resuspend cellulose or chromatin. When partially purified (R-TA) was used, bovine serum albumin was added to a final concentration of 0.1%. At the end of the incubation, the tubes were centrifuged at 10,OOOg for 2 min and the pellets washed twice with cold incubation medium of the appropriate ionic strength. Radioactivity measurements were performed by resuspending the pellets in 7 ml of Bray’s solution (Bray, 1960). Determination of DNA content was performed in a parallel assay by the procedure of Burton (1956). When BSA was used, the corresponding blanks in the absence of the receptor and with similar amounts of [3H] TA were performed. The kinetics of association and dissociation for the reaction of the partially purified (R-TA) with chromatin and DNA were determined as described in the legend to fig. 7. Effect of NaCl concentration The influence of NaCl on the binding of (R-TA) to chromatin and DNA was investigated with partially purified receptor preparations. In one set of experiments (R-TA) was incubated with chromatin, DNA-cellulose or cellulose in buffer containing 25 mM NaCl for 30 min at 22°C and after centrifugation the pellets were washed twice in incubation buffer containing the appropriate NaCl concentration. For each wash, the resuspended samples were allowed to
~l~c~cortic~id receptor minding to the genome
53
stand in the ice-bath for 10 min before centrifugation. The final pellet was used for radioactivity determination. In another set of experiments the incubation of the receptor with chromatin or DNA-cellulose was carried out at different ionic strengths and the washing of the pellets was performed at the same NaCl concentration as was present during the incubation. determination of the receptor birzditzgcapacity of chromutin and DIVA For these experiments increasing amounts of a suspension of chromatin, DNAcellulose or cellulose alone were resuspended in 550 $ of incubation medium containing 0.15 M NaCl, the partially purified (R-TA) and 0.5 mg BSA. After incuba” tion at 22°C for 30 min the samples were centrifuged, the pellets washed twice and the radioactivity and DNA content determined as described above.
Rat liver chromatin was resuspended by sonication in 50 mM sodium phosphate buffer, pH 7.0, and centrifuged at 10,OOOg for 10 min. To the supernatant (4.90 ml containing 5 X 10V4 M nucleotides) 100 111aliquots of polylysine solution in 50 mM sodium phosphate buffer, pH 7.0, (2 X 10m3 M lysine) were added. After standing for 10 min at 0°C aliquots of 50 111were taken for incubation with (R-TA) and for the determination of the amounts of DNA in chromatin which was complexed with polylysine, as described below. This procedure was repeated 11 times after addition each time of 100 ,ul of pdylysine. The binding of the receptor to the polylysine-treated chromatin was measured by incubating 0.5 ml of (R-TA) with a 50 4 aliquot of polylysine-treated chromatin at 22°C for 1 h in 50 mM sodium phosphate buffer, pH 7.0. The samples were then centrifuged at 10,OOOg for 10 min and the pellets washed twice with 1.5 ml of incubation medium. The final pellets were resuspended by sonication in 200 4 of incubation medium and aliquots were taken for the determination of DNA and for radioactivity measurements. The determination of the amount of chromatin DNA which was precipitated by polylysine was performed by mixing a 50 &l aliquot of polylysine-treated chromatin with 1 ml incubation medium and centrifuging at 10,OOOg for 10 min. The DNA content in the supernatant was determined, and the difference between this value and the total DNA content of the chromatin gave the amount of DNA precipitated by polylysine.
RESULTS Binding of the receptor-steroid complex to chromatin The binding of (R-TA) to chromatin was first investigated using as receptor preparation either the crude cytosol labelled with [3H]TA or the ammonium sulphate fraction containing the receptor-triamcinolone complex. Under these con-
H. Bugany, M. Beato
54
ditions incubation of a given amount of chromatin with increasing concentrations of receptor-steroid complex leads to a saturation curve, comparable to that obtained with isolated rat liver nuclei (Kalimi et al., 1973). The extent of binding is very sensitive to the ionic conditions of the incubation medium (fig. 1). In medium containing 25 mM NaCl and no divalent cations a high binding capacity is observed and half saturation is reached at concentrations of (R-TA) in the order of lo-* M, as calculated by Scatchard analysis. Raising the concentration of NaCl to 0.15 M results in a considerable decrease in the amount of bound (R-TA), and half saturation is reached at lower concentrations of (R-TA). Addition of MgCla (3 mM) to a medium containing low concentration of NaCl(25
bl
.3mM
1
MgC12
2.0
LIVER.25mM
1.0
LIVER.lSOrnM
LIVER.lSOmM
J
Fig. 1. Effect of ions on the binding of crude (R-TA) to chromatin. A crude (R-TA) was prepared from liver cytosol incubated with [3H] TA (50 nM) by precipitation with 30% saturated ammonium sulphate, as indicated in Methods. Increasing amounts of this (R-TA) preparation were incubated at 20°C for 1 h with a constant amount of either rat liver (125 Mg DNA) or duck erythrocyte (405 pg DNA) chromatin in a final volume of 0.55 ml, containing different final concentrations of NaCl and MgClZ. The amount of chromatin-bound (R-TA) and the DNA content of the chromatin pellets were determined as described in Methods. (a) Incubations performed in the absence of divalent cations: at 25 mM NaCl with liver (0) and erythrocyte (0) chromatin; or at 150 mM NaCl, with liver (e) and erythrocyte chromatin (0); (b) incubation performed in the presence of 3 mM MgCls. All other conditions and symbols as in (a).
Glucocorticoid
receptor
binding to the genome
55
mM) also results in a reduction of the binding capacity of chromatin for (R-TA). However, when the concentration of NaCl in the incubation mixture is 0.15 M, MgCl, has little effect on either the affinity or the number of binding sites for the receptor in isolated chromatin (compare Fig. 1, a and b). Under these more physiological conditions (0.15 M NaCI) the number of molecules of (R-TA) bound per haploid genome, calculated by Scatchard analysis, is in the order of 4-7 X 103, but this number varies considerably with the amount of DNA used in the different experiments. When a small amount of DNA in chromatin is used and (R-TA) is added in large excess, the number of receptor molecules bound at saturation is low (range of 4 experiments: 600-2400 molecules per haploid genome). Raising the amount of chromatin-DNA leads to a higher number of bound molecules of (RTA) and the apparent affinity is lower. These latter results already indicate that some components of the cytosol preparation influence the binding of the receptor to the chromatin and prevent a precise quantitation of the binding parameters. Nevertheless, under these conditions a considerable tissue specificity is observed as demonstrated by a comparison of the amount of (R-TA) bound to rat liver versus erythrocyte chromatin (Fig. la). The form of the saturation curve, however, is very similar for both chromatins indicating that the binding process is not essentially different, but just that there are fewer acceptor sites in the erythrocyte chromatin. In order to explore the behaviour of the saturation curve under different incubation conditions, and following the suggestion of Chamness et al. (1974), a series of control experiments were performed, in which the concentration of cytosol proteins was maintained constant and the concentration of receptor-steroid complex was the only variable. This was achieved in one set of experiments by incubating a constant amount of cytosol with different concentrations of [3H] TA and determining concentration of (R-TA) by the charcoal procedure. Under these conditions the binding of receptor-steroid complex to chromatin was linearly dependent on the concentration of free (R-TA). Similar results were obtained when increasing amounts of cytosol labelled with [3H]TA were incubated with chromatin and the volume was maintained constant by the addition of non-labelled cytosol or cytosol heated at 50°C for IO min before the incubation (data not shown). This latter control eliminates the possibility, that the apparent saturation is due to free receptor molecules present in non-labelled cytosol, as they will be inactivated by the heating step (Koblinsky et al., 1972) A linear dependence on (R-TA) concentration was also observed when a receptor preparation enriched by fractionation with ammonium sulphate was used and the volume was kept constant by adding an ammonium sulphate fraction prepared from cytosol which was not incubated with [3H] TA (fig. 2). In all the experiments with added cytosol fractions the values obtained at low concentrations of (R-TA) are lower than those observed with a conventional assay (fig. 2) suggesting the existence in the cytosol preparation, and in the ammonium sulphate fraction of heat-stable inhibitors of the binding of the receptor to chromatin. A similar conclusion can be drawn from the experiments with partially purified (R-TA), in
H. Bugany, M. Beato
IR-TA
I
FREE’
nM
Fig. 2. Effect of cytosol. components on the binding of (R-TA) to rat liver chromatin. Constant amounts of rat liver chromatin (140 pg DNA) were incubated with increasing amounts of a crude preparation of (R-TA) obtained by precipitation of the cytosol with ammonium sulphate as indicated in Methods. Conditions of incubation were 22°C for 1 h in buffer containing 0.15 M NaCl. In one series of experiments the final voiume of 0.55 ml was maintained by addition of incubation buffer (=), whereas in another series of experiments the final volume and protein concentration were kept constant by the addition of a 30% ammonium sulphate fraction prepared from unlabelled cytosol (0). Also shown are the results obtained with a partially purified preparation of (R-TA) incubated with chromatin under similar conditions (0). The determination of chromatin-bound (R-TA) and DNA was performed as described in Methods.
which the amount of receptor bound to chromatin was also a linear function of the concentration of free (R-TA), and the values obtained were considerably higher than those observed with the crude cytosol preparations or the ~monium sulphate fraction (fig. 3). Under these conditions, the binding capacity of rat liver chromatin for the partially purified (R-TA) is 3.fold higher than that of chicken erythrocyte chromatin (fig. 3). biding of receptor-steroid complex to DIVA Incubation of calf thymus DNA-cellulose with increasing
amounts
of cytosol
Glucocorticoid
receptor binding to the genome
0.25
0.5 IR -TA IFREE n M
Fig. 3. Binding of partially purified (R-TA) to chromatin. Constant amounts of chromatin prepared either from rat liver (211 pg DNA) or from chicken erythrocytes (625 Hg DNA) were incubated in buffer containing 0.15 M NaCl, with increasing concentrations of partially purified (R-TA), prepared as described in Methods. The incubations were performed in a final volume of 0.55 ml for 1 h, at 22”C, and the amount of (R-TA) bound to rat liver (9) and erythrocyte (0) chromatin, as well as the DNA content were determined as described in Methods.
labelled with [3H] TA leads to an apparent saturation of the DNA binding capacity (fig. 4). However, determination of the DNA content in the pellet obtained after incubation shows that the crude cytosol contains considerable DNase activity. When the values of (R-TA) bound are expressed per mg DNA recovered in the pellet, a linear dependence on the concentration of free (R-TA) in the incubation medium is observed. It is interesting to note that the apparent saturation of DNA with crude cytosol receptor preparations is only detected in the presence of MgCla. This observation is in agreement with the finding that the DNase activity of the cytosol, as measured in the DNA-cellulose assay, is dependent on Mg*+. In the absence of MgCla, even using crude cytosol, there is no indication of saturation of the DNA-cellulose with (R-TA). Similar linear relationship between free and bound (R-TA) was observed when partially purified receptor preparations were
H. Bugany, M. Beato
Fig. 4. Binding of crude (R-TA) to calf thymus DNA. A constant amount of calf thymus DNA-cellulose (30 Irg DNA) or an equivalent amount of plain cellulose were incubated at 22°C for 1 h with increasing amounts of rat liver cytosol, labeled with [ %I] TA. The final volume was maintained at 0.55 ml by addition of buffer containing NaCl and MgCIz to give fii concentrations of 0.15 M and 3 mM, respectively. The amount of (R-TA) bound to DNA (a) was determined by subtracting from the DNA-cellulose values, the values obtained with cellulose alone. In parallel analysis the DNA content of the cellulose was determined after incubation with the same amount of crude (R-TA), and used to calculate the amount of (R-TA) bound per mg of DNA (s).
used in the binding assays, both in the absence and presence of Mg’+. There is little specificity in respect to the source of the DNA, as only quantitative differences are detected in the binding capacity of rat liver, calf thymus and salmon sperm (fig. 5). Similar binding was also observed with DNA prepared from E. coli (data not shown). Comparison between receptor binding to chromatin and DNA The comparison of the values in figs. 3 and 5 clearly shows that the capacity of DNA for binding (R-TA) is much higher than that of chromatin (Climent et al.,
Glucocorticoid
receptor
binding to the genome
1
RAT
:
59
LIVER
CALF
THYMUS
Q e \ 2aI
SALMON
50-
=. P 2 -_Q : E
25-
I
I
2.5
5.0
‘R-‘“IFREE , n
M
Fig. 5. Binding of partially purified (R-TA) to DNA of various sources. A constant amount (30 pg DNA) of DNA-cellulose, or an equivalent amount of plain cellulose, was incubated at 22°C for 1 h with increasing concentrations of partially purified (R-TA) in buffer containing 0.15 M NaCl. The amount of (R-TA) bound to DNA of rat liver (a), calf thymus (o), and salmon sperm (e), was calculated by subtracting the blank values obtained with plain cellulose. The DNA content was determined in parallel assays and shown to be constant, independent of the amount of (R-TA) added.
1976). A direct comparison of relative capacities of rat liver chromatin and calf thymus DNA-cellulose for receptor binding was performed by exposing a constant amount of partially purified (R-TA) to increasing concentration of either chromatin or DNA-cellulose (fig. 6). From the initial slopes of the saturation curves, it can be calculated that DNA has at least 10 times higher binding capacity than chromatin for the partially purified (R-TA). The results presented above indicate quantitative differences in the binding of (R-TA) to chromatin and DNA. In order to detect the possible existence of qualitative differences, the kinetics of association and dissociation, as well as the sensitivity to ionic strength of the binding of the receptor to chromatin and DNA were compared. As depicted in fig. 7a the time kinetic of association is very similar for receptor binding to chromatin and DNA, and reaches a plateau after 1 h of incubation at O’C. At 22°C the kinetics are also very similar, but the binding is much faster and cannot be measured precisely. The rate of dissociation of the receptor from chromatin and DNA at 22°C follows first order kinetics (fig. 7b). After subtraction of the blank values obtained
60
H. Bugany, M. Beat0
y
j 2,o
1.0
DNA.
mg
Fig. 6. Titration of DNA and chromatin with partially purified (R-TA). A constant amount of partially purified (R-TA) was incubated with increasing amounts of either rat liver chromatin, calf thymus DNA-cellulose or equivalent amounts of plain cellulose. Incubation was performed at 22°C for 30 min in a final volumeof 0.55 ml, containing 0.15 M NaCl. The amount of (R-TA) bound to either chromatin (0) or DNA (m) was determined after subtraction of the blank values obtained with plain cellulose (see Methods).
with pure cellulose, the half-times of the dissociation of (R-TA) from DNA and chromatin are practically indistinguishable (trip N 90 min). The sensitivity to salt extraction of the receptor-steroid complex bound to, DNA or chromatin is depicted in fig. 8. In these experiments the incubations were performed in 25 mM NaCl and the washing of the pellets at the indicated concentrations of NaCl. It is obvious that the course of both curves is very similar, with most of the extractable (R-TA) being removed at 0.3 M NaCl. However, even at 0.4 M NaCl around 10% of the originally bound (R-TA) remains associated with the chromatin pellet. This percentage of salt-res~tant binding was also found with chicken erythrocyte chromatin, indicating that it is not an exclusive property of target tissue chromatin. A similar influence of ionic strength was observed in experiments in which the incubations, and not only the washing steps, were performed at different concentrations of NaCl.
Polylysine was used to cover the so called free or accessible DNA stretches in chromatin, in order to decide whether these accessible DNA sequences are involved in the binding of (R-TA) ln fact, as the amount of polylysine bound to chromatin increases, the ability of the chromatin to bind the receptor decreases in parallel (fig. 91. As the number of lysin residues per nucleotide reaches 0.5, most of the
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Fig. 7. Binding of (R-TA) to DNA and chromatin: kinetics of association and dissociation. Le.& Kinetics of association. A constant amount of rat liver chromatin (374 E.cgDNA), calf thymus DNA-cellulose (232 pg DNA) or an equivalent amount of plain cellulose were incubated at 0°C with a constant concentration of partially purified (R-TA) in a final volume of 0.55 ml containing 0.15 M NaCl. The reaction was started by the addition of (R-TA) and stopped after different time intervals by dilution with 1 ml incubation buffer, immediately followed by centrifugation. The value obtained at time 0, that means, by centrifugation immediately after adding (R-TA), was subtracted from all experimental values. To calculate the values for calf thymus DNA, the blanks obtained with plain cellulose were subtracted from the DNA-cellulose values. The values are expressed as percentage of the (R-TA) bound to chromatin (0) and DNA (m) in the plateau region of the curve, after 180 min of incubation. Right, kinetics of dissociation. A constant amount of rat liver chromatin (82 pg DNA), calf thymus DNA-cellulose (66 pg DNA) or an equivalent amount of plain cellulose were incubated for 30 min at 22°C with a high concentration (5 X lop9 M) of partially purified (R-TA) in a final volume of 0.55 ml containing 0.15 M NaCl. After centrifugation and two washes with 1 ml of incubation buffer at O”C, the pellets were resuspended in 0.45 ml incubation buffer containing 0.15 M NaCl, and incubated at 22°C. At the indicated time intervals, the reaction was stopped by centrifugation, and the amount of radioactivity in the pellet was measured. The values are expressed as perce.ttage of the amount of (R-TA) bound at time 0, that means, centrifugation immediately after resuspension of the pellet. All the values are corrected for the inactivation of (R-TA) under the incubation conditions, which was determined in parallel assays. The value obtained after incubation for 12 h has been subtracted from all the experimental values-, as this was shown to represent the value for the new equilibrium reached after 9-10 h of incubation. Chromatin (0) and DNA (u) were calculated after subtracting the cellulose blank.
H. Bugany, hf. Beato
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Fig. 8. Effect of NaCl on the binding of (R-TA) to chromatin and DNA. A constant amount of rat liver chromatin (273 wg DNA), calf thymus DNA-cellulose (224 fig DNA) or an equivalent weight of plain cellulose were incubated at 22°C for 30 min with a constant amount of partially purified (R-TA) in a final volume of 0.55 ml containing 25 mM NaCl. After centrifugation the pellet was washed twice with 1 ml cold incubation buffer containing the indicated concentration of NaCl, and used for radioactivity determination. The values are expressed a’s percentage of the value obtained with 50 mM NaCl in the washing buffer. Chromatin (0) and DNA (m) were calculated after subtraction of the blanks obtained with plain cellulose.
DNA in chromatin has been covered with polylysine and only a small residual binding of (R-TA) to chromatin is detected, comparable to that observed at high ionic strength, in the absence of polylysine (fig. 8).
DISCUSSION The results presented above clearly demonstrate the importance of the degree of purification of the receptor preparations for binding studies to DNA or chromatin. The comparison of the results obtained with crude cytosol preparations and with partially purified (R-TA) suggests the existence in the cytosol of inhibitors of receptor binding to chromatin. The existence of such inhibitors is responsible for the apparent saturation of the nuclear and chromatin acceptor sites, which is ob-
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Fig. 9. Effect of poly-(D)-lysine on the binding of (R-TA) to chromatin. Aliquots of sonicated rat liver chromatin were incubated with increasing amounts of polylysine as described in Methods. The amount of DNA not precipitated by polylysine was determined by the procedure of Burton (1956) in the supernatant obtained after centrifugation of an’aliquot at 10,OOOgfor 10 min. This value was subtracted from the total DNA, to give the amount of DNA in the precipitated chromatin, which is expressed as a percentage of total DNA (m). Another aliquot of the polylysine-treated chromatin was used to determine the binding capacity for partially purified (R-TA) as described in Methods. The values are expressed as radioactivity bound per mg DNA in the pellet (*).
sulphate fractions are used as from control experiments in which the concentration of cytosol protein was maintained constant in the entire range of (R--TA) concentration tested. Similar results have been initially reported by Chamness et al. (1974) and more recently by Milgrom et al. (1975) and Simons et al. (1976). A physiological significance of these inhibitory factors in cytosol can, however, not be excluded. Using the partially purified receptor preparation, a linear relationship was observed between the concentration of free (R-TA) in the incubation medium and the amount of receptor bound to chromatin. Even at concentrations of receptorsteroid complex 3-4 times higher than those present in cytosol, no saturation of the chromatin acceptor sites could be detected. Under these conditions, chromatin prepared from avian erythrocytes binds considerably less .(R-TA) than liver chromatin. As we will discuss below, this finding probably reflects the more condensed structure of the physiologically inactive erythrocyte chromatin. served when crude cytosol preparations or ammonium a source of (R-TA). This interpretation is derived
64
H. Bugany, M. Beat0
The study of the interaction of (R-TA) with purified DNA is also markedly affected by the purity of the receptor preparation. The use of crude cytosol as the source of the receptor leads to an apparent saturation curve, which is dependent on the presence of Mg2+. This apparent saturability of the DNA with (R-TA) is, however, due to the presence of DNases in crude cytosol. If this activity is taken into consideration, a linear relationship is observed between the concentration of (RTA) in the incubation medium and the amount of receptor bound to DNA-cellulose. A comparison of the binding of partially purified (R-TA) to chromatin and DNA shows that calf thymus DNA has at least 10 times higher binding capacity than liver chromatin, and about 50 times higher binding capacity than erythrocyte chromatin. These figures are in good agreement with estimates of the amount of DNA in these two types of chromatins that can be transcribed by bacterial DNA-dependent RNA polymerases (Seligy and Miyagi, 1974). It is therefore conceivable that the binding of the (R-TA) to chromatin takes place by interaction with those sequences of DNA which are also accessible to other proteins and to RNA polymerases. This assumption is supported by the finding that around 90% of the receptor binding capacity of the chromatin can be inhibited by titration with polylysine, which is known to interact primar~y with the accessible DNA stretches (Clark and Felsenfeld, 1971). The similarities observed in the kinetic experiments, as well as in the sensitivity of the binding to the concentration of NaCl also suggests that the majority of the (R-TA) molecules bound to chromatin are interacting with the so called free DNA stretches. We cannot confirm the report by Simons et al. (1976), that the receptor dissociates more slowly from chromatin that it does from DNA. In our experiments the rates of dissociation of (R-TA) from chromatin and DNA were indistinguishable (fig. 7b). The results presented here indicate that there is a relatively unspecific binding of the receptor-steroid complex to the DNA in DNA-cellulose and in chromatin, but do not exclude the possible existence of a limited number of specific receptor binding sites in the genome of the target tissue. A similar difficulty in detecting specific binding to DNA has been observed in studies on the interaction of the cyclic AMP binding protein with the lac operon in E. coli. Only after a sequence of about 200 bases containing the binding site for the cyclic AMP binding protein was obtained with restriction endonucleases, could the binding of this protein be demonstrated to be sequence specific (Majors, 1975). In fact, a general affinity for DNA appears to be an important property of all regulatory proteins which recognize specific DNA sequences, including the lac repressor (von Hippel et al., 1974; Lin and Riggs, 1975). We Cannot exclude that the weak interaction of (R-TA) with DNA is physiologically significant. Under in vivo conditions, after injection of radioactive glucocorticoids, a considerable amount of the radioactivity can be removed in the nucleus bound to chromatin. Calculations of the number of receptor molecules per haploid genome give values in the order of 5-10 X lo3 indicating a weak inter-
Glucocorticoid receptor binding to the genome
65
action with a large number of acceptor sites within the nucleus, similar to that observed using crude cytosol preparation (fig. 1). In addition to the interaction with free DNA, we have to take into consideration the possibility that the (R-TA) recognizes other components of the chromatin, such as the chromosomal proteins. Reports from other steroid target tissues have shown an interaction of the corresponding receptors with basic and acidic proteins of the chromatin (O’Malley et al., 1972; Puca et al., 1975). Such an interaction could be responsible for the residual binding to chromatin observed at high salt concentrations or after titration of chromatin with polylysine. The clarification of these points, awaits the development of procedures able to distinguish between specific and non-specific binding of the receptor to DNA and chromatin.
REFERENCES Baxter, J.D., Rousseau, G.G., Benson, M.C., Garcea, R.L., Ito, J. and Tomkins, G.M. (1972) J. Biol. Chem. 69,1892-1896. Beato, M. and Doenecke, D. (1976) Metabolic effects and modes of action of glucocorticoids. In: General, Comparative and Clinical Endocrinology, Vol. HI, Eds.: I. Chester Jones and I.W. Henderson (Academic Press) in press. Beato, M. and Feigelson, P. (1972) J. Biol. Chem. 247, 7890-7896. Beato, M., Kalimi, M., Konstam, M. and Feigelson, P. (1973) Biochemistry 12,3372-3379. Beato, M,, Seifart, K.H. and Sekeris, C.E. (1970) Arch. Biochem. Biophys. 138,272-284. Bray, GA. (1960) Anal, Biochem. 1,279-285. Burton, K. (1956) Biochem. J. 62,315-321. Chamness, G.C., Jennings, A.W. and McGuire, W.L. (1974) Biochemistry 13,327-331. Clark, R.J. and Felsenfeld, G. (1971) Nature New Biol. 229, 101-106. Climent, F., Bugany, H. and Beato, M. (1976) FEBS Lett. 66, 317-321. Hamana, K. and Iwai, K. (1973) Gunma Symp. Endocrinol. 10,77-88. Higgins, S.J., Rousseau, G.G., Baxter, J.D. and Tomkins, G.M. (1973a) J. Biol. Chem. 248, 5866-5872. Higgins, S.J., Rousseau, G.G., Baxter, J.D. and Tomkins, G.M. (1973b) J. Biol. Chem. 58735879. Kalimi, M., Beato, M. and Feigelson, P. (1973) Biochemistry 12, 3365-3371. Koblinsky, M., Beato, M., Kalimi, M. and Feigelson, P. (1972) J. Biol. Chem. 247, 7897-7904. Lin, S. and Riggs, A.D. (1975) Cell 4,107-l 11. Lippman, M.E. and Thompson, E.B. (1974) J. Biol. Chem. 249,2483--2488. Litman, R.M. (1968) J. Biol. Chem. 243,6222-6231. Majors, J. (1975) Nature 256,672-674. Marmur, J. (1961) J. Mol. Biol. 3, 208-220. Milgrom, E. and Atger, M. (1975) J. Steroid Biochem. 6,487-492. Milgrom, E., Atger, M. and Baulieu, E.-E. (1973) Biochemistry 12,5198-5205. O’Malley, B.W., Spelsberg, Th.C., Schrader, W.T., Chytil, F. and Steggles, A.W. (1972) Nature 235,141-145. Puca, G.A., Nola, E., Hibner, U., Cicala, G. and Sica, V. (1975) J. Biol. Chem. 250, 64526459. Rousseau, G.G., Higgins, S.J., Baxter, J.D., Gelfant, D. and Tomkins, G.M. (1975) J. Biol. Chem. 250,6015-6021.
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Rousseau, G.G., Higgins, S.J., Baxter, J.D. and Tomkins, G.M. (1974) J. Steroid Biochem. 5, 935-939. Seligy, V.L. and Miyagi, M. (1974) Eur. J. Biochem. 46,259-269. Simons, S.S., Martinez, H.M., Garcea, R.L., Baxter, J.D. and Tomkins, G.M. (1976) J. Biol. Chem. 251,334-343. Von Hippel, P.H., Renin, A., Gross, CA. and Wang, A.M. (1974) Proc. Acad. Sci. U.S.A. 7 I, 4808-4812.
