568th MEETING, ABERDEEN
633
nuclear membrane, since most is removed during the Triton X-100 treatment (Aaronson & Blobel, 1974, 1975). Indeed the same effects can be obtained by using crude prelabelled nuclei isolated in the absence of Triton X-100, which in fact appear to have nuclear membranes. Aaronson, R.P. & Blobel, G. (1974) J. Cell Biol. 62, 746754 Aaronson, R. P. &Blobel, G. (1975) Proc. Natl. Acad. Sci. U.S.A.72,1007-1011 Acheson, N. H. (1976) Cell 8 , 1 4 2 Augenlicht, L. H. & Lipkin, M. (1976)J. Biol. Chem. 254,2592-2599 Getz, M. J., Birnie, G. D., Young, B. D., MacPhail, E. &Paul, J. (1975) Ce114,121-129 Green, M. R., Green, M. & Mackey, J. K. (1976) Nature (London) 261,340-342 Herman, R. C., Williams, J. G. & Penman, S. (1976) Cell 7,429-437 Perry, R. P. (1976) Annu. Rev. Biochem. 45,605429 Pederson, T. (1974) J. Mol. Biol. 83, 163-183 Sarma, M. H., Ferman, E. R. & Baglioni, C. (1976) Biochim. Biophys. Acta 418,29-38 Spohr, G., Granboulan, M., Morel, C. & Scherrer, K. (1970) Eur. J. Biochem. 17,296318
STEROID BINDING AND OLIGOMERIC PROTEINS : a Colloquium organized on behalf of the Steroid Biochemistry Group of the Biochemical Society and the Society for Endocrinology by J. Jeffery (Aberdeen) Glucocorticoid-Receptor Interactions in Rat Liver
JAN-AKE
GUSTAFSSON, JAN CARLSTEDT-DUKE and dRJAN WRANGE
Department of Chemistry, Karolinska Institute, S-104 01 Stockholm 60, Sweden
The oestradiol-receptor complex in rat and calf uterus (Puca et al., 1972; Notides & Nielsen, 1974; Rat et al., 1974) and the progesterone-receptor complex in chick oviduct (Sherman et al., 1974, 1976) have been shown to occur in different forms with different molecular weights. Similar reports on different molecular forms of the dexamethasone-receptor complex are not available in the literature. We have been interested in various aspects of mechanismsof glucocorticoidaction in rat liver (Wrange, 1976; Carlstedt-Duke et al., 1977~).By using [l ,2,4-3H]dexamethasone (specific radioactivity 27Ci/mmol) as ligand, we have studied the size of the glucocorticoidreceptor complex in rat liver cytosol and nuclear extract after administration in vivo or incubation in vitro of [3H]dexamethasone (Carlstedt-Duke ei al., 19776). By using sucrose-gradient centrifugation and Sephadex-gel filtration at high ionic strength, the sedimentation coefficient and Stokes radius of the dexamethasone-receptor complex have been measured. The molecular weight has been calculated as described in Table 1. The liver cytosol complex has a Stokes radius of 6.1nm and a sedimentation coefficient of 4.0S, yielding a mol.wt. of approx. 102000 (Table 1). The O.~M-KCI extract from liver cell nuclei, however, contains protein-bound dexamethasone with a Stokes radius of 3 - 3 . 6 ~ 1and a sedimentation coefficient of 3.2S, yielding a mol.wt. of approx. 46OOO (Table 1). The same result is obtained both after administration in uivo of [3H]dexamethasone as after incubation of purified cell nuclei with [3H]dexamethasone labelled rat liver cytosol at 25°C. Further investigation has revealed that when prepared in buffers of low osmolarity, the cytosol dexamethasone receptor has similar physical characteristics to the nuclear complex. Referring to the two forms of the dexamethasone-receptor complex as the
Vol. 5
634
BIOCHEMICAL SOCJETY TRANSACTIONS
Table 1. Molecular weights and frictional ratios of the dzyerent [3H]dexamethasonereceptor complexes The molecular weights were calculated as described by Siege1 & Monty (1966) and the frictional ratios as described by Notides & Nielsen (1974):
15
-
t-Fe+
+Hb+
1977
635
568th MEETING, ABERDEEN
6.1 n m
t
30
I
3 - 3 . 6 nm
d
5
- 0.2
3 >
Ei
4 20
h
3
3
.-x> .-
5
Y
-0.1
Y
V
g
k%
10
1-
a
2 I
0
10
1 20
I
30
40
50
Fraction no. Fig. 2. Chromatography on DNA-cellulose of the 6.1nm and 3-3.6nm forms of the [3H]dexamethasone-receptor complex Liver cytosol was labelled with [3H]dexamethasoneand approximately one-half of the amount of the 6.1 nm form of the [3H]dexametha~~ne-receptorcomplex was transformed to the 3-3.6nm form by limited trypsin digestion. After an activation step (25”C, 30min) the cytosol was chromatographed on a Sephadex G-25 column and the protein-bound radioactivity applied to a lOml DNA-cellulose column. The column was first washed with buffer of low ionic strength and then the column was ( 0 ;total volume 16Oml). Radioactivity (0) eluted with a linear O ~ . ~ M - Kgradient CI and conductivity were measured and samples of fractions 30 and 38 were subjected to gel filtration on Sephadex G-200 and G-150 columns respectively, calibrated for determination of Stokes radius. ‘6.1 nmform’and the‘3.6nmform’respectively,it is possible to transformthe6.1 nmform into the 3.6nm form by treatment with a hypo-osmoticextract of the pellet sedimenting between 1000and 1OOOOg on centrifugation of rat liver homogenate.This transformation is also obtained when the 6.1 nm form is subjected to limited proteolysis with trypsin. When increasing concentrations of trypsin are used the dexamethasone-receptor complex is further transformed to a 1.9nm form with a sedimentation coefficient of 2.5s and a mol.wt. of approx. 19000 (Fig. 1, Table 1). This form of the dexamethasonereceptor complex can also be obtained by incubation of the 6.1 nm form or the 3.6nm form with the hypo-osmotic extract of the 1 ~ 1 O O O O gsediment from rat liver homogenate mentioned above. The transformation of the 6.1 nm form into the 3.6nm and 1.9 nm forms of the dexamethasone-receptor complex occurs without significant loss of dexamethasone-binding sites. The 6.1 nm form of the dexamethasone-receptor complex is a highly asymmetrical protein with a frictional ratio of approx. 1.84 (Table 1). The 3.6nm and 1.9nm forms are more symmetrical and have frictional ratios of 1.38 and 1.OO respectively. The affinity of the different forms of dexamethasone-receptor complex to DNA in terms of ionic strength needed for elution has been tested on DNA-cellulose columns (Alberts & Herrick, 1971) containing double-stranded calf thymus DNA and eluted with a linear M . ~ M - K Cgradient I (Yamamoto, 1974). The 6.lnm and 3.6nm forms bind to DNA-cellulose after an activation step (incubation at 25°C for 30min) (Giannopoulos, 1975; Kalimi et al., 1975; Rousseau et al., 1975) and are eluted at 0.10-0.12~-KC1and at 0.15-0.17~-KC1respectively (Fig. 2). The 1.9nm form does not bind to DNA-cellulose. Gel filtration of the different forms of the dexamethasone-receptor complex after chromatography on DNA-cellulose shows that this procedure does not result in transformation of one form into any of the other two forms. Experiments have also been performed with purified rat liver nuclei VOl. 5
22
636
BIOCHEMICAL SOCIETY TRANSACTIONS
that have been incubated with each of the three forms of the [3H]dexamethasonereceptor complex whereafter the nuclear uptake of radioactivity has been measured. In these experiments, the 6.1 and 3.6nm forms are taken up by the nuclei, whereas the 1.9nm form is not retained. All these data indicate that the DNA-binding site and the dexamethasonebinding site on the glucocorticoid receptor are located on separate parts of the receptor molecule. The significance of the 3.6nm form of the dexamethasone-receptor complex present in liver cell nuclei cannot yet be completely evaluated. The ready formation of the 3.6nm form from the 6.1 nm form by proteolytic digestion indicates that the nuclear 3.6nm form may possibly arise as an artifact by the action of nuclear proteinase activated during extraction of nuclei with hyperosmotic salt solution (Carter & Chac, 1976). Repeated trials to inhibit this suspected proteinase activity with 2m~-di-isopropyl phosphorofluoridate during nuclear extraction have not been successful. With the reservation mentioned above in mind, it is tempting to speculate that the higher affinity of the 3.6nm form to DNA compared with that of the 6.1nm form of the dexamethasone-receptor complex could have some physiological significance, perhaps reflecting potentiation of glucocorticoid-receptor-complex-mediated biological response by intracellular proteinase activity. It may be argued, however, that the binding of the diflerent forms of the dexamethasone-receptor complex of DNA assayed as described above only reflects unspecific binding to polyanionic molecules and that further experiments are needed to test the presented hypothesis. Under all circumstances, the demonstration of the different characteristics of the various forms of the dexamethasone-receptor complex in terms of DNA binding implies that studies of uptake of steroid-receptor complexes by DNA, chromatin or nuclei are improved in validity by physical characterization of the steroid-receptor complexes in question. This work was supported by grants from the Swedish Medical Research Council (no. 13X2819) and from the Leo Research Foundation. Alberts, B. & Hemck, G. (1971)MefhodsEnzyml. 21D, 198-217 Carlstedt-Duke,J., Gustafsson,J.-A., Gustafsson,S. A. & Wrange, 6. (1977~) Eur. J . Biochem.
in the press Carlstedt-Duke,J., Gustafsson, J.-A. & Wrange, 6.(19776)Biochim. Biophys. Acta in the press Carter, D. B. & Chac, C.-B. (1976) Biochemisfry 15, 180-185 GiaMopoulos, G. (1975)J. Biol. Chem. 250,2896-2903 Kalimi, M., Colman, P. 19Feigelson, P. (1975) J. Biol. Chem. 250, 1080-1086 Notides, A. C. & Nielsen, S. (1974)J. Biol. Chem. 249, 1866-1873 Puca, G. A.,Nola, E., Sica, V. & Bresciani, F. (1972)Biochemisfry 11,4157-4165 Rat, R. L.,Vallet-Strouve, C. & Erdos, T. (1974)Biochimie 56, 1387-1394 Rousseau,G. G.,Higgins, S.J.,Baxter,J. D.,Gelfand,D. &Tomkins,G. M. (1975)J. Biol. Chem. 250,6015-6021 Sherman, M. R., Atienza, S. P., Shansky, J. R. & Hoffman, L. M. (1974)J. Biol. Chem. 249, 5351-5363 Sherman, M. R., Tuazon, F. B., Diaz, S.D. &Miller, L. K. (1976)Biochemisfry15,980-988 Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acfa 112, 346-362 Wrange, 6.(1976)Biochinr. Biophys. Acfa 434, 483-489 Yarnamoto, K.R. (1974)J. Biol. Chem. 249, 7068-7075
Alcohol Dehydrogenase: Structural Studies, Functional Aspects and Evolutionary Conclusions in Relation to Steroid Binding HANS JGRNVALL Department of Chemistry, Karolinska Institute, S-104 01 Stockholm 60,Sweden
The activity of alcohol dehydrogenase towards certain steroid substrates is of interest from different aspects, e.g. the functional role of this enzyme activity in metabolic conversions, the relationship between enzyme structure and substrate specificity, and 1977
633
nuclear membrane, since most is removed during the Triton X-100 treatment (Aaronson & Blobel, 1974, 1975). Indeed the same effects can be obtained by using crude prelabelled nuclei isolated in the absence of Triton X-100, which in fact appear to have nuclear membranes. Aaronson, R.P. & Blobel, G. (1974) J. Cell Biol. 62, 746754 Aaronson, R. P. &Blobel, G. (1975) Proc. Natl. Acad. Sci. U.S.A.72,1007-1011 Acheson, N. H. (1976) Cell 8 , 1 4 2 Augenlicht, L. H. & Lipkin, M. (1976)J. Biol. Chem. 254,2592-2599 Getz, M. J., Birnie, G. D., Young, B. D., MacPhail, E. &Paul, J. (1975) Ce114,121-129 Green, M. R., Green, M. & Mackey, J. K. (1976) Nature (London) 261,340-342 Herman, R. C., Williams, J. G. & Penman, S. (1976) Cell 7,429-437 Perry, R. P. (1976) Annu. Rev. Biochem. 45,605429 Pederson, T. (1974) J. Mol. Biol. 83, 163-183 Sarma, M. H., Ferman, E. R. & Baglioni, C. (1976) Biochim. Biophys. Acta 418,29-38 Spohr, G., Granboulan, M., Morel, C. & Scherrer, K. (1970) Eur. J. Biochem. 17,296318
STEROID BINDING AND OLIGOMERIC PROTEINS : a Colloquium organized on behalf of the Steroid Biochemistry Group of the Biochemical Society and the Society for Endocrinology by J. Jeffery (Aberdeen) Glucocorticoid-Receptor Interactions in Rat Liver
JAN-AKE
GUSTAFSSON, JAN CARLSTEDT-DUKE and dRJAN WRANGE
Department of Chemistry, Karolinska Institute, S-104 01 Stockholm 60, Sweden
The oestradiol-receptor complex in rat and calf uterus (Puca et al., 1972; Notides & Nielsen, 1974; Rat et al., 1974) and the progesterone-receptor complex in chick oviduct (Sherman et al., 1974, 1976) have been shown to occur in different forms with different molecular weights. Similar reports on different molecular forms of the dexamethasone-receptor complex are not available in the literature. We have been interested in various aspects of mechanismsof glucocorticoidaction in rat liver (Wrange, 1976; Carlstedt-Duke et al., 1977~).By using [l ,2,4-3H]dexamethasone (specific radioactivity 27Ci/mmol) as ligand, we have studied the size of the glucocorticoidreceptor complex in rat liver cytosol and nuclear extract after administration in vivo or incubation in vitro of [3H]dexamethasone (Carlstedt-Duke ei al., 19776). By using sucrose-gradient centrifugation and Sephadex-gel filtration at high ionic strength, the sedimentation coefficient and Stokes radius of the dexamethasone-receptor complex have been measured. The molecular weight has been calculated as described in Table 1. The liver cytosol complex has a Stokes radius of 6.1nm and a sedimentation coefficient of 4.0S, yielding a mol.wt. of approx. 102000 (Table 1). The O.~M-KCI extract from liver cell nuclei, however, contains protein-bound dexamethasone with a Stokes radius of 3 - 3 . 6 ~ 1and a sedimentation coefficient of 3.2S, yielding a mol.wt. of approx. 46OOO (Table 1). The same result is obtained both after administration in uivo of [3H]dexamethasone as after incubation of purified cell nuclei with [3H]dexamethasone labelled rat liver cytosol at 25°C. Further investigation has revealed that when prepared in buffers of low osmolarity, the cytosol dexamethasone receptor has similar physical characteristics to the nuclear complex. Referring to the two forms of the dexamethasone-receptor complex as the
Vol. 5
634
BIOCHEMICAL SOCJETY TRANSACTIONS
Table 1. Molecular weights and frictional ratios of the dzyerent [3H]dexamethasonereceptor complexes The molecular weights were calculated as described by Siege1 & Monty (1966) and the frictional ratios as described by Notides & Nielsen (1974):
15
-
t-Fe+
+Hb+
1977
635
568th MEETING, ABERDEEN
6.1 n m
t
30
I
3 - 3 . 6 nm
d
5
- 0.2
3 >
Ei
4 20
h
3
3
.-x> .-
5
Y
-0.1
Y
V
g
k%
10
1-
a
2 I
0
10
1 20
I
30
40
50
Fraction no. Fig. 2. Chromatography on DNA-cellulose of the 6.1nm and 3-3.6nm forms of the [3H]dexamethasone-receptor complex Liver cytosol was labelled with [3H]dexamethasoneand approximately one-half of the amount of the 6.1 nm form of the [3H]dexametha~~ne-receptorcomplex was transformed to the 3-3.6nm form by limited trypsin digestion. After an activation step (25”C, 30min) the cytosol was chromatographed on a Sephadex G-25 column and the protein-bound radioactivity applied to a lOml DNA-cellulose column. The column was first washed with buffer of low ionic strength and then the column was ( 0 ;total volume 16Oml). Radioactivity (0) eluted with a linear O ~ . ~ M - Kgradient CI and conductivity were measured and samples of fractions 30 and 38 were subjected to gel filtration on Sephadex G-200 and G-150 columns respectively, calibrated for determination of Stokes radius. ‘6.1 nmform’and the‘3.6nmform’respectively,it is possible to transformthe6.1 nmform into the 3.6nm form by treatment with a hypo-osmoticextract of the pellet sedimenting between 1000and 1OOOOg on centrifugation of rat liver homogenate.This transformation is also obtained when the 6.1 nm form is subjected to limited proteolysis with trypsin. When increasing concentrations of trypsin are used the dexamethasone-receptor complex is further transformed to a 1.9nm form with a sedimentation coefficient of 2.5s and a mol.wt. of approx. 19000 (Fig. 1, Table 1). This form of the dexamethasonereceptor complex can also be obtained by incubation of the 6.1 nm form or the 3.6nm form with the hypo-osmotic extract of the 1 ~ 1 O O O O gsediment from rat liver homogenate mentioned above. The transformation of the 6.1 nm form into the 3.6nm and 1.9 nm forms of the dexamethasone-receptor complex occurs without significant loss of dexamethasone-binding sites. The 6.1 nm form of the dexamethasone-receptor complex is a highly asymmetrical protein with a frictional ratio of approx. 1.84 (Table 1). The 3.6nm and 1.9nm forms are more symmetrical and have frictional ratios of 1.38 and 1.OO respectively. The affinity of the different forms of dexamethasone-receptor complex to DNA in terms of ionic strength needed for elution has been tested on DNA-cellulose columns (Alberts & Herrick, 1971) containing double-stranded calf thymus DNA and eluted with a linear M . ~ M - K Cgradient I (Yamamoto, 1974). The 6.lnm and 3.6nm forms bind to DNA-cellulose after an activation step (incubation at 25°C for 30min) (Giannopoulos, 1975; Kalimi et al., 1975; Rousseau et al., 1975) and are eluted at 0.10-0.12~-KC1and at 0.15-0.17~-KC1respectively (Fig. 2). The 1.9nm form does not bind to DNA-cellulose. Gel filtration of the different forms of the dexamethasone-receptor complex after chromatography on DNA-cellulose shows that this procedure does not result in transformation of one form into any of the other two forms. Experiments have also been performed with purified rat liver nuclei VOl. 5
22
636
BIOCHEMICAL SOCIETY TRANSACTIONS
that have been incubated with each of the three forms of the [3H]dexamethasonereceptor complex whereafter the nuclear uptake of radioactivity has been measured. In these experiments, the 6.1 and 3.6nm forms are taken up by the nuclei, whereas the 1.9nm form is not retained. All these data indicate that the DNA-binding site and the dexamethasonebinding site on the glucocorticoid receptor are located on separate parts of the receptor molecule. The significance of the 3.6nm form of the dexamethasone-receptor complex present in liver cell nuclei cannot yet be completely evaluated. The ready formation of the 3.6nm form from the 6.1 nm form by proteolytic digestion indicates that the nuclear 3.6nm form may possibly arise as an artifact by the action of nuclear proteinase activated during extraction of nuclei with hyperosmotic salt solution (Carter & Chac, 1976). Repeated trials to inhibit this suspected proteinase activity with 2m~-di-isopropyl phosphorofluoridate during nuclear extraction have not been successful. With the reservation mentioned above in mind, it is tempting to speculate that the higher affinity of the 3.6nm form to DNA compared with that of the 6.1nm form of the dexamethasone-receptor complex could have some physiological significance, perhaps reflecting potentiation of glucocorticoid-receptor-complex-mediated biological response by intracellular proteinase activity. It may be argued, however, that the binding of the diflerent forms of the dexamethasone-receptor complex of DNA assayed as described above only reflects unspecific binding to polyanionic molecules and that further experiments are needed to test the presented hypothesis. Under all circumstances, the demonstration of the different characteristics of the various forms of the dexamethasone-receptor complex in terms of DNA binding implies that studies of uptake of steroid-receptor complexes by DNA, chromatin or nuclei are improved in validity by physical characterization of the steroid-receptor complexes in question. This work was supported by grants from the Swedish Medical Research Council (no. 13X2819) and from the Leo Research Foundation. Alberts, B. & Hemck, G. (1971)MefhodsEnzyml. 21D, 198-217 Carlstedt-Duke,J., Gustafsson,J.-A., Gustafsson,S. A. & Wrange, 6. (1977~) Eur. J . Biochem.
in the press Carlstedt-Duke,J., Gustafsson, J.-A. & Wrange, 6.(19776)Biochim. Biophys. Acta in the press Carter, D. B. & Chac, C.-B. (1976) Biochemisfry 15, 180-185 GiaMopoulos, G. (1975)J. Biol. Chem. 250,2896-2903 Kalimi, M., Colman, P. 19Feigelson, P. (1975) J. Biol. Chem. 250, 1080-1086 Notides, A. C. & Nielsen, S. (1974)J. Biol. Chem. 249, 1866-1873 Puca, G. A.,Nola, E., Sica, V. & Bresciani, F. (1972)Biochemisfry 11,4157-4165 Rat, R. L.,Vallet-Strouve, C. & Erdos, T. (1974)Biochimie 56, 1387-1394 Rousseau,G. G.,Higgins, S.J.,Baxter,J. D.,Gelfand,D. &Tomkins,G. M. (1975)J. Biol. Chem. 250,6015-6021 Sherman, M. R., Atienza, S. P., Shansky, J. R. & Hoffman, L. M. (1974)J. Biol. Chem. 249, 5351-5363 Sherman, M. R., Tuazon, F. B., Diaz, S.D. &Miller, L. K. (1976)Biochemisfry15,980-988 Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acfa 112, 346-362 Wrange, 6.(1976)Biochinr. Biophys. Acfa 434, 483-489 Yarnamoto, K.R. (1974)J. Biol. Chem. 249, 7068-7075
Alcohol Dehydrogenase: Structural Studies, Functional Aspects and Evolutionary Conclusions in Relation to Steroid Binding HANS JGRNVALL Department of Chemistry, Karolinska Institute, S-104 01 Stockholm 60,Sweden
The activity of alcohol dehydrogenase towards certain steroid substrates is of interest from different aspects, e.g. the functional role of this enzyme activity in metabolic conversions, the relationship between enzyme structure and substrate specificity, and 1977
