627
PROTAMINE-PRECIPITATEDESTROGEN RECEPTOR: A SOLID-PHASE LIGAND EXCHANGE ASSAY
Gary C. Chamness, Karen Huff, and William L. McGuire
Department of Medicine University of Texas Health Science Center San Antonio, Texas 78284 Emuscript
received:
2/7/75 ABSTRACT
Cytoplasm&z estrogen receptor can exist either free (R) or bound to estradiol-176 (RR). Both forms can be precipitated from cytosols by protamine sulfate. After protamine precipitation R binds 3H-estradiol178 quantitatively at either 0' or 30", while precipitated RE binds 3Hestradiol-178 only at 30" by exchanging with previously bound hormone. Using these observations, we have developed a method for separate determination of both cytoplasmic R and RE. This method should also be applicable for assay of other steroid receptors, especially in cases where interfering components are present in the whole cytosol.
Estrogen receptor in the cytoplasm of target cells can exist either free (R) or bound to estradiol-17$ (RR). It is RE which enters the nucleus to initiate the nuclear responses to hormone. To quantitate RR in the nucleus, a ligand exchange assay has been developed by Anderson et al. (I). In the cytosol, however, RX is frequently found to be rather unstable under the incubation conditions required for exchange. We have found that if R and RB are first precipitated from the cytosol by protamine sulfate (2), R is still able to bind added E quantitatively, while RE can exchange its ligand without loss of activity. Using protamine precipitation and ligand exchange, we have developed a method for separate determination of cytoplasmic R and RE.
VoZwne 25, Number 5
S
~EIEOXDU
May, 2975
S
TBEOXDI METHODS
Cytosol Preparation Uteri provided by Dr. A.F. Parlow from adult Sprague-Dawley rats were stored at -76'. The frozen tissue was pulverized in a Thermovac tissue pulverizer, thawed to 4" and homDgeni.zedusing a Polytron PT-lOST with four volumes TED buffer (0.01 M Tris-HCl, pH 7.4 at 4', 1.5 mM EDTA, 0.5 mM dithiothreitol). In certain experiments where noted, fresh uteri taken from mature Sprague-Dawley rats at least two days after ovariectomy were used instead. These were minced and then homogenized as before. Homogenates were centrifuged at 105,000 g for 30 minutes and the cytosol was collected. Two volumes of dextran-coated charcoal suspension (DCC: 2.5 g/l Norit A, 25 mgfl dextran, 0.01 M Tris-HCl, pH 8.0 at 4') were centrifuged 10 minutes at 2000 g. The pellet was suspended in the cytosol for 15 minutes at 0" and then centrifuged again for 10 minutes at 2000 g to remove endogenous free steroids. The eytosol was diluted 1:5 to give one to two mg protein per ml. The final protein concentration was later measured by the method of Lowry. Protamine Precipitation and Assay All assays were performed in triplicate. Acid washed 12 x 75 mm glass tubes were incubated at 30" for 10 minutes with 1 ml TE buffer (0.01 M Tris-HCl, pW 7.4 at 4", 1.5 mM EDTA) containing 0.1% bovine serum albumin. The tubes were then rinsed with 1 ml ice-cold TE buffer, and subsequent procedures were carried out at 0" unless otherwise stated. Protamine sulfate (USP injection, without phenol preservative, Eli Lilly Co.) was diluted to 1 mgfml in TED buffer and 0.25 ml was added to each tube. After addition of 0.2 ml cytosol, the mixtures were vortexed and the precipitates were sedimented by centrifugation at 2000 g for 10 minutes. The precipitates firmly coated the bottoms of the tubes and could be incubated, washed, etc., without further centrifugation. E* (3H-estradiol-178,105 Ci/mmole, New England Nuclear) at 10-g M or as specified was added in 0.5 ml TED to the precipitated receptor; parallel incubations included 5 x 10-7 M E" (unlabeled estradiof-17B) to measure nonspecific E* binding. 'Ihetubes were incubated at 0" or 30' for the times indicated, then chilled to 0'. After three washes with 1 ml cold TE buffer, bound radioactivity was extracted twice with 1 ml 100% ethanol at room temperature. The extracts were dried in scintillation vials and counted in 5 ml fluor (4.0 gm PPO, 0.05 gm POPOP, 1 liter toluene) in a Beckman LS233 counter. Dextran-Coated Charcoal Assay Cytosof was prepared as above. 0.2 ml cytosol and 0.2 ml TED buffer containing E* at the indicated concentrations were incubated three hours at 0". 0.6 ml DCC suspension (described above) was added and the mixture was shaken intermittently for 15 minutes and then centrifuged 10 minutes at 2000 g. 0.5 ml of the supernatant was transferred to a scintillation vial for counting with 5 ml BBS fluor (6 gm PPO, 150 ml Beckman Biosolve BBS-3 plus toluene to 1 liter). Parallel samples also contained a 100fold excess of nonradioactive estradiol for determination of nonspecific binding.
S
TISEOXDI
629
RESULTS Estradiol Binding at 0" We first showed that specific E* binding to protamine-precipitated R at 0“ is very nearly complete within three hours (Figure 1).
By ton-
trast, RE prepared by preincubatfng a portion of the same cytosol with Eo binds very little E* at 0".
RE __-e-----0
-o------~
2
3
4
5
Hours Figure 1. A portion (RE) of a cytosol receptor preparation was charged with 2 x 10-g M Eo for 4.5 hours at 0' while the remainder (R) was held at 0' before both were treated with charcoal. Samples of both were precipitated and incubated with 5 x 10-S M E* at 0' for the times indicated. Nonspecific binding was subtracted from all points.
Estradiol Binding at 30" At 30*, E* is able to replace Eo on protamine-precipitatedRE (Figure 2).
The exchange is complete within two to three hours.
Spe-
cifically bound E* then remains nearly constant during continued incubation at 30“, showing little loss even up to 24 hours in some experiments. As an internal control, the same RE preparation incubated at 0" with E* binds very little E*.
To confirm that E* bound at x)" remains
exchangeable, we showed that an excess of Eo added after three hours rapidly replaces the bound E*.
S
630
TEIIICOXDIDI
0 0
I
2
3
4
5
6
Figure 2. Binding of E* at 30" by precharged protamine precipitated receptor (RE). Uterine cytosol receptor was charged with 2 x 10-g M Eo for two hours before charcoal treatment, precipitation, and incubation at 30" (see methods) for the times indicated with 5 x 10-g M E*. Excess E" (final concentration 10-6 M) was added after three hours where indicated. A control was incubated at 0'. Nonspecific binding was subtracted in all cases.
Quantitative Determination The
of R and RE
preceding experiments showed completion of E* binding by R at
O* and by RE at 30" within three hours. concentration was 5 x 10-g M.
In these experiments, the E*
We chose to use 10-g M E* in our routine
assays in order to be even more certain of reaching completion during three hour incubations, which were then established as standard. A number of experiments confirmed that a given R preparation shows essentially the same specific E* binding whether assayed at 0' or 30" after protamine precipitation or at 0' by the standard dextran-coated charcoal procedure (3,4). Table 1.
One of these experiments is presented in
The table also shows that RE prepared from the same cytosol
gives about the same value as free R at 30" but, as expected, a very low value at 0".
This 0" value, similar in both assays, represents
residual R sites uncharged during the preparation of RE. To demonstrate the application of this assay to mixtures of R and RE, a single uterine cytosol was prepared and half was charged with E"
S
631
!I?-EOIDI
TABLE 1 Comparison of Protamine and Dextranxoated Charcoal Assays of Free (R) and Precharged (RE) Uterine Cytosol Receptor, Prepared as in Figure 3, from Ovariectomized Rats.
Receptor Assayed (fm/mg protein, 2 S.E.M.) RE R
Dextran-Coated Charcoal, 0" Protamine, 0" Protamine, 30"
to form RE.
796 k 8 716 t 26 807 + 37
595 9 65 k 10 702 _+54
R and RE were then mixed in various proportions, precipi-
tated with protamine, and assayed with E* at 0' and at 30". The result appears in Figure 3.
As expected, the 0" value is a linear function of
the amount of free R present since RE does not exchange at this temperature. The 30" value measures the total amunt of receptor present,
20,000
Figure 3. Assay of mixtures of R and RE at 0' and 30". Half of a cytosol receptor preparation from uteri of ovariectomized rats was precharged with 2 x 10-8 M Eo for three hours at 0". Both halves were treated with charcoal and then mixed in various proportions. After protamine precipitation, aliquots of each mixture were incubated at either 0" or 30" for three hours with 10-g M E*. Nonspecific binding was subtracted in all cases.
0 KR_ %RE
I#
,
&3 60
& 60
so 40
& 20
S
632
TBEOXDE
independent of the ratio of R to RE, so that RE can be determined by subtracting the R value. The linearity of this assay with varying protein concentrations from 0.3 to 5 m&ml
(60-1000 1_rg total protein) is shown in Figure 4.
Lower concentrations have not been examined. As before, with uncharged R there is little difference between 0" and 30" assays. 60,000,
mg Protein
Figure 4. Assay of uncharged receptor at several concentrations, 0' and 30". Uterine cytosol from ovariectomized rats was diluted to various protein concentrations from 0.3 to 5 mg/ml, precipitated in 200 ul aliquots, and incubated three hours at O" or 30' with 10-S M E*. Nonspecific binding, which was subtracted in all cases, averaged 34% of total binding at 0" and 40% at 30“. At the lowest protein concentration, nonspecific binding was 44% at 0' and 53% at 30'.
Binding Constants We then compared the affinity of E for protamine precipitated R with its affinity for R in solution. The Scatchard pl.otsin Figure 5 show that the affinity at 0' appears to be the same in both states, with a Kd of about 2 x 10-10 M.
Of course, the determination assumes
complete equilibration between E* in solution and the precipitate, but the identity of the Kd values would seem to support such an assumption.
S
TIEIEOXDI
633
The rate of approach to equilibrium may be different, as suggested by the report of Sala-Trepat and Reti (5) of a somewhat faster dissociation of estradiol from receptor after protamine precipitation. At 30' the affinity of R for E is probably less, but precdse determination is not possible since we find that the instability of uncharged R during long incubations at this temperature causes erroneously low E* binding values at the lower E* concentrations.
E* Specific Binding (cpm) Figure 5. Scatchard plots of specific E* binding to R in a protamine precipitate (left) and in a solution (right). Separate uterine cytosols were either precipitated with protamine and incubated with E* (2 x lo-10 to 1 x 10-S M) three hours at 0", or incubated in solution with E* (final concentrations 1 x lo-10 to 5 x 10-g M) three hours at 0' before DCC treatment. Counts were corrected for the small difference in counting efficiency between the two systems. Nonspecific binding was subtracted in all cases.
DISCUSSION Quantitative precipitation of RE* with protamine sulfate was used by Steggles and King (2) to separate the RE* from free E*.
The special
virtue of the method was that many proteins which bound E* nonspecifically did not precipitate, so that errors were reduced.
S
634
7?-EOXDI
We have now shown that R can bind and exchange E* quantitatively even after precipitation with protamine, and have devised an assay based on this fact to determine separately cytoplasmic R and RE.
The
assay thus derived retains the advantage of R separation from other potential binding components as well as from many possibly destructive enzymes present in a number of tissues and tumors. used where cytosol exchange methods
It can therefore be
(6-8) may be subject to interference.
One such application could be the determination of R and RE in human breast tumors where endogenous estrogen may be significant
(9) and
where cytosol composition varies widely. Since other steroid receptors seem to share the property of protamine precipitability,
the procedure presented here for estrogen re-
ceptor may be even more useful when adapted to other systems; e.g., assay of androgen receptor when 3-ketoreductase
in the whole cytosol
attacks the ligand (lo), or assay of progesterone receptor when the cytosol receptor is unstable and CBG interferes with the determination of binding.
ACKNOWLEDGMENTS The expert technical assistance of Miss Linda Mitchell is gratefully acknowledged. This work was supported in part by the USPHS CA-11378, CB-23862, the American Cancer Society BC-23D, and the Robert A. Welch Foundation.
REFERENCES 1.
2. 3. 4. 5.
Anderson, J.N., Clark, J.H. and Peck, E.J., Jr. BIOCHEM. J. 126, 561 (1972). Steggles, A.W. and King, R.J.B. BIOCHEM. J. 118, 695 (1970). Korenman, S.G. STEROIDS 13, 163 (1969). McGuire, W.L. and De La Garza, M. J. CLIN. ENDOCRINOL. METAB. 37, 986 (1973). Sala-Trepat, J.M. and Reti, E. BIOCHIM. ET BIOPHYS. ACTA 338, 92 (1974).
S 6. 7. 8. 9.
10.
TDEOXDb
635
Katzenellenbogen, J.A., Johnson, H.J., Jr., and Carlson, K.E. BIOCHEMISTRY 12, 4092 (1973). Truong, H., Geynet, C., Millet, C., Soulignac, O., Bucourt, R., Vignau, M., Torelli, V. and Baulieu, E.-E. FEBS LET. 35, 289 (1973). Daehnfeldt, J.L. PROC. SOC. EKP. BIOL. MED. 146, 159 (1974). McGuire, W.L., Chamness, G-C., Costlow, M.E. and Shepherd, R.E. In HORMONES AND CANCER (K.W. McKerns, ed.). Academic Press, %w York, pp. 75-101 (1974). Bardin, C.W., Bullock, L.P., Sherins, R.J., Mowszowicz, I. and Blackburn, W.R. REC. PROGR. HORMONE RES. 29, 65 (1973).
PROTAMINE-PRECIPITATEDESTROGEN RECEPTOR: A SOLID-PHASE LIGAND EXCHANGE ASSAY
Gary C. Chamness, Karen Huff, and William L. McGuire
Department of Medicine University of Texas Health Science Center San Antonio, Texas 78284 Emuscript
received:
2/7/75 ABSTRACT
Cytoplasm&z estrogen receptor can exist either free (R) or bound to estradiol-176 (RR). Both forms can be precipitated from cytosols by protamine sulfate. After protamine precipitation R binds 3H-estradiol178 quantitatively at either 0' or 30", while precipitated RE binds 3Hestradiol-178 only at 30" by exchanging with previously bound hormone. Using these observations, we have developed a method for separate determination of both cytoplasmic R and RE. This method should also be applicable for assay of other steroid receptors, especially in cases where interfering components are present in the whole cytosol.
Estrogen receptor in the cytoplasm of target cells can exist either free (R) or bound to estradiol-17$ (RR). It is RE which enters the nucleus to initiate the nuclear responses to hormone. To quantitate RR in the nucleus, a ligand exchange assay has been developed by Anderson et al. (I). In the cytosol, however, RX is frequently found to be rather unstable under the incubation conditions required for exchange. We have found that if R and RB are first precipitated from the cytosol by protamine sulfate (2), R is still able to bind added E quantitatively, while RE can exchange its ligand without loss of activity. Using protamine precipitation and ligand exchange, we have developed a method for separate determination of cytoplasmic R and RE.
VoZwne 25, Number 5
S
~EIEOXDU
May, 2975
S
TBEOXDI METHODS
Cytosol Preparation Uteri provided by Dr. A.F. Parlow from adult Sprague-Dawley rats were stored at -76'. The frozen tissue was pulverized in a Thermovac tissue pulverizer, thawed to 4" and homDgeni.zedusing a Polytron PT-lOST with four volumes TED buffer (0.01 M Tris-HCl, pH 7.4 at 4', 1.5 mM EDTA, 0.5 mM dithiothreitol). In certain experiments where noted, fresh uteri taken from mature Sprague-Dawley rats at least two days after ovariectomy were used instead. These were minced and then homogenized as before. Homogenates were centrifuged at 105,000 g for 30 minutes and the cytosol was collected. Two volumes of dextran-coated charcoal suspension (DCC: 2.5 g/l Norit A, 25 mgfl dextran, 0.01 M Tris-HCl, pH 8.0 at 4') were centrifuged 10 minutes at 2000 g. The pellet was suspended in the cytosol for 15 minutes at 0" and then centrifuged again for 10 minutes at 2000 g to remove endogenous free steroids. The eytosol was diluted 1:5 to give one to two mg protein per ml. The final protein concentration was later measured by the method of Lowry. Protamine Precipitation and Assay All assays were performed in triplicate. Acid washed 12 x 75 mm glass tubes were incubated at 30" for 10 minutes with 1 ml TE buffer (0.01 M Tris-HCl, pW 7.4 at 4", 1.5 mM EDTA) containing 0.1% bovine serum albumin. The tubes were then rinsed with 1 ml ice-cold TE buffer, and subsequent procedures were carried out at 0" unless otherwise stated. Protamine sulfate (USP injection, without phenol preservative, Eli Lilly Co.) was diluted to 1 mgfml in TED buffer and 0.25 ml was added to each tube. After addition of 0.2 ml cytosol, the mixtures were vortexed and the precipitates were sedimented by centrifugation at 2000 g for 10 minutes. The precipitates firmly coated the bottoms of the tubes and could be incubated, washed, etc., without further centrifugation. E* (3H-estradiol-178,105 Ci/mmole, New England Nuclear) at 10-g M or as specified was added in 0.5 ml TED to the precipitated receptor; parallel incubations included 5 x 10-7 M E" (unlabeled estradiof-17B) to measure nonspecific E* binding. 'Ihetubes were incubated at 0" or 30' for the times indicated, then chilled to 0'. After three washes with 1 ml cold TE buffer, bound radioactivity was extracted twice with 1 ml 100% ethanol at room temperature. The extracts were dried in scintillation vials and counted in 5 ml fluor (4.0 gm PPO, 0.05 gm POPOP, 1 liter toluene) in a Beckman LS233 counter. Dextran-Coated Charcoal Assay Cytosof was prepared as above. 0.2 ml cytosol and 0.2 ml TED buffer containing E* at the indicated concentrations were incubated three hours at 0". 0.6 ml DCC suspension (described above) was added and the mixture was shaken intermittently for 15 minutes and then centrifuged 10 minutes at 2000 g. 0.5 ml of the supernatant was transferred to a scintillation vial for counting with 5 ml BBS fluor (6 gm PPO, 150 ml Beckman Biosolve BBS-3 plus toluene to 1 liter). Parallel samples also contained a 100fold excess of nonradioactive estradiol for determination of nonspecific binding.
S
TISEOXDI
629
RESULTS Estradiol Binding at 0" We first showed that specific E* binding to protamine-precipitated R at 0“ is very nearly complete within three hours (Figure 1).
By ton-
trast, RE prepared by preincubatfng a portion of the same cytosol with Eo binds very little E* at 0".
RE __-e-----0
-o------~
2
3
4
5
Hours Figure 1. A portion (RE) of a cytosol receptor preparation was charged with 2 x 10-g M Eo for 4.5 hours at 0' while the remainder (R) was held at 0' before both were treated with charcoal. Samples of both were precipitated and incubated with 5 x 10-S M E* at 0' for the times indicated. Nonspecific binding was subtracted from all points.
Estradiol Binding at 30" At 30*, E* is able to replace Eo on protamine-precipitatedRE (Figure 2).
The exchange is complete within two to three hours.
Spe-
cifically bound E* then remains nearly constant during continued incubation at 30“, showing little loss even up to 24 hours in some experiments. As an internal control, the same RE preparation incubated at 0" with E* binds very little E*.
To confirm that E* bound at x)" remains
exchangeable, we showed that an excess of Eo added after three hours rapidly replaces the bound E*.
S
630
TEIIICOXDIDI
0 0
I
2
3
4
5
6
Figure 2. Binding of E* at 30" by precharged protamine precipitated receptor (RE). Uterine cytosol receptor was charged with 2 x 10-g M Eo for two hours before charcoal treatment, precipitation, and incubation at 30" (see methods) for the times indicated with 5 x 10-g M E*. Excess E" (final concentration 10-6 M) was added after three hours where indicated. A control was incubated at 0'. Nonspecific binding was subtracted in all cases.
Quantitative Determination The
of R and RE
preceding experiments showed completion of E* binding by R at
O* and by RE at 30" within three hours. concentration was 5 x 10-g M.
In these experiments, the E*
We chose to use 10-g M E* in our routine
assays in order to be even more certain of reaching completion during three hour incubations, which were then established as standard. A number of experiments confirmed that a given R preparation shows essentially the same specific E* binding whether assayed at 0' or 30" after protamine precipitation or at 0' by the standard dextran-coated charcoal procedure (3,4). Table 1.
One of these experiments is presented in
The table also shows that RE prepared from the same cytosol
gives about the same value as free R at 30" but, as expected, a very low value at 0".
This 0" value, similar in both assays, represents
residual R sites uncharged during the preparation of RE. To demonstrate the application of this assay to mixtures of R and RE, a single uterine cytosol was prepared and half was charged with E"
S
631
!I?-EOIDI
TABLE 1 Comparison of Protamine and Dextranxoated Charcoal Assays of Free (R) and Precharged (RE) Uterine Cytosol Receptor, Prepared as in Figure 3, from Ovariectomized Rats.
Receptor Assayed (fm/mg protein, 2 S.E.M.) RE R
Dextran-Coated Charcoal, 0" Protamine, 0" Protamine, 30"
to form RE.
796 k 8 716 t 26 807 + 37
595 9 65 k 10 702 _+54
R and RE were then mixed in various proportions, precipi-
tated with protamine, and assayed with E* at 0' and at 30". The result appears in Figure 3.
As expected, the 0" value is a linear function of
the amount of free R present since RE does not exchange at this temperature. The 30" value measures the total amunt of receptor present,
20,000
Figure 3. Assay of mixtures of R and RE at 0' and 30". Half of a cytosol receptor preparation from uteri of ovariectomized rats was precharged with 2 x 10-8 M Eo for three hours at 0". Both halves were treated with charcoal and then mixed in various proportions. After protamine precipitation, aliquots of each mixture were incubated at either 0" or 30" for three hours with 10-g M E*. Nonspecific binding was subtracted in all cases.
0 KR_ %RE
I#
,
&3 60
& 60
so 40
& 20
S
632
TBEOXDE
independent of the ratio of R to RE, so that RE can be determined by subtracting the R value. The linearity of this assay with varying protein concentrations from 0.3 to 5 m&ml
(60-1000 1_rg total protein) is shown in Figure 4.
Lower concentrations have not been examined. As before, with uncharged R there is little difference between 0" and 30" assays. 60,000,
mg Protein
Figure 4. Assay of uncharged receptor at several concentrations, 0' and 30". Uterine cytosol from ovariectomized rats was diluted to various protein concentrations from 0.3 to 5 mg/ml, precipitated in 200 ul aliquots, and incubated three hours at O" or 30' with 10-S M E*. Nonspecific binding, which was subtracted in all cases, averaged 34% of total binding at 0" and 40% at 30“. At the lowest protein concentration, nonspecific binding was 44% at 0' and 53% at 30'.
Binding Constants We then compared the affinity of E for protamine precipitated R with its affinity for R in solution. The Scatchard pl.otsin Figure 5 show that the affinity at 0' appears to be the same in both states, with a Kd of about 2 x 10-10 M.
Of course, the determination assumes
complete equilibration between E* in solution and the precipitate, but the identity of the Kd values would seem to support such an assumption.
S
TIEIEOXDI
633
The rate of approach to equilibrium may be different, as suggested by the report of Sala-Trepat and Reti (5) of a somewhat faster dissociation of estradiol from receptor after protamine precipitation. At 30' the affinity of R for E is probably less, but precdse determination is not possible since we find that the instability of uncharged R during long incubations at this temperature causes erroneously low E* binding values at the lower E* concentrations.
E* Specific Binding (cpm) Figure 5. Scatchard plots of specific E* binding to R in a protamine precipitate (left) and in a solution (right). Separate uterine cytosols were either precipitated with protamine and incubated with E* (2 x lo-10 to 1 x 10-S M) three hours at 0", or incubated in solution with E* (final concentrations 1 x lo-10 to 5 x 10-g M) three hours at 0' before DCC treatment. Counts were corrected for the small difference in counting efficiency between the two systems. Nonspecific binding was subtracted in all cases.
DISCUSSION Quantitative precipitation of RE* with protamine sulfate was used by Steggles and King (2) to separate the RE* from free E*.
The special
virtue of the method was that many proteins which bound E* nonspecifically did not precipitate, so that errors were reduced.
S
634
7?-EOXDI
We have now shown that R can bind and exchange E* quantitatively even after precipitation with protamine, and have devised an assay based on this fact to determine separately cytoplasmic R and RE.
The
assay thus derived retains the advantage of R separation from other potential binding components as well as from many possibly destructive enzymes present in a number of tissues and tumors. used where cytosol exchange methods
It can therefore be
(6-8) may be subject to interference.
One such application could be the determination of R and RE in human breast tumors where endogenous estrogen may be significant
(9) and
where cytosol composition varies widely. Since other steroid receptors seem to share the property of protamine precipitability,
the procedure presented here for estrogen re-
ceptor may be even more useful when adapted to other systems; e.g., assay of androgen receptor when 3-ketoreductase
in the whole cytosol
attacks the ligand (lo), or assay of progesterone receptor when the cytosol receptor is unstable and CBG interferes with the determination of binding.
ACKNOWLEDGMENTS The expert technical assistance of Miss Linda Mitchell is gratefully acknowledged. This work was supported in part by the USPHS CA-11378, CB-23862, the American Cancer Society BC-23D, and the Robert A. Welch Foundation.
REFERENCES 1.
2. 3. 4. 5.
Anderson, J.N., Clark, J.H. and Peck, E.J., Jr. BIOCHEM. J. 126, 561 (1972). Steggles, A.W. and King, R.J.B. BIOCHEM. J. 118, 695 (1970). Korenman, S.G. STEROIDS 13, 163 (1969). McGuire, W.L. and De La Garza, M. J. CLIN. ENDOCRINOL. METAB. 37, 986 (1973). Sala-Trepat, J.M. and Reti, E. BIOCHIM. ET BIOPHYS. ACTA 338, 92 (1974).
S 6. 7. 8. 9.
10.
TDEOXDb
635
Katzenellenbogen, J.A., Johnson, H.J., Jr., and Carlson, K.E. BIOCHEMISTRY 12, 4092 (1973). Truong, H., Geynet, C., Millet, C., Soulignac, O., Bucourt, R., Vignau, M., Torelli, V. and Baulieu, E.-E. FEBS LET. 35, 289 (1973). Daehnfeldt, J.L. PROC. SOC. EKP. BIOL. MED. 146, 159 (1974). McGuire, W.L., Chamness, G-C., Costlow, M.E. and Shepherd, R.E. In HORMONES AND CANCER (K.W. McKerns, ed.). Academic Press, %w York, pp. 75-101 (1974). Bardin, C.W., Bullock, L.P., Sherins, R.J., Mowszowicz, I. and Blackburn, W.R. REC. PROGR. HORMONE RES. 29, 65 (1973).
