[17]
CHICK OVIDUCT PROGESTERONE RECEPTOR
187
any form can be deliberately produced by appropriate manipulation.The interrelationships of these forms and their physiological significance are not yet understood.
Acknowledgments This work was supported by The National Cancer Institute, The American Cancer Society, and The Robert A. Welch Foundation.
[17] Methods for Extraction and Quantification of Receptors B y WILLIAM T. SCHRADER I. I n t r o d u c t i o n
The chick oviduct has been used as a model system for the study of progesterone action in target cells. 1-3 In this tissue, estrogen administration induces growth and differentiation; the estrogen-primed oviduct cells then respond to progesterone by synthesizing the egg protein avidin. 4,~ Studies on the mechanism of this progesterone response have shown that the hormone is first bound to intracellular macromolecules ~,7 and is subsequently transported to the nuclear compartment s,9 where the hormone-macromolecular complex becomes tightly associated with the 1B. W. 0'Malley, Biochemistry 6, 2546 (1967). 2 B. W. O'Malley, W. L. McGuire, P. O. Kohler, and S. G. Korenman, Recent Progr. Horm. Res. 25, 105 (1969). ' B. W. O'Malley, W. T. Schrader, and T. C. Spelsberg, in "Receptors for Reproductive Hormones" (B. W. O'Malley and A. R. Means, eds.), p. 174. Plenum, New York, 1973. 4 B. W. O'Malley and P. O. Kohler, Proc. Nat. Acad. Sci. U.S. 58, 2359 (1967). B. W. O'Malley, G. C. Rosenfeld, J. P. Comstock, and A. R. Means, Nature (London), New Biology 240, 45 (1972). M. R. Sherman, P. L. Corvol, and B. W. O'Malley, J. Biol. Chem. 245, 6085 (1970). 7B. W. O'Malley, M. R. Sherman, D. O. Toft, T. C. Spelsberg, W. T. Schrader, and A. W. Steggles, Advan. Biosci. 7, 213 (1971). 8B. W. O'Malley, D. O. Toft, and M. R. Sherman, J. Biol. Chem. 246, 1117 (1971). 9B. W. O'Malley, M. R. Sherman, and D. O. Toft, Proc. Nat. Acad. Sci. U.S. 67, 501 (1970).
188
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
[17]
cell chromatin. 1°-13 These studies have been facilitated by use of partially purified progesterone receptors from the oviduct prepared as described below.14, ~5 I t is i m p o r t a n t to keep in mind t h a t there is as yet no true end-point assay for the functional form of a steroid receptor protein in any tissue. At least five p a r a m e t e r s have been defined for the progesterone receptor system which m a y serve as substitutes for such an end-point assay. ~6 These p a r a m e t e r s should be monitored throughout all manipulations, in order to avoid working with materials which are so altered t h a t they do not represent the starting material. The p a r a m e t e r s used in this labor a t o r y are (1) steroid-binding kinetics and specificity, ~7 (2) molecular size and charge, 6,1s,19 (3) uptake and retention by nuclei, s,9,~° (4) binding to D N A , z~-~3 and (5) binding to chromatin. Whenever a manipulation results in a deviation in one of these parameters, it should serve to prejudice the investigation toward defining the nature of the changes. No step should be adopted into the protocol which results in the isolation of an altered receptor form without a clear realization t h a t the product m a y be biologically inactive. II. Tissue Sources
Progesterone receptors can be isolated and identified from chick oviduct of any age or hormonal history. I m m a t u r e oviducts of average weights of about 10 mg can be pooled for extraction of receptors. I t is more convenient, however, to work with immature female chicks injected ~oT. C. Spelsberg, A. W. Steggles, and B. W. O'Malley, J. Biol. Chem. 246, 4188 (1971). 11A. W. Steggles, T. C. Spelsberg, and B. W. O'Malley, Biochem. Biophys. Res. Commun. 43, 20 (1971). 12A. W. Steggles, T. C. Spelsberg, S. R. Glasser, and B. W. O'Malley, Proc. Nat. Aead. Sci. U.S. 68, 1479 (1971). 1~T. C. Spelsberg, A. W. Steggles, F. Chytil, and B. W. O'Malley, J. Biog. Chem. 247, 1368 (1972). 14W. T. Schrader and B. W. O'Malley, J. Biol. Chem. 247, 51 (1972). ~sW. T. Sehrader, D. O. Toft, and B. W. O'Malley, J. Biol. Chem. 247, 2401 (1972). ~eW. T. Schrader, this Vol. [17]. 1TS. G. Korenman, Endocrinology 87, 1119 (1970). is G. A. Puca, E. Nola, V. Sica, and F. Bresciani, Biochemistry 10, 3769 (1971). ~9W. I. P. Mainwaring and F. R. Mangan, Advan. B/osc/. 7 (1971). 20R. E. Buller and D. O. Toft, J. Biog. Chem. (in press). = K. R. Yamamoto and B. M. Alberts, Proc. Nat. Acad. Sci. U.S. 69, 2105 (1972). = D. O. Toft, in "Receptors for Reproductive Hormones" (B. W. O'Malley and A. R. Means, eds.), p. 85. Plenum, New York: 1973. = J. D. Baxter, D. G. Rousseau, N. C. Benson, R. L. Garcea, J. Ito, and G. M. Tomkins, Proc. Nat. Acad. Sei. U.$. 69, 1892 (1972).
[17]
CHICK OVIDUCT PROGESTERONE RECEPTOR
189
subcutaneously for 7-21 days with 5 mg per day of diethylstilbestrol in sesame oil. The oviducts grow to 1-2 g each and contain no progesterone. There is no indication that the diethylstilbestrol competes for and masks any progesterone receptor sites. Mature hens can also be used; a typical laying hen oviduct weighing 20-40 g provides a large amount of receptors. However, due to the presence of high titers of endogenous progesterone the concentration of free receptor sites is normally only 10-25% that of the estrogenized immature oviduct. Attempts to free the progesterone sites by treatment of extracts with dextran-charcoal have met with mixed success at best. Hens which have recently stopped laying eggs undergo rapid regression of the oviduct, accompanied by rapid shut-off of progesterone secretion. Thus, recently laying hens having oviducts weighing 5-10 g frequently have high titers of free receptor sites. It must be pointed out, however, that the difficulty of working with hens makes the chick oviduct the tissue of choice. Receptor preparations are best made from fresh tissue, rather than from stored or frozen sources since the frozen materials yield receptor preparations which tend to be highly aggregated. The progesterone-binding capacity of the tissues is not markedly decreased by freezing, however. No attempt has been made in our laboratory to preserve tissue in liquid nitrogen, nor to extract powdered oviducts. Progesterone receptors are present in equal titers along the length of the oviduct, including the shell gland. In normal practice the oviduct is cut off anteriorly where it constricts and posteriorly at the broadening, marking the transition to shell gland. It is assumed that the receptors are distributed throughout the oviduct, although to our knowledge no study has been published to measure receptor titers in epithelial vs. stromal or glandular elements.
IIl. Preparation of Crude Cytoplasmic Receptors A. Buffers
The standard buffer used is 10 mM Tris-HCl, pH 7.4, containing 1 mM Na2EDTA and 12 mM 1-thioglycerol (Buffer A). This is supplemented with various amounts of KC1 as indicated below. Receptor preparations have also been made using 10 mM phosphate buffer with no differences in properties. Thioglycerol is added to stabilize one receptor form obtained at the DEAE-cellulose step 14 (see below), fl-Mercaptoethanol can be used equivalently, but glycerol alone (0-30%) is not effective in stabilizing the DEAE eluates.
190
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
[171
B. Tissue Removal and Homogenization Oviducts are cut from chicks freshly killed by cervical dislocation and are obtained by a ventral incision. The process takes less than a minute for each chick. The oviducts should be clipped free of mesentery, blotted, and placed immediately in ice-cold 0.9% NaCl saline solution. The tissue is then blotted, weighed, minced in the cold room, and homogenized using a Polytron (Brinkmann Instruments, PT-10). It is important to maintain the solution at 0 °. This is best done by homogenizing always in ice-water slush rather than crushed ice alone, and by using bursts (5-10 seconds) of the Polytron with cooling between bursts. For preparative studies a few strokes of a large Teflon-glass homogenizer run at low speed in an ice-water bath will improve the yield. The crude homogenate is then centrifuged in a swinging-bucket rotor (Sorvall HB-4) for 10 minutes at 10,000 rpm. The layer of floating lipid is drawn off and the crude low-speed cytoplasmic material decanted. The crude nuclear pellet is stored frozen for preparation of DNA.
C. Preparation of Cytoplasmic Soluble Fraction (Cytosol) The low-speed supernatant fraction is then centrifuged at 150,000 g for I hour in a Spinco swinging-bucket rotor (SW-50.I, SW-27, or SW-40 depending on volume) to prepare the cytosol. Another small fat plug is found after this centrifugation and is carefully drawn off in the cold room by aspiration. The cytosol prepared in this manner is largely free of lipid material and gives much cleaner preparations later on. It has been found useful to avoid angle rotors at all times, even for large batches because of the cleanliness of the resultant solutions. D. Labeling of Cytoplasmic Extracts with Radioactive Steroids One milliliter of cytosol prepared as described above will contain about 20 mg of protein and will contain about 10 nmoles of progesterone receptor sites. This is about 75 ng of steroid bound at saturation, or 2 X 10~ dpm of [3H]progesterone at 50 Ci/mmole. To saturate these preparations of cytosol with progesterone it is usually convenient to add about a 10-fold excess of [3H]progesterone. This steroid is soluble at about 5/~g/ml and is generally sold as a benzene solution at about 5 /~g/ml. To prepare a suitable stock solution, the [3H]progesterone is evaporated under N.,, redissolved in 100% ethanol, diluted 1:10 with Buffer A, and stored frozen. One milliliter of cytosol is then labeled by adding 10/~l of diluted [~H]progesterone. At 0 ° com-
[17]
CHICK OVIDUCT P R O G E S T E R O N E R E C E P T O R
191
plete equilibrium requires about 12-18 hours. However, for preparative studies, labeling times of 2-6 hours are usually sufficient. It is also possible to label the preparations by adding [~H]progesterone to the low-speed supernatant fraction before preparation of the cytosol. However, substantial metabolism of steroids can occur in these perticulate preparations even at 0 °. Therefore, labeling of cytosol itself is routinely used. Under these conditions, about 90% of the input steroid is still authentic progesterone after 6 hours. Progesterone complexed to receptor is even more stable; there is essentially no metabolism of the hormone in purified preparations when the receptor-bound material is assayed.
E. Stability of Cytosol Receptors 1. Time Cytosol receptor preparations generally can be stored at 0 ¢ for about 24 hours before the receptor properties dramatically deteriorate. The state of aggregation of the receptor is affected by storage, and sucrose gradient analysis of aged material shows no loss of label from binding sites, but progressively more appearance of receptor at the bottom of the gradient. Gel filtration on Sephadex G-75 also shows no increase in free hormone but a loss of label in the bound fractions, indicative of conversion of the receptor to an altered state which adsorbs to the gel or to the apparatus. Small amounts of detergents (Triton X-100 or deoxycholate, 0.1%) have been tried to prevent this aggregation without success. The aggregation phenomenon is time-dependent. Cytosol has been frozen and thawed repeatedly on the same day with no loss of activity or aggregation, whereas storage in the frozen state caused aggregation within 5 days. The frozen cytosol can be used for steroid-binding competition studies even in the aggregated state. Apparently there is no deterioration of the steroid-binding sites themselves.
2. Temperature In general, elevated temperature, particularly in the absence of bound steroid, promotes denaturation of progesterone receptors. Bound progesterone labeled to saturation has a half-life as receptor complex of many days at 0 °, 6 hours at 23 °, and only about 30 minutes at 37 °. The process is irreversible at the higher temperatures, as evidenced by the failure of hormone to re-form a complex during subsequent storage at 0 °. This denaturation is not merely protease activity, however, since the process has also been observed in highly purified preparations.
192
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
[17]
A most useful storage method has been simply to lyophilize the labeled receptor preparations. If known volumes are freeze-dried, they can be stored for months and redissolved by adding cold H20. Further purification of material stored in this way has shown the receptors to be intact after lyophilization; they have not been tested in the crude state for uptake by nuclei.
3. pH The progesterone-receptor complexes show a marked pH optimum for stability. They are stable over a broad pH range from pH 7-9, but over half the binding activity is destroyed at pH 6. This is found whether intact progesterone-receptor complexes are lowered at pH 6, or whether nascent receptors are treated at this pH and then assayed for binding activity. The process is also essentially irreversible, since aliquots treated at pH 6 and then incubated at pH 7.4 do not reform the steroid-receptor complex. Finally, if cytosol is brought to pH 4.5, a precipitate forms consisting of ovomucoids. Assay of the precipitate showed no receptor activity in the sedimented material. It should be pointed out that no attempts have been made to raise pH gradually, such as by dialysis which might allow some of the complexes to re-form.
4. Salt and Urea High concentrations of either KC1 or urea cause dissociation of progesterone-receptor complexes. Exposure to 2.0 M KC1 for 1-2 hours causes a 50% loss of bound progesterone. This is apparently not an irreversible effect since progesterone-receptor complexes precipitated at 50% saturation of ammonium sulfate (2.0 M) remain complexed during brief exposure to the salt. Low concentrations of KC1 (0.15, 0.3 M) are favorable solutes to include in receptor preparations since receptors are maintained in a disaggregated state above about 0.15 M KC1. The relationship between receptor aggregation and the concentration of divalent cations such as Ca ~÷ remains to be established. Stabilization of slowly sedimenting receptor forms by the addition of 4 mM CaC12 has been found to occur with the oviduct progesterone receptor as with other receptors. But these calcium effects have not been correlated with the isolation of the receptor forms reported here. Urea also dissociates the progesterone-receptor complexes in a reversible process. Exposure of labeled eytosol to 1.0 M urea for 6 hours had no effect on the concentration of bound hormone. However, a 50% drop in binding (as determined by chromatography on Sephadex G-75) oc-
[17l
CHICK OVIDUCT P R O G E S T E R O N E R E C E P T O R
193
curred at 2.0 M urea. If the preparation was diluted 10-fold, the [3H]progesterone did not rebind to the receptors. However, if cytosol treated overnight at 3.0 M urea was dialyzed to remove the urea, the dialyzed receptor preparation could rebind about 50% of the original concentration of progesterone. Thus the urea denaturant appears to be similar to that observed for other proteins; gradual removal of the agent causes at least some recovery of activity. F. Characteristics of the Cytosol Receptors Progesterone receptors prepared in this way sediment on 5-20% sucrose gradients in Buffer A as a 6-8 species showing a broad peak and considerable aggregated material at the tube bottom. If the gradients are run in 0.15 M KC1 or higher, a sharper peak at about 4 S is seen. This pattern can also be observed if the eytosol is sedimented without added hormone, and each fraction is subsequently assayed for binding sites. Thus the 4 S-8 S relationship is not progesterone-dependent. Furthermore, the pattern can be seen in either unprimed or estrogenized oviduct extracts, and thus is not directly estrogen-dependent either. The receptors are acidic proteins, having isoeleetric points of between pH 4 and pH 4.5. At neutral pH they have an equilibrium constant for progesterone binding of 1 X 10-~° M and a half-life of 12 hours under pseudo first-order conditions. 14 The binding constants are summarized in Table I. IV. Binding-Site Measurements A. Assays for Bound Hormone
1. Ammonium Sulfate Precipitation If a [3H]progesterone-labeled receptor solution is brought to 50% saturation in ammonium sulfate, the receptor-hormone complexes precipitate and can be collected by centrifugation. This method is convenient because the reagent is soluble rather than particulate (as in the charcoal precipitation method) and because both the pellet (receptor-hormone complex) fraction and supernatant (free hormone) fraction can be counted easily for radioactivity. The method is gentle and can be used with purified receptor preparations. In contrast, the charcoal technique seems to dissociate the complexes or adsorb receptor molecules, especially when used to assay purified preparations. It is advisable to use polypropylene tubes for steroid receptor assays since the receptors have a tendency to adhere to glass, especially when
194
[17]
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
TABLE I APPARENT KINETIC CONSTANTS FOR OVIDUCT P~ECEPToR--PRoGESTERONE INTERACTION a
Receptor fraction
Binding site eoneentration b (nM)
Crude cytosol Component A Component B
4.6 0.7 0.8
Association Dissociation rate rate constant c constant d 10-5 X ka 10~ X k~ (M -1 sec-1) (sec-1) 4.0 2.8 6.3
1.9 1.9 2.4
Equilibrium constant e 1011 X kd/k~ (M)
Equilibrium constantf 109 × k~ (M)
4.8 7.0 3.8
4.4 ---
From Schrader and O'Malley. TM b Determined from saturation concentration of receptor-bound 3H. Second-order rate constant obtained from slopes of plots in Fig. 5A. 14 First-order constant obtained from slopes of plots in Fig. 5B. 14 Calculated as ratio of rate constants for the dissociation reaction RH* --~ R -f- H*. / Calculated from Scatchard plot slope. purified. This holds true for D N A and chromatin-binding assays as well. Tubes 12 X 75 mm (Falcon Plastics ~2053) are suitable. The protocol used for the ammonium sulfate assay is as follows: 10-100 ~l of a receptor preparation is diluted in 0.5 ml with buffer and precipitated by rapid addition of an equal volume of 100% saturated ammonium sulfate in Buffer A. After 30 minutes the tube is centrifuged at 2000 g for 15 minutes. The supernatant fraction is decanted, and an aliquot counted for ~H to determine free steroid in the preparation. The tube is washed with 2 ml of 50% saturated ammonium sulfate in Buffer A, recentrifuged, and the supernatant fraction discarded. The small pellet is dissolved in 700 ~l H20 and counted for aH to determine the receptor-hormone complex concentration in the preparation. 2. Charcoal M i x e d w i t h D e x t r a n
Charcoal mixed with dextran has been used extensively for steroid receptor determinations. This method is described in detail elsewhere. ~7,24 I t is rapid and precise when used to assay crude cytosol receptor levels or when used for steroid competition studies. However, during assays of purified preparations even brief exposure to charcoal causes loss of bound hormone and a concomitant loss of receptor sites in solution. The addition of egg-white protein or bovine serum albumin does not reverse this deleterious effect. The method is thus best used with great caution for assay of purified materials. 2, S. G. Korenman, this Vol. [4].
[17]
CHICK OVIDUCT PROGESTERONE RECEPTOR
195
3. Sucrose-Gradient Analysis
Sucrose-gradient analysis can be used to determine the binding of hormones to receptors. Due to the time required (9-16 hours) and the fact that it is under nonequilibrium conditions the technique undoubtedly gives estimates of receptor-bound hormone which are too low. Other drawbacks include poor (
CHICK OVIDUCT PROGESTERONE RECEPTOR
187
any form can be deliberately produced by appropriate manipulation.The interrelationships of these forms and their physiological significance are not yet understood.
Acknowledgments This work was supported by The National Cancer Institute, The American Cancer Society, and The Robert A. Welch Foundation.
[17] Methods for Extraction and Quantification of Receptors B y WILLIAM T. SCHRADER I. I n t r o d u c t i o n
The chick oviduct has been used as a model system for the study of progesterone action in target cells. 1-3 In this tissue, estrogen administration induces growth and differentiation; the estrogen-primed oviduct cells then respond to progesterone by synthesizing the egg protein avidin. 4,~ Studies on the mechanism of this progesterone response have shown that the hormone is first bound to intracellular macromolecules ~,7 and is subsequently transported to the nuclear compartment s,9 where the hormone-macromolecular complex becomes tightly associated with the 1B. W. 0'Malley, Biochemistry 6, 2546 (1967). 2 B. W. O'Malley, W. L. McGuire, P. O. Kohler, and S. G. Korenman, Recent Progr. Horm. Res. 25, 105 (1969). ' B. W. O'Malley, W. T. Schrader, and T. C. Spelsberg, in "Receptors for Reproductive Hormones" (B. W. O'Malley and A. R. Means, eds.), p. 174. Plenum, New York, 1973. 4 B. W. O'Malley and P. O. Kohler, Proc. Nat. Acad. Sci. U.S. 58, 2359 (1967). B. W. O'Malley, G. C. Rosenfeld, J. P. Comstock, and A. R. Means, Nature (London), New Biology 240, 45 (1972). M. R. Sherman, P. L. Corvol, and B. W. O'Malley, J. Biol. Chem. 245, 6085 (1970). 7B. W. O'Malley, M. R. Sherman, D. O. Toft, T. C. Spelsberg, W. T. Schrader, and A. W. Steggles, Advan. Biosci. 7, 213 (1971). 8B. W. O'Malley, D. O. Toft, and M. R. Sherman, J. Biol. Chem. 246, 1117 (1971). 9B. W. O'Malley, M. R. Sherman, and D. O. Toft, Proc. Nat. Acad. Sci. U.S. 67, 501 (1970).
188
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
[17]
cell chromatin. 1°-13 These studies have been facilitated by use of partially purified progesterone receptors from the oviduct prepared as described below.14, ~5 I t is i m p o r t a n t to keep in mind t h a t there is as yet no true end-point assay for the functional form of a steroid receptor protein in any tissue. At least five p a r a m e t e r s have been defined for the progesterone receptor system which m a y serve as substitutes for such an end-point assay. ~6 These p a r a m e t e r s should be monitored throughout all manipulations, in order to avoid working with materials which are so altered t h a t they do not represent the starting material. The p a r a m e t e r s used in this labor a t o r y are (1) steroid-binding kinetics and specificity, ~7 (2) molecular size and charge, 6,1s,19 (3) uptake and retention by nuclei, s,9,~° (4) binding to D N A , z~-~3 and (5) binding to chromatin. Whenever a manipulation results in a deviation in one of these parameters, it should serve to prejudice the investigation toward defining the nature of the changes. No step should be adopted into the protocol which results in the isolation of an altered receptor form without a clear realization t h a t the product m a y be biologically inactive. II. Tissue Sources
Progesterone receptors can be isolated and identified from chick oviduct of any age or hormonal history. I m m a t u r e oviducts of average weights of about 10 mg can be pooled for extraction of receptors. I t is more convenient, however, to work with immature female chicks injected ~oT. C. Spelsberg, A. W. Steggles, and B. W. O'Malley, J. Biol. Chem. 246, 4188 (1971). 11A. W. Steggles, T. C. Spelsberg, and B. W. O'Malley, Biochem. Biophys. Res. Commun. 43, 20 (1971). 12A. W. Steggles, T. C. Spelsberg, S. R. Glasser, and B. W. O'Malley, Proc. Nat. Aead. Sci. U.S. 68, 1479 (1971). 1~T. C. Spelsberg, A. W. Steggles, F. Chytil, and B. W. O'Malley, J. Biog. Chem. 247, 1368 (1972). 14W. T. Schrader and B. W. O'Malley, J. Biol. Chem. 247, 51 (1972). ~sW. T. Sehrader, D. O. Toft, and B. W. O'Malley, J. Biol. Chem. 247, 2401 (1972). ~eW. T. Schrader, this Vol. [17]. 1TS. G. Korenman, Endocrinology 87, 1119 (1970). is G. A. Puca, E. Nola, V. Sica, and F. Bresciani, Biochemistry 10, 3769 (1971). ~9W. I. P. Mainwaring and F. R. Mangan, Advan. B/osc/. 7 (1971). 20R. E. Buller and D. O. Toft, J. Biog. Chem. (in press). = K. R. Yamamoto and B. M. Alberts, Proc. Nat. Acad. Sci. U.S. 69, 2105 (1972). = D. O. Toft, in "Receptors for Reproductive Hormones" (B. W. O'Malley and A. R. Means, eds.), p. 85. Plenum, New York: 1973. = J. D. Baxter, D. G. Rousseau, N. C. Benson, R. L. Garcea, J. Ito, and G. M. Tomkins, Proc. Nat. Acad. Sei. U.$. 69, 1892 (1972).
[17]
CHICK OVIDUCT PROGESTERONE RECEPTOR
189
subcutaneously for 7-21 days with 5 mg per day of diethylstilbestrol in sesame oil. The oviducts grow to 1-2 g each and contain no progesterone. There is no indication that the diethylstilbestrol competes for and masks any progesterone receptor sites. Mature hens can also be used; a typical laying hen oviduct weighing 20-40 g provides a large amount of receptors. However, due to the presence of high titers of endogenous progesterone the concentration of free receptor sites is normally only 10-25% that of the estrogenized immature oviduct. Attempts to free the progesterone sites by treatment of extracts with dextran-charcoal have met with mixed success at best. Hens which have recently stopped laying eggs undergo rapid regression of the oviduct, accompanied by rapid shut-off of progesterone secretion. Thus, recently laying hens having oviducts weighing 5-10 g frequently have high titers of free receptor sites. It must be pointed out, however, that the difficulty of working with hens makes the chick oviduct the tissue of choice. Receptor preparations are best made from fresh tissue, rather than from stored or frozen sources since the frozen materials yield receptor preparations which tend to be highly aggregated. The progesterone-binding capacity of the tissues is not markedly decreased by freezing, however. No attempt has been made in our laboratory to preserve tissue in liquid nitrogen, nor to extract powdered oviducts. Progesterone receptors are present in equal titers along the length of the oviduct, including the shell gland. In normal practice the oviduct is cut off anteriorly where it constricts and posteriorly at the broadening, marking the transition to shell gland. It is assumed that the receptors are distributed throughout the oviduct, although to our knowledge no study has been published to measure receptor titers in epithelial vs. stromal or glandular elements.
IIl. Preparation of Crude Cytoplasmic Receptors A. Buffers
The standard buffer used is 10 mM Tris-HCl, pH 7.4, containing 1 mM Na2EDTA and 12 mM 1-thioglycerol (Buffer A). This is supplemented with various amounts of KC1 as indicated below. Receptor preparations have also been made using 10 mM phosphate buffer with no differences in properties. Thioglycerol is added to stabilize one receptor form obtained at the DEAE-cellulose step 14 (see below), fl-Mercaptoethanol can be used equivalently, but glycerol alone (0-30%) is not effective in stabilizing the DEAE eluates.
190
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
[171
B. Tissue Removal and Homogenization Oviducts are cut from chicks freshly killed by cervical dislocation and are obtained by a ventral incision. The process takes less than a minute for each chick. The oviducts should be clipped free of mesentery, blotted, and placed immediately in ice-cold 0.9% NaCl saline solution. The tissue is then blotted, weighed, minced in the cold room, and homogenized using a Polytron (Brinkmann Instruments, PT-10). It is important to maintain the solution at 0 °. This is best done by homogenizing always in ice-water slush rather than crushed ice alone, and by using bursts (5-10 seconds) of the Polytron with cooling between bursts. For preparative studies a few strokes of a large Teflon-glass homogenizer run at low speed in an ice-water bath will improve the yield. The crude homogenate is then centrifuged in a swinging-bucket rotor (Sorvall HB-4) for 10 minutes at 10,000 rpm. The layer of floating lipid is drawn off and the crude low-speed cytoplasmic material decanted. The crude nuclear pellet is stored frozen for preparation of DNA.
C. Preparation of Cytoplasmic Soluble Fraction (Cytosol) The low-speed supernatant fraction is then centrifuged at 150,000 g for I hour in a Spinco swinging-bucket rotor (SW-50.I, SW-27, or SW-40 depending on volume) to prepare the cytosol. Another small fat plug is found after this centrifugation and is carefully drawn off in the cold room by aspiration. The cytosol prepared in this manner is largely free of lipid material and gives much cleaner preparations later on. It has been found useful to avoid angle rotors at all times, even for large batches because of the cleanliness of the resultant solutions. D. Labeling of Cytoplasmic Extracts with Radioactive Steroids One milliliter of cytosol prepared as described above will contain about 20 mg of protein and will contain about 10 nmoles of progesterone receptor sites. This is about 75 ng of steroid bound at saturation, or 2 X 10~ dpm of [3H]progesterone at 50 Ci/mmole. To saturate these preparations of cytosol with progesterone it is usually convenient to add about a 10-fold excess of [3H]progesterone. This steroid is soluble at about 5/~g/ml and is generally sold as a benzene solution at about 5 /~g/ml. To prepare a suitable stock solution, the [3H]progesterone is evaporated under N.,, redissolved in 100% ethanol, diluted 1:10 with Buffer A, and stored frozen. One milliliter of cytosol is then labeled by adding 10/~l of diluted [~H]progesterone. At 0 ° com-
[17]
CHICK OVIDUCT P R O G E S T E R O N E R E C E P T O R
191
plete equilibrium requires about 12-18 hours. However, for preparative studies, labeling times of 2-6 hours are usually sufficient. It is also possible to label the preparations by adding [~H]progesterone to the low-speed supernatant fraction before preparation of the cytosol. However, substantial metabolism of steroids can occur in these perticulate preparations even at 0 °. Therefore, labeling of cytosol itself is routinely used. Under these conditions, about 90% of the input steroid is still authentic progesterone after 6 hours. Progesterone complexed to receptor is even more stable; there is essentially no metabolism of the hormone in purified preparations when the receptor-bound material is assayed.
E. Stability of Cytosol Receptors 1. Time Cytosol receptor preparations generally can be stored at 0 ¢ for about 24 hours before the receptor properties dramatically deteriorate. The state of aggregation of the receptor is affected by storage, and sucrose gradient analysis of aged material shows no loss of label from binding sites, but progressively more appearance of receptor at the bottom of the gradient. Gel filtration on Sephadex G-75 also shows no increase in free hormone but a loss of label in the bound fractions, indicative of conversion of the receptor to an altered state which adsorbs to the gel or to the apparatus. Small amounts of detergents (Triton X-100 or deoxycholate, 0.1%) have been tried to prevent this aggregation without success. The aggregation phenomenon is time-dependent. Cytosol has been frozen and thawed repeatedly on the same day with no loss of activity or aggregation, whereas storage in the frozen state caused aggregation within 5 days. The frozen cytosol can be used for steroid-binding competition studies even in the aggregated state. Apparently there is no deterioration of the steroid-binding sites themselves.
2. Temperature In general, elevated temperature, particularly in the absence of bound steroid, promotes denaturation of progesterone receptors. Bound progesterone labeled to saturation has a half-life as receptor complex of many days at 0 °, 6 hours at 23 °, and only about 30 minutes at 37 °. The process is irreversible at the higher temperatures, as evidenced by the failure of hormone to re-form a complex during subsequent storage at 0 °. This denaturation is not merely protease activity, however, since the process has also been observed in highly purified preparations.
192
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
[17]
A most useful storage method has been simply to lyophilize the labeled receptor preparations. If known volumes are freeze-dried, they can be stored for months and redissolved by adding cold H20. Further purification of material stored in this way has shown the receptors to be intact after lyophilization; they have not been tested in the crude state for uptake by nuclei.
3. pH The progesterone-receptor complexes show a marked pH optimum for stability. They are stable over a broad pH range from pH 7-9, but over half the binding activity is destroyed at pH 6. This is found whether intact progesterone-receptor complexes are lowered at pH 6, or whether nascent receptors are treated at this pH and then assayed for binding activity. The process is also essentially irreversible, since aliquots treated at pH 6 and then incubated at pH 7.4 do not reform the steroid-receptor complex. Finally, if cytosol is brought to pH 4.5, a precipitate forms consisting of ovomucoids. Assay of the precipitate showed no receptor activity in the sedimented material. It should be pointed out that no attempts have been made to raise pH gradually, such as by dialysis which might allow some of the complexes to re-form.
4. Salt and Urea High concentrations of either KC1 or urea cause dissociation of progesterone-receptor complexes. Exposure to 2.0 M KC1 for 1-2 hours causes a 50% loss of bound progesterone. This is apparently not an irreversible effect since progesterone-receptor complexes precipitated at 50% saturation of ammonium sulfate (2.0 M) remain complexed during brief exposure to the salt. Low concentrations of KC1 (0.15, 0.3 M) are favorable solutes to include in receptor preparations since receptors are maintained in a disaggregated state above about 0.15 M KC1. The relationship between receptor aggregation and the concentration of divalent cations such as Ca ~÷ remains to be established. Stabilization of slowly sedimenting receptor forms by the addition of 4 mM CaC12 has been found to occur with the oviduct progesterone receptor as with other receptors. But these calcium effects have not been correlated with the isolation of the receptor forms reported here. Urea also dissociates the progesterone-receptor complexes in a reversible process. Exposure of labeled eytosol to 1.0 M urea for 6 hours had no effect on the concentration of bound hormone. However, a 50% drop in binding (as determined by chromatography on Sephadex G-75) oc-
[17l
CHICK OVIDUCT P R O G E S T E R O N E R E C E P T O R
193
curred at 2.0 M urea. If the preparation was diluted 10-fold, the [3H]progesterone did not rebind to the receptors. However, if cytosol treated overnight at 3.0 M urea was dialyzed to remove the urea, the dialyzed receptor preparation could rebind about 50% of the original concentration of progesterone. Thus the urea denaturant appears to be similar to that observed for other proteins; gradual removal of the agent causes at least some recovery of activity. F. Characteristics of the Cytosol Receptors Progesterone receptors prepared in this way sediment on 5-20% sucrose gradients in Buffer A as a 6-8 species showing a broad peak and considerable aggregated material at the tube bottom. If the gradients are run in 0.15 M KC1 or higher, a sharper peak at about 4 S is seen. This pattern can also be observed if the eytosol is sedimented without added hormone, and each fraction is subsequently assayed for binding sites. Thus the 4 S-8 S relationship is not progesterone-dependent. Furthermore, the pattern can be seen in either unprimed or estrogenized oviduct extracts, and thus is not directly estrogen-dependent either. The receptors are acidic proteins, having isoeleetric points of between pH 4 and pH 4.5. At neutral pH they have an equilibrium constant for progesterone binding of 1 X 10-~° M and a half-life of 12 hours under pseudo first-order conditions. 14 The binding constants are summarized in Table I. IV. Binding-Site Measurements A. Assays for Bound Hormone
1. Ammonium Sulfate Precipitation If a [3H]progesterone-labeled receptor solution is brought to 50% saturation in ammonium sulfate, the receptor-hormone complexes precipitate and can be collected by centrifugation. This method is convenient because the reagent is soluble rather than particulate (as in the charcoal precipitation method) and because both the pellet (receptor-hormone complex) fraction and supernatant (free hormone) fraction can be counted easily for radioactivity. The method is gentle and can be used with purified receptor preparations. In contrast, the charcoal technique seems to dissociate the complexes or adsorb receptor molecules, especially when used to assay purified preparations. It is advisable to use polypropylene tubes for steroid receptor assays since the receptors have a tendency to adhere to glass, especially when
194
[17]
CYTOPLASMIC RECEPTORS FOR STEROID HORMONES
TABLE I APPARENT KINETIC CONSTANTS FOR OVIDUCT P~ECEPToR--PRoGESTERONE INTERACTION a
Receptor fraction
Binding site eoneentration b (nM)
Crude cytosol Component A Component B
4.6 0.7 0.8
Association Dissociation rate rate constant c constant d 10-5 X ka 10~ X k~ (M -1 sec-1) (sec-1) 4.0 2.8 6.3
1.9 1.9 2.4
Equilibrium constant e 1011 X kd/k~ (M)
Equilibrium constantf 109 × k~ (M)
4.8 7.0 3.8
4.4 ---
From Schrader and O'Malley. TM b Determined from saturation concentration of receptor-bound 3H. Second-order rate constant obtained from slopes of plots in Fig. 5A. 14 First-order constant obtained from slopes of plots in Fig. 5B. 14 Calculated as ratio of rate constants for the dissociation reaction RH* --~ R -f- H*. / Calculated from Scatchard plot slope. purified. This holds true for D N A and chromatin-binding assays as well. Tubes 12 X 75 mm (Falcon Plastics ~2053) are suitable. The protocol used for the ammonium sulfate assay is as follows: 10-100 ~l of a receptor preparation is diluted in 0.5 ml with buffer and precipitated by rapid addition of an equal volume of 100% saturated ammonium sulfate in Buffer A. After 30 minutes the tube is centrifuged at 2000 g for 15 minutes. The supernatant fraction is decanted, and an aliquot counted for ~H to determine free steroid in the preparation. The tube is washed with 2 ml of 50% saturated ammonium sulfate in Buffer A, recentrifuged, and the supernatant fraction discarded. The small pellet is dissolved in 700 ~l H20 and counted for aH to determine the receptor-hormone complex concentration in the preparation. 2. Charcoal M i x e d w i t h D e x t r a n
Charcoal mixed with dextran has been used extensively for steroid receptor determinations. This method is described in detail elsewhere. ~7,24 I t is rapid and precise when used to assay crude cytosol receptor levels or when used for steroid competition studies. However, during assays of purified preparations even brief exposure to charcoal causes loss of bound hormone and a concomitant loss of receptor sites in solution. The addition of egg-white protein or bovine serum albumin does not reverse this deleterious effect. The method is thus best used with great caution for assay of purified materials. 2, S. G. Korenman, this Vol. [4].
[17]
CHICK OVIDUCT PROGESTERONE RECEPTOR
195
3. Sucrose-Gradient Analysis
Sucrose-gradient analysis can be used to determine the binding of hormones to receptors. Due to the time required (9-16 hours) and the fact that it is under nonequilibrium conditions the technique undoubtedly gives estimates of receptor-bound hormone which are too low. Other drawbacks include poor (
