Progesterone Receptor from Chick Oviduct:. Purification of Molybdate-Stabilized Form and Preliminary Characterization

Abstract

A molydate-stabilized, 'non-activated' form of the progesterone receptor from the cytosol of oestrogenstimulated chick oviduct has been purified to homogeneity by a three-step procedure. The first step, affinity chromatography using a N-(l2-amino-dodecyl)-3-oxo-4-androsten-17~-carboxamide-substituted Sepharose gel, purified the receptor 1500 -2700-fold with z 50 % recovery. In the second step, ion-exchange chromatography through a DEAE-cellulose column, progesterone receptor was eluted as a single peak at 0.1 M KCI. Purification after this step was >6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification z 7400-fold with overall recovery z 25 of pure receptor on the basis of 1 binding site/molecule of M, 85000. The purified molybdate-stabilized receptor had a sedimentation coefficient = 7.9 S & 0.1 ( n = 4) in 0.15 M or 0.4 M KCI containing sucrose 5-20% gradient and z 8.9 S k 0.2 (n = 6) in 0.15 M KCI containing glycerol 10-35 "/, gradient, and its Stokes radius was 7.05 k 0.10 nm (n = 3) (calculated M , between 240000 and 280000). Binding specificity of the purified receptor was the same as that found in crude cytosol. SDS-PAGE revealed a single band migrating as a polypeptide of MI z 85000 -+ 2300 (n = 9). PAGE under nondenaturing conditions at total acrylamide concentrations 5 "/,, 7 % and 9 "/, showed a single [3H]ORG 2058-protein band (ORG 2058 is a high-affinity analogue more suitable than progesterone for electrophoretic studies). The data suggest that the high molecular weight molybdate-stabilized progesterone receptor purified from oestrogen-primed chick oviduct is composed of only = 85000-M, polypeptide chains. [5] have recently described a 'non-activated' or 'native' form of the progesterone receptor from chick oviduct cytosol, stabilized and prepared in the presence of sodium molybdate. This form of the receptor sedimented at 8-9 S on sucrose gradients containing 0.15 M KCI, and was eluted from DEAE-cellulose as a single peak at 0.15 M KCI 151. Its Stokes radius R, was z 7.9 nm and data permitted calculation of M , z 290000. These studies prompted us to purify the molybdate-stabilized receptor using an appropriate biospecific affinity material. The chick oviduct progesterone receptor has been extensively studied by Schrader, O'Malley and coworkers during the past decade (for a review, see [6]), and two non-identical binding components have been characterized and separately purified from the cytosol of hen or of oestrogen-primed chick oviduct. These components were reported to be polypeptides of M, z 79000 ('subunit A') and "N I10000 ('subunit B'), respectively. They are readily separated by ion-exchange chromatography. In contrast, using crude cytosol, Murayama et al. [7] found MI = 84000 for a 'native' unit and Miller et al.[8] M , = 87000 for a 'fast' component of progesterone receptor from chick oviduct.We have developed a method to purify the molybdatestabilized form of the progesterone receptor. This involves a three-step procedure including a new aff...

Intracellular glucocorticoid receptors in spleen, but not skin, vary seasonally in wild house sparrows (Passer domesticus)

“…After subtracting out non-specific binding, MR binding can be calculated directly from test tubes containing RU486; GR binding can be calculated by subtracting MR binding from total binding. Although RU486 also binds with high affinity to progesterone receptors (PR), CORT only binds avian PR at very high concentrations (about 1 mM [39]). Based on affinity estimates derived from previous equilibrium saturation analyses [14], mass action predicts that 10 nM [ 3 H]CORT should occupy more than 95% of MR and approximately 63% of GR.…”

Antibodies against Progesterone Receptor from Chick Oviduct. Cross-Reactivity with Mammalian Progesterone Receptors

“…When purified, molybdate-stabilized 8 -9-S receptor is stored at 0-4 "C for several days, partial transformation is observed, as indicated by the appearance of 4-S-sedimenting form [14]. The mixture of 8 -9-S and 4-S forms of the receptor thus obtained was incubated with IgG-G3 and IgG-R3 (not shown).…”

“…After labelling with 20 nM [3H]progesterone (2 at 0 "C) and removal of free steroid by charcoal treatment [14], 0.5-ml aliquots were frozen, and thawed just before incubation with antibodies. Specific binding activity was 1.5 pmol progestin/mg cytosol protein (0.13 pmol/ 5 PI).…”

“…Aliquots of 0.2 ml of incubation mixtures were layered on top of preformed 4.2-ml gradients and centrifuged 15 h at 0-4"C, in a L8-80 ultracentrifuge (Beckman). Horseradish peroxidase (3.6 S), fungal glucose oxidase (7.9 S), and catalase from bovine liver (11.3 S) (references in [14]) were used as internal standards in each tube. After centrifugation, two-drop fractions were collected, adjusted to 350p1 with water, and enzymes were assayed and radioactivity measured with counting efficiency = 50 %.…”

Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.

“…The transformed progesterone receptor was purified from hen oviducts by a procedure similar to that of Chang et al (21), to be described in detail elsewhere. The final steps in the procedure were affinity chromatography on N-(12-aminododecyl)-3-keto-4-androsten-17,3-carboxamide-Sepharose (22) Laemmli (26 (14).…”