Characterization of the progesterone receptor of rabbit uterus with the synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione

Fleischmann, Gerhard; Beato, Miguel

doi:10.1016/0304-4165(78)90180-0

Cited by 30 publications

(3 citation statements)

References 20 publications

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“…In the presence of 0.3 M KC1, a single 3.5 S receptor was observed on sucrose gradient analysis, irrespective of the presence of sodium molybdate. A similar salt-dependent reduction in the sedimentation coefficient was observed for rabbit uterine progesterone receptor by Fleischmann & Beato (1978). We have further analyzed molybdate-dependent increases in cRp size by gel filtration chromatography.…”

Section: Discussionsupporting

confidence: 69%

Uterine progesterone receptor: stabilization and physicochemical alterations produced by sodium molybdate

Chen¹,

MacDonald²,

Leavitt³

1981

Biochemistry

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Incubation of hamster uterine cytosol with millimolar concentrations of sodium molybdate prior to addition of labeled steroid increased recovery of progesterone receptor 2-fold. This stabilizing effect of molybdate was also manifest on gel electrophoresis of the receptor. In the absence of molybdate, no specific [3H]progesterone binding was detectable on polyacrylamide gel electrophoresis. But, in the presence of 5 mM sodium molybdate, a [3H]progesterone-binding species was clearly evident on the gels. The radioactivity associated with this binding was displaceable by unlabeled progesterone but not by cortisol and depended on the concentration of [3H]progesterone employed, suggesting that this binding species is a progesterone receptor. Molybdate treatment produced a small increase in receptor size on low-salt sucrose gradients (from 6-7 to S to 7.5 S). There was no effect of molybdate of receptor sedimentation in the presence of high salt (0.3 M KCl). Further analysis of this phenomenon by gel filtration suggested that this molybdate-mediated increase in receptor size was due to receptor aggregation. In low-salt buffers, molybdate treatment markedly increased the proportion of receptors contained in large aggregates (Stokes radius greater than 8.0 nm). Again, this effect was abolished in the presence of high salt. In conjunction with receptor stabilization, molybdate prevented binding of uterine progesterone receptor to DNA--cellulose. These findings suggest that sodium molybdate stabilizes the unliganded, unactivated form of the receptor. Moreover, these effects seem to be mediated through a direct interaction of molybdate with the receptor, one which results in receptor aggregation.

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Section: Discussionsupporting

confidence: 69%

Uterine progesterone receptor: stabilization and physicochemical alterations produced by sodium molybdate

Chen¹,

MacDonald²,

Leavitt³

1981

Biochemistry

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“…The specificity of ORG 2058 for the cytoplasmic progesterone receptor has been established previously (Jänne, Kontula & Vihko, 1976;Fleischmann & Beato, 1978) and the present data show that the nuclear progesterone receptor satisfies the criteria of high chemical specificity and high affinity.…”

Section: Measurement Ofprogesterone Receptor Sites In Rat Uterine Cytsupporting

confidence: 61%

Progesterone Receptors and Rna Polymerase Activity in the Rat Uterus During the Oestrous Cycle

Kurl¹,

Borthwick²

1979

Journal of Endocrinology

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The concentrations of progesterone receptors in uterine cytoplasm and nuclei were measured during the oestrous cycle of the rat. The concentration of cytoplasmic progesterone receptors was highest at pro-oestrus and declined at oestrus to reach lowest levels at metoestrus before rising at dioestrus. Similar changes were observed in the concentration of nuclear progesterone receptors, the highest levels being present at pro-oestrus and dioestrus. In addition, both activities A and B of RNA polymerase mirrored these alterations in nuclear receptor levels.

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“…The affinity toward the hormone could only be studied in steroid-free receptor preparations. As shown in Figure 4, the purified aporeceptor exhibited the same affinity for its ligand (Kq = 5 nM) as when receptor was studied in crude cytosol (Fleischmann & Beato, 1978).…”

Section: Purification Of Receptor (A) Purification Of Steroid-mentioning

confidence: 68%

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit

Logeat¹,

Pamphile²,

Loosfelt³

et al. 1985

Biochemistry

118

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A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

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Characterization of the progesterone receptor of rabbit uterus with the synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione

Cited by 30 publications

References 20 publications

Uterine progesterone receptor: stabilization and physicochemical alterations produced by sodium molybdate

Uterine progesterone receptor: stabilization and physicochemical alterations produced by sodium molybdate

Progesterone Receptors and Rna Polymerase Activity in the Rat Uterus During the Oestrous Cycle

One-step immunoaffinity purification of active progesterone receptor. Further evidence in favor of the existence of a single steroid binding subunit

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