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

Renoir, Jack‐Michel; Radanyi, Christine; Yang, Chang-Ren; Baulieu, Étienne-Émile

doi:10.1111/j.1432-1033.1982.tb06840.x

Cited by 47 publications

(25 citation statements)

References 19 publications

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“…No such effect was observed when the "2I-labeled PR was incubated with nonimmune goat IgG (IgG-GO). There was an increase of radioactivity at the bottom of the tube in the presence of IgG-G3, as described previously for the native noniodinated receptor (4).…”

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confidence: 62%

“…Steroid receptors are immunogenic: antisera have been obtained after injection to rabbits and goats of calf uterus estradiol receptor (15)(16)(17)(18), human myometrium receptor (19), rat liver glucocorticosteroid receptor (20,21), rabbit uterus PR (22), and chicken oviduct PR (4). It is surprising that previous attempts to generate antibodies against purified chicken oviduct PR were unsuccessful (2).…”

Section: Discussionmentioning

confidence: 99%

“…1), initial attempts to obtain PR antibodies were unsuccessful (2). Now, however, a homogeneous molybdate-stabilized "8-9S" form of PR has become available (3) and we have obtained anti-PR antibodies in a rabbit and a goat (4). The goat antiserum crossreacted not only with mammalian PRs (4) but, more surprisingly, with chicken glucocorticosteroid receptor (5).…”

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confidence: 93%

“…After the iodination process, the sedimentation coefficient was determined to be 6 A 'B 7.9 1B L~~~~1 400 E l00 5001~~~~~~I S. This difference between S values of the two receptors is discussed below. Because the native form and the iodinated PR had different sedimentation coefficients and no other radioiodinated steroid receptor has yet been found to be immunoreactive, we carried out experiments using goat anti-PR IgG "IgG-G3" (4). The sedimentation coefficient of the radioactive receptor shifted from =6 to =16 S in 10-35% glycerol gradient ultracentrifugation, as shown in Fig.…”

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confidence: 99%

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Monoclonal antibody to chicken oviduct progesterone receptor.

Radanyi¹,

Joab²,

Renoir³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Antibodies to molybdate-stabilized chicken oviduct progesterone receptor were raised in a Wistar rat and detected by interaction with homogeneous radioiodinated progesterone receptor. Spleen cells of this rat were then fused with mouse Sp2/0-Agl4 myeloma cells and the antibodies produced by the hybrid cells were detected by double immunoprecipitation using the '5I-labeled receptor. Cells of one of the positive cultures were then cloned by limiting dilution and one hybridoma cell line was studied. The monoclonal antibody produced was an IgG2b, and it reacted with the molybdate-stabilized "8-9S" form of the chicken oviduct progesterone receptor, labeled with either [H] Although the chicken oviduct progesterone receptor (PR) has been extensively studied (for review, see ref. 1), initial attempts to obtain PR antibodies were unsuccessful (2). Now, however, a homogeneous molybdate-stabilized "8-9S" form of PR has become available (3) and we have obtained anti-PR antibodies in a rabbit and a goat (4). The goat antiserum crossreacted not only with mammalian PRs (4) but, more surprisingly, with chicken glucocorticosteroid receptor (5). The latter unexpected result prompted us to obtain monoclonal antibodies (6) Exponentially growing mouse myeloma cells were fused with spleen cells by using 50% (wt/vol) polyethylene glycol 4000 and 5% dimethyl sulfoxide according to a published procedure (10) with minor modifications: 5 x 107 myeloma cells were exposed to 4 x 108 spleen cells and plated in selective medium (50 pkM hypoxanthine/10 tuM azaserine). The next day, surviving cells were counted and 0.1-ml aliquots containing 104 cells were plated in the presence of 4 X 105 rat macrophages. The medium was changed once a week. Two to 3 wk later, when the cell concentration had reached 105/ml, growing cells were tested for antibody production.The antibody-secreting hybrids were cloned by limiting dilution in the presence of 2 x 106 mouse thymocytes per ml.Wells containing a single cell cluster were assayed for anti-PR antibody production. Labeling of Chicken Oviduct Progesterone Receptor. Iodination. Highly purified (>90% pure) molybdate-stabilized PR (6 Ag) was labeled with 1 mCi of 125I (specific activity, -2,200Ci/mmol; 1 Ci = 37 GBq; New England Nuclear) by using the Bolton and Hunter reagent (11). Human Mammary Cancer Cytosol. PR-containing cytosol was prepared from MCF-7 cells (12) and total tumor, as described (4 2854The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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confidence: 62%

Section: Discussionmentioning

confidence: 99%

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confidence: 93%

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confidence: 99%

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Monoclonal antibody to chicken oviduct progesterone receptor.

Radanyi¹,

Joab²,

Renoir³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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“…The purified molybdate-stabilized receptor was injected into rabbit and goat, and specific anti-(progesterone receptor) antisera were obtained in both [48]. Characteristics of these antisera are reported elsewhere [48,49].…”

Section: Discussionmentioning

confidence: 99%

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

Renoir

Yang

Formstecher³

et al. 1982

Eur J Biochem

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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...

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