Progesterone-binding Components of Chick Oviduct: I. PRELIMINARY CHARACTERIZATION OF CYTOPLASMIC COMPONENTS

Merry Rubin Sherman; Pierre L. Corvol; Bert W. O'Malley

doi:10.1016/S0021-9258(18)62667-5

CONTROL MECHANISMS AND BIOCHEMICAL GENETICS| Volume 245, ISSUE 22, P6085-6096, November 25, 1970

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Progesterone-binding Components of Chick Oviduct

I. PRELIMINARY CHARACTERIZATION OF CYTOPLASMIC COMPONENTS

Merry Rubin Sherman
Merry Rubin Sherman
Affiliations
From the National Institute of Dental Research and the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014
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Pierre L. Corvol
Pierre L. Corvol
Affiliations
From the National Institute of Dental Research and the National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014
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Bert W. O'Malley
Bert W. O'Malley
Affiliations
From the Departments of Obstetrics-Gynecology, Medicine and Biochemistry, Vanderbilt School of Medicine, Nashville, Tennessee 37203
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Open AccessPublished:November 25, 1970DOI:https://doi.org/10.1016/S0021-9258(18)62667-5

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Induction of the synthesis of the specific protein avidin by a single administration of progesterone has been demonstrated previously in chicks in vivo and in tissue minces and monolayer cultures of chick oviduct. To investigate the mechanism of induction, macromolecular components of oviduct cytoplasm which bind ³H-progesterone in vitro were isolated and characterized by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, enzymatic digestion, and gel filtration on Agarose (Bio-Rad Laboratories, Richmond, California).

The radioactive steroid in the isolated complex was identified as progesterone, not a metabolite, by paper chromatography. The interaction with ³H-progesterone has an apparent dissociation constant kd ≃ 8 x 10^-10m in 0.3 m KCl at 1° and is reversed by mild heating and by unlabeled progesterone > testosterone > 20α-hydroxy-4-pregnene-3-one > 17β-estradiol > cortisol > estrone > androstenedione. The participation of protein in the steroid-binding site was inferred from the destruction of the complex by 10^-3m p-hydroxymercuribenzoate and by Pronase, but not by ribo- or deoxyribonucleases.

The apparent number and size of the cytoplasmic binding components vary with the concentration of KCl and the technique of isolation and detection. In the absence of KCl, the major components are characterized by sedimentation coefficients, s⁰_20,w, of about 5 S and 8 S, molecular weights of about 1.0 and 3.6 x 10⁵ (estimated from the variation of electrophoretic mobility with gel concentration), and sufficiently large effective radii to be eluted in, or near, the void volume of columns of Agarose A-0.5m. Under the same conditions the corticosteroid-binding globulin (CBG) of chick plasma behaves as a single component with s⁰_20,w ≃ 3.7 S and mol wt ≃ 6.0 x 10⁴. In solutions containing 0.3 m KCl, the cytoplasmic components and CBG all sediment at about the same rate but may be distinguished from each other by the distribution coefficients on Agarose A-0.5m. From the latter results, molecular Stokes radii of 55 and 63 A can be calculated for the cytoplasmic components, compared with 37 A for CBG. The progesterone-binding components of chick oviduct cytoplasm are thus distinguishable from CBG by all physicochemical methods tested.

A functional role for these components in the induction of avidin synthesis by progesterone is supported by (a) the parallel order of effectiveness of various steroids in competing with progesterone binding and in potency as inducers, and (b) the analogous effects of treating the chicks with diethylstilbestrol on progesterone-binding activity and on avidin induction.

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Published online: November 25, 1970

Received: June 25, 1970

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DOI: https://doi.org/10.1016/S0021-9258(18)62667-5

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