Progesterone-binding Components of Chick Oviduct

Abstract

Abstract 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 10-10 m in 0.3 m KCl at 1° and is reversed by mild heating and by unlabeled progesterone g testosterone g 20α-hydroxy-4-pregnene-3-one g 17β-estradiol g cortisol g estrone g androstenedione. The participation of protein in the steroid-binding site was inferred from the destruction of the complex by 10-3 m 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, s020,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 s020,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.