Further characterization of the second oestrogen-binding species of the rat granulosa cell

Abstract

ABSTRACT Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A–Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4°C (1·72 ± 0·27(s.e.m.) × 108 mol/h) and 25°C (4·50 ± 0·36 × 108 mol/h) was faster than with the oestrogen receptor (7·20 ± 0·15 × 107 mol/h and 1·23 ± 0·15 × 108 mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25°C (rate constants k−1 = 0·30±0·07/min and k−2 = 3·73±0·57 × 10−3/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10·3S, Stokes radius >5·2 nm) in low ionic strength buffers and as a small species (4·4S, Stokes radius 3·5 nm) in buffers containing 0·4 m-KCl. However, the SOB fractionated as 2–3S, Stokes radius 3·7–4·0 nm at low ionic strength and as 5·8S, Stokes radius 3·5 nm in 0·4 m-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4·6S, mol. wt 69–72 000 on SDG. J. Endocr. (1984) 102, 93–102