A high-affinity oestrogen-binding protein in cockerel liver cytosol

Abstract

In contrast with several earlier reports, cytosol from cockerel liver contains a significant concentration of a protein that binds oestradiol with high affinity. To demonstrate the activity, certain alterations in the conventional method of preparation of cytosol must be made. Homogenization in sucrose-containing buffer at pH 8.4 in the presence of proteinase inhibitors and rapid fractionation of the cytosol with (NH4)2SO4 enables demonstration of a single class of oestradiol-binding sites with a Kd of about 1 nM and specificity only for oestrogens. The concentration is about 300 sites per cell in liver from 2-week-old cockerels. Oestradiol treatment in vivo decreases the number of exchangeable cytosol oestradiol-binding sites by about 80% for 1--4h, after which time it is gradually restored. Gel filtration of the cytosol preparation in the presence of high salt concentrations reveals that most of the oestradiol-binding activity is in high-molecular-weight aggregates, but a mild trypsin treatment generates a specific binding protein with an approximate mol.wt. of 40 000. This protein may be an oestrogen receptor.

Feminization in common terns (Sterna hirunda) : relationship to persistent organic contaminants

“…Results at the highest [3H]estradiol incubation concentration (15 nM) in chicken ovary cytosol suggest the binding curve may be approaching a maximum (Figure 3). Literature binding curves for estradiol to avian estrogen receptor suggest that a maximum should be reached by 1 -3 nM estradiol (Lazier and Haggarty 1979;Janz and Bellward 1996;Stevens 1996). Furthermore, the present results show the Kd values determined from double reciprocal and Scatchard plots of binding data are between 9 and 11.5 nM for chicken ovary cytosol and are either 5.3 or too high to be determined for tern hepatic cytosol.…”

“…Furthermore, the present results show the Kd values determined from double reciprocal and Scatchard plots of binding data are between 9 and 11.5 nM for chicken ovary cytosol and are either 5.3 or too high to be determined for tern hepatic cytosol. Literature values for avian estrogen receptor Kds are usually from 0.1 to 1 nM, and only seldom are values above 2.0 nM reported (Lazier and Haggarty 1979;MacLaughlin, Hutson et al 1983;Janz and Bellward 1996;Stevens 1996). The unusually high Kd values and the lack of a maximum observed in the binding curves suggest that binding may not be specific for the estrogen receptor.…”

“…A non-receptor steroid binding protein was found in chicken hepatic cytosol; removal of this protein was required before estrogen receptor binding in preparations could be detected (Lazier and Haggarty 1979;Lazier, Lonergan et al 1985). In experiments examining unfractionated chicken hepatic cytosol for high-affinity [3H]estradiol binding, copious quatities of bound hormone were detected, but less than 5% suppression of total binding by inclusion of 100-1000-fold excess of unlabelled estradiol or DES in the assay mixture was found (Lazier and Haggarty 1979).…”

“…Human pregnancy 1) 2 plasma 2) lower affinity form (has SHBG and aFP)g DES very low affinity rat pregnancy plasmag 2.5 DES very low affinity salmon plasmaf 13 DES very low affinity a this study b Stevens (1996) c Maclaughlin, Hutson, and Donahoe (1983) d Janz and Bellward (1996) e Lazier and Haggarty (1979) f Lazier, Lonergan, and Mommsen (1985) g Sheehan and Young (1979) (Janz and Bellward 1996), other studies with cytosol preparations suggest that it is not unusual for the presence of other hormone binding proteins to mask estrogen receptor binding, or for estrogen receptor binding to be too low for detection. A non-receptor steroid binding protein was found in chicken hepatic cytosol; removal of this protein was required before estrogen receptor binding in preparations could be detected (Lazier and Haggarty 1979;Lazier, Lonergan et al 1985).…”

“…binding studies, including studies in chicken, great blue heron, and pigeon (Lazier and Haggarty 1979;Janz and Bellward 1996). Furthermore, high affinity binding of DES to estrogen binding sites often is used as a check that binding is specific for the estrogen receptor, and is used across vetebrate species including mammals, birds, turtles, alligators, sting rays, and fish (Lazier and Haggarty 1979;Lazier, Lonergan et al 1985;Giannoukos and Callard 1996;Yamamoto, Suzuki et al 1996;Vonier, Guillette Jr. et al 1997). All previous work indicates that DES should bind to the estrogen receptor of chicken with an affinity equivalent to estradiol.…”