PMID: 429374May 10, 1979Paper

Isolation of estrogen receptor in complex with a discrete nuclear subfraction from hen oviduct.

The Journal of Biological Chemistry
R T Franceschi, K H Kim

Abstract

A nuclear subfraction containing bound estrogen receptor in presumed complex with its nuclear acceptor site has been partially purified from hen oviduct. Sucrose density gradient ultracentrifugation was used to separate mechanically sheared chromatin (i.e. lysed nuclei) into several fractions which differed in protein to DNA ratio as well as in vitro template activity. Gradient fractions were then examined for the presence of bound estrogen receptors. Care was taken to use physiological ionic strength buffers when preparing nuclei since the number of estrogen receptors per nucleus decreased from 5600 to 1600 when nuclei prepared in low ionic strength (mu = 0.013 M) were compared with nuclei prepared in physiological ionic strength (mu = 0.2 M). [3H]Estradiol was introduced into nuclear estrogen receptors by exposing minced oviduct to labeled hormone in tissue culture or by exchanging nuclear estrogen receptor complexes formed in vivo with labeled hormone. In all cases, receptor was found in a fast sedimenting nuclear subfraction of low in vitro template activity. Sodium dodecyl sulfate-gel electrophoresis revealed no differences between proteins from receptor-containing and slower sedimenting fractions. Hybrdization experiments...Continue Reading

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