Polynucleotides associated with histone preparations

Greenaway, Peter; Murray, Kenneth

doi:10.1042/bj1310585

Cited by 5 publications

(1 citation statement)

References 24 publications

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“…These contaminants are, however, effectively removed from basic proteins by ion exchange chromatography (43,44). Furthermore, specific binding of estradiol receptors persists even after incubation and washing of the acceptoragarose derivatives with 6 M guanidine* HCl at pH 1.7, a treatment that should remove any noncovalently-bound cofactor.…”

Section: Resultsmentioning

confidence: 99%

Identification of a High Affinity Nuclear Acceptor Site for Estrogen Receptor of Calf Uterus

Puca

Sica

Nola

1974

Proc. Natl. Acad. Sci. U.S.A.

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By means of affinity chromatography, specific nuclear acceptor sites for estradiol receptors are identified in a fraction that can be solubilized from purified nuclei with 2 M NaCl. Interaction between these acceptor sites and crude or partially purified estradiol receptor shows a high association constant (over 109 M). Receptor-acceptor interaction is dependent on physiological concentrations of 1710-estradiol; it is disrupted by high ionic strength.The nuclear acceptor sites appear to be protein in nature and exist in 5-to 10-fold excess over the estrogen binding sites present in the cytosol. Single-or double-stranded DNA does not bind estrogen-receptor complexes. Acceptor sites appear to be associated with basic nuclear proteins as judged by hydroxyapatite chromatography. The nuclear acceptor sites probably represent less than 0.1% of the purified basic proteins from the nucleus.It is currently believed that the mechanism of action of a variety of steroid hormones is essentially identical. Steroids interact with high affinity specific proteins (receptors) coinfined to the cytoplasm of the target cell, and the formed hormone-recep)tor complex moves rapidly to the nucleus where it interacts with some chromatin component (nuclear acceptor) from which it can be released with 0.3 or 0.4 M KCl (1-7). We reported (8-10) on the partial purification and characterization of the receptor proteins for 17f-estradiol of calf uterus, and on the interrelationships, based on physicochemical measurements, which exist between the cytoplasmic (8.6-5.3S) native system and the smaller (4.5S) receptor obtained after activation of a specific hydrolytic enzyme found in cytosol or in nuclei incubated with 17f-estradiol. The strict correlation which exists between nuclear binding of the estrogen-receptor comlplex with uterotropic response in vivo (11), or with induction of de novo synthesis of a slecific uterine protein in vivo and in vitro (12,13) indicates that nuclear localization is involved in initiating estrogen-dependent events. In this paper, affinity chromatography is used to identify and, as a first step, characterize a nuclear comlponent which has the properties of a sl)ecific acceptor for estrogen receptor complexes.MATERIALS AND METHODS [6,7-3H ] et al. (14). To one volume of purified nuclei were added 20 volumes of 2 M NaCl in 50 mM sodium phosphate buffer (pH 7.1), 1 mM NaHSO3, and, after mixing (Ultraturrax, low speed), the suspension was incubated for 120 min at 4°. The sediment (48,000 X g, 30 min) consisted of nuclear material not solubilized by 2 M NaCl (fraction A); it was resuspended (Ultraturrax, high speed) in TKED 0.12 M. Material extracted from the nuclei with 2 M NaCl was dialyzed with two changes for 20 hr at 40 against 10 volumes of 25 mM sodium phosphate buffer (pH 7.1) and centrifuged at 46,000 X g for 20 min. The supernatant contained material soluble at low ionic strength (fraction B). The sediment contained nuclear DNA associated with basic and some acidic nuclear proteins (fraction C). Fra...

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Section: Resultsmentioning

confidence: 99%