Characterization of trypsin-treated forms of the estrogen receptor from rat and lamb uterus

Carlson, Kathryn E.; Sun, Lee; Katzenellenbogen, John A.

doi:10.1021/bi00638a025

Cited by 39 publications

(13 citation statements)

References 53 publications

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“…Several investigators (Sherman et al, 1978;Puca et al, 1971Puca et al, ,1972Wrange & Gustafsson, 1978;Carlson et al, 1977) have produced small forms of steroid-receptor complexes by either Ca2+ treatment or protease digestion which are similar in size to those which we have described here for pyridoxal phosphate treated receptors. The 2.9S form of the glucocorticoid receptor that we observe is an endogenous component of both activated and unactivated cytosols, and is the sole product observed on sucrose gradients after pyridoxal phosphate treatment and NaBH4 reduction.…”

Section: Discussionsupporting

confidence: 59%

Pyridoxal phosphate induced alterations in glucocorticoid receptor conformation

Cidlowski¹,

Thanassi²

1979

Biochemistry

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Section: Discussionsupporting

confidence: 59%

Pyridoxal phosphate induced alterations in glucocorticoid receptor conformation

Cidlowski¹,

Thanassi²

1979

Biochemistry

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“…In addition, the cytoplasmic receptor that after deaggregation with 400 mM KC1 sediments at ~4 S and the small form of the cytoplasmic receptor (~3.6 S) generated by exposure to trypsin (40 Mg/uterine equiv, 1 h, 0-2 °C; reaction terminated with soybean trypsin inhibitor, 2.5 ug/ug of trypsin) have been resolved on this column (Figure ID). [For reference to sedimentation analysis, see Jensen & DeSombre (1972), Carlson et al (1977), and Katzenellenbogen et al (1980).] Moreover, the relationship between these forms previously established by sucrose-gradient analysis persists on this column: cytoplasmic estrogen receptor > extracted nuclear estrogen receptor > KCl-deaggregated cytoplasmic estrogen receptor > trypsintreated cytoplasmic estrogen receptor.…”

Section: Resultsmentioning

confidence: 99%

“…The partition coefficients (K¿) were determined as defined by Ackers (1975); K¿ = (Vc -V0)/ VT -V0) on at least three replicate determinations. Estimates of Stokes' radii were referenced for soybean trypsin inhibitor, ovalbumin, bovine serum albumin (BSA) (Carlson et al, 1977), 7-globulin (Click, 1970), human breast estrogen receptor, molybdate stabilized (Miller et al, 1981), and trypsin, calculated from///0 (Walsh, 1970) and molecular weight (Click, 1970) according to the relationship f/fQ = a/ [3 /)/(4 )]1/3; C = 0.725 cm2 g'1 (Siegel & Monty, 1966). The dashed line is the best fit estimate determined by linear-regression analysis from points including ovalbumin, BSA, 7-globulin, and human breast estrogen receptor.…”

Section: Analysis Of Estrogen and Progesterone Receptors Frommentioning

confidence: 99%

“…Steroid-receptor determinations can be based upon principally quantitative analysis by employing assays involving dextrancoated charcoal, hydroxylapatite, or protamine sulfate (Garola & McGuire, 1977a,b, 1978Rosner et al, 1980;Zava et al, 1976). Moreover, receptor assays involving sucroseor glycerol-gradient analysis, gel filtration, column chromatography, 0006-2960/82/0421-0139$01.25/0 © 1982 American Chemical Society and electrophoresis can also provide a qualitative characterization (Sherman et al, 1980;Miller et al, 1975Miller et al, , 1981Tilzer et al, 1981;Carlson et al, 1977;Katzenellenbogen et al, 1980). Time and convenience are generally limiting factors in the application of qualitative receptor analysis.…”

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confidence: 99%

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Rapid analysis of estrogen and progesterone receptors using gel-exclusion high-performance liquid chromatography

Pavlik¹,

Nagell²,

Muncey³

et al. 1982

Biochemistry

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Estrogen and progesterone receptors prepared from mouse, rat, and human uteri, as well as from human breast cancers, have been characterized by gel-exclusion high-performance liquid chromatography. The qualitative relationships previously established by sedimentation analysis between the cytoplasmic [aggregated (approximately 8S), deaggregated (approximately 4S), and trypsinized (approximately 3.6S)] and nuclear (approximately 5S) forms of the rat uterine estrogen receptor were maintained by this technique. Differences in the partition of estrogen and progesterone receptors from the same species as well as interspecies differences in these receptors were reproducibly observed. Multiple forms of human estrogen and progesterone receptors could clearly be resolved in a single analysis and were distinct from serum steroid binding tissue contaminants. Separation analyses, performed at flow rates up to 2 mL min-1, were capable of resolving all receptor forms in 10--12 min with the column returning to base line in 25 min. With this exclusion gel column (TSK-G3000SW) as a background upon which to reference different receptor forms, eight distinct partitions or elution positions have been enumerated. This approach has considerable promise for the rapid characterization of different forms of steroid-receptor proteins. Moreover, it should provide a critical advantage in minimizing the opportunities for receptor modification during separation analysis and in maximizing the opportunity to study short-lived interactions between receptors and physiologic or pharmacologic ligands.

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“…Such short-range interactive forces (e.g., charge-transfer) at the ligand-binding site can be dramatically altered by urea (Ochoa et al, 1980). Limited trypsin digestion of the native and transformed estrogen receptor forms to remove the DNA-binding structural domains (Carlson et al, 1977;Sherman et al, 1978) resulted in a significant decrease in receptor interaction with the immobilized Zn2+, Cu2+ and Ni2+ ions. These data suggest that a large percentage of receptor interacted with the immobilized metals via binding sites located on the DNA-binding domain of the receptor.…”

Section: Discussionmentioning

confidence: 99%

Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn²⁺, Cu²⁺ and Ni²⁺ and separation of receptor isoforms

Hutchens

1988

J of Molecular Recognition

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We have utilized iminodiacetate (IDA) gels with immobilized Zn2+, Cu2+ and Ni2+ ions to evaluate the metal binding properties of uterine estrogen receptor proteins. Soluble (cytosol) receptors labeled with [3H]estradiol were analyzed by immobilized metal affinity chromatography (IMAC) before as well as after (1) 3 M urea-induced transformation to the DNA-binding form, and (2) limited trypsin digestion to separate the steroid- and DNA-binding domains. Imidazole (2-200 mM) affinity elution and pH-dependent (pH 7-3.6) elution techniques were both evaluated and found to resolve several receptor isoforms differentially in both the presence and absence of 3 M urea. Individual receptor forms exhibited various affinities for immobilized Zn2+, Cu2+ and Ni2+ ions, but all intact receptor forms were strongly adsorbed to each of the immobilized metals (Ni2+ greater than Cu2+ much greater than Zn2+) at neutral pH. Generally, similar results were obtained with IDA-Cu2+ and IDA-Ni2+ in the absence of urea. Receptors were tightly bound and not eluted before 100 mM imidazole or pH 3.6. Different results were obtained using IDA-Zn2+; at least four receptor isoforms were resolved on IDA-Zn2+. Receptor-metal interaction heterogeneity and affinity for IDA-Zn2+ and IDA-Cu2+, but not IDA-Ni2+, were substantially decreased in the presence of 3 M urea. The receptor isoforms identified and separated by IDA-Zn2+ chromatography were not separable using high-performance size-exclusion chromatography, density gradient centrifugation, chromatofocusing or DNA-affinity chromatography. The affinity of trypsin-generated (mero)receptor forms for each of the immobilized metals was decreased relative to that of intact receptor. High-affinity metal-binding sites were mapped to the DNA-binding domain, but at least one of the metal-binding sites is located on the steroid-binding domain. Recovery of all receptor forms from the immobilized metal ion columns was routinely above 90%. These results demonstrate the differential utility of various immobilized metals to characterize and separate individual receptor isoforms and domain structures. Receptor-metal interactions warrant further investigation to establish their effects on receptor structure/function relationships. In addition to the biological implications, recognition of estrogen receptor proteins as metal-binding proteins suggests new and potentially powerful receptor immobilization and purification regimes previously unexplored by those in this field.

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Characterization of trypsin-treated forms of the estrogen receptor from rat and lamb uterus

Cited by 39 publications

References 53 publications

Pyridoxal phosphate induced alterations in glucocorticoid receptor conformation

Pyridoxal phosphate induced alterations in glucocorticoid receptor conformation

Rapid analysis of estrogen and progesterone receptors using gel-exclusion high-performance liquid chromatography

Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn²⁺, Cu²⁺ and Ni²⁺ and separation of receptor isoforms

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Characterization of trypsin-treated forms of the estrogen receptor from rat and lamb uterus

Cited by 39 publications

References 53 publications

Pyridoxal phosphate induced alterations in glucocorticoid receptor conformation

Pyridoxal phosphate induced alterations in glucocorticoid receptor conformation

Rapid analysis of estrogen and progesterone receptors using gel-exclusion high-performance liquid chromatography

Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn2+, Cu2+ and Ni2+ and separation of receptor isoforms

Contact Info

Product

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Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn²⁺, Cu²⁺ and Ni²⁺ and separation of receptor isoforms