A novel class of inactive steroid-binding sites in female rat liver nuclei

1986 ◽  
Vol 110 (1) ◽  
pp. 27-36 ◽  
Author(s):  
D. M. Bechet ◽  
B. N. Perry
Keyword(s):  
Rat Liver ◽  
Binding Sites ◽  
Specific Binding ◽  
Gel Filtration ◽  
Nuclear Extract ◽  
Female Rat ◽  
Rat Liver Nuclei ◽  
Heat Stable ◽  
Nuclear Extracts ◽  
Liver Nuclei

ABSTRACT Nuclear salt extracts from intact female rat liver showed insignificant levels of progesterone-, oestradiol-, testosterone- or dexamethasone-specific binding. However, brief exposure of nuclear extracts to dextran-coated charcoal (DCC) induced binding for all the above classes of steroids. This 'DCC-effect', which was reproduced neither by gel filtration nor by extensive dialysis of the nuclear extract, could not be ascribed to removal of endogenous free or loosely bound steroids. We show that rat liver nuclei contain a class of secondary binding sites (BsII), which exhibit moderate or low affinity for steroid ligands, positive co-operativity, and cross-reaction between classes of steroids. The capacity of BsII sites to bind steroids depends strictly on prior neutralization by DCC of endogenous heat-stable non-dialysable inhibitor(s). The putative roles of these BsII binding sites are discussed in relation to component(s) probably responsible for inhibitory activity. J. Endocr. (1986) 110, 27–36

Effect of thiol blocking agents on the binding of T3 and T4 in rat liver nuclear extract

1981 ◽  
Vol 98 (1) ◽  
pp. 68-72 ◽  
Author(s):  
J. Knopp ◽  
J. Brtko
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Nuclear Extract ◽  
Blocking Agent ◽  
Binding Affinities ◽  
Structural Configuration ◽  
Blocking Agents ◽  
Nuclear Extracts ◽  
Liver Nuclei ◽  
Apparent Association

Abstract. Liver nuclei bind thyroid hormones (T3 and T4) with different binding affinities. In our studies it was estimated that the apparent association constant (Ka) in the liver nuclei for T3 was 8.8 × 1010 l mol−1 and for T4 was 4.1 × 109 l mol−1. We have found that the binding sites for T4 are also saturated by an excess of unlabelled T4 and that saturation was dependent on the purification step of liver binding proteins. In liver nuclear extracts with 0.4 mol l−1 KCl the specific binding of T3 and T4 was blocked with a typical -SH blocking agent N-ethylmaleinimide (NEM) and by new types of -SH blocking agents such as p-bromphenylisothiocyanate (p-BPI) and 2,3 dicyano-1,4-dithio-9,10 antrachinone (Delan). NEM and p-BPI increased the non-specific binding of T4 and completely abolished specific binding. All these agents blocked the specific binding of T3. These results demonstrate that T3 and T4 binding sites in the liver nuclei may not be altogether identical and that the different effects of -SH blocking agents on the binding of T3 and T4 is probably associated with the structural configuration of these drugs.

scholarly journals Binding of dihydrotestosterone to a nuclear-envelope fraction from the male rat liver

1982 ◽  
Vol 202 (1) ◽  
pp. 225-230 ◽  
Author(s):  
Y A Lefebvre ◽  
S J Morante
Keyword(s):  
Heat Treatment ◽  
Nuclear Envelope ◽  
Binding Site ◽  
Rat Liver ◽  
Binding Sites ◽  
Specific Binding ◽  
Male Rat ◽  
Scatchard Analysis ◽  
Female Rat ◽  
Similar Class

Intact nuclear ‘ghosts’ containing small amounts of DNA were obtained from rat liver. Incubation of radiolabelled dihydrotestosterone with isolated nuclear-envelope fraction from male rat liver resulted in specific binding of the dihydrotestosterone to the membranes. Optimal binding occurred at 20 degrees C after 20h incubation. Storage for 2 weeks at -80 degrees C resulted in little loss of specific binding. Scatchard analysis revealed a class of binding sites with a KD of 23.2 nM. Pronase and heat treatment destroyed the binding site. Androgens and glucocorticoids competed for labelled dihydrotestosterone binding to the ghosts, whereas oestrogens did not compete. Castration 24h before preparation of ghosts did not alter the binding site, and a similar class of binding sites was identified on female rat liver nuclear envelopes.

scholarly journals Nuclear polyadenylate-binding protein.

1985 ◽  
Vol 5 (8) ◽  
pp. 1993-1996 ◽  
Author(s):  
A B Sachs ◽  
R D Kornberg
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Rat Liver ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Single Peak ◽  
Nuclear Extracts ◽  
Dodecyl Sulfate ◽  
Liver Nuclei ◽  
High Degree

Polyadenylate-binding activity can be detected in eluates from sodium dodecyl sulfate gels by a nitrocellulose filter-binding assay. Nuclear extracts from rat liver show a single peak of binding activity at 50 to 55 kilodaltons; cytoplasmic extracts show a single peak at 70 to 80 kilodaltons, corresponding to a 75-kilodalton protein previously described. Similar results are obtained with yeast and mouse fibroblasts, indicating a high degree of conservation of both nuclear and cytoplasmic polyadenylate-binding proteins. The activity from rat liver nuclei has been purified 125-fold on the basis of specific binding to polyadenylate and shows two main bands in sodium dodecyl sulfate gels at 53 and 55 kilodaltons.

Nuclear polyadenylate-binding protein

1985 ◽  
Vol 5 (8) ◽  
pp. 1993-1996
Author(s):  
A B Sachs ◽  
R D Kornberg
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Rat Liver ◽  
Specific Binding ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Single Peak ◽  
Nuclear Extracts ◽  
Dodecyl Sulfate ◽  
Liver Nuclei ◽  
High Degree

Polyadenylate-binding activity can be detected in eluates from sodium dodecyl sulfate gels by a nitrocellulose filter-binding assay. Nuclear extracts from rat liver show a single peak of binding activity at 50 to 55 kilodaltons; cytoplasmic extracts show a single peak at 70 to 80 kilodaltons, corresponding to a 75-kilodalton protein previously described. Similar results are obtained with yeast and mouse fibroblasts, indicating a high degree of conservation of both nuclear and cytoplasmic polyadenylate-binding proteins. The activity from rat liver nuclei has been purified 125-fold on the basis of specific binding to polyadenylate and shows two main bands in sodium dodecyl sulfate gels at 53 and 55 kilodaltons.

Two Forms of Histone–Acetyltransferase in High Salt Extracts of Rat Liver Nuclei

1973 ◽  
Vol 51 (8) ◽  
pp. 1177-1194 ◽  
Author(s):  
Peter F. Lue ◽  
A. G. Gornall ◽  
C. C. Liew
Keyword(s):  
Rat Liver ◽  
Histone Acetyltransferase ◽  
Extraction Procedure ◽  
Gel Filtration ◽  
Electrophoretic Analysis ◽  
High Salt ◽  
Sephadex Column ◽  
Nuclear Extracts ◽  
Calf Thymus ◽  
Liver Nuclei

Two forms of 'histone' acetyltransferase (AT I and AT II) were separated by DEAE-Sephadex chromatography of rat liver nuclear extracts prepared by sonication in 0.3 M (NH4)2SO4. A 12-fold greater amount of AT I was not adsorbed on the DEAE-Sephadex column, whereas AT II was adsorbed and was eluted with 0.15–0.2 M (NH4)2SO4. Chromatography of AT I on Sephadex G-200 and cellulose phosphate indicated the presence of additional forms of the enzyme. Optimal conditions for the assay of the enzyme have been determined.In vitro acetylation of calf thymus histone fractions by AT I or AT II showed that at low histone concentration (313 μg/ml), the "arginine-rich" fractions (f2a1 and f3) were acetylated to a greater extent than were the "lysine-rich" fractions (f1 and f2b). However, at a higher concentration of histone (1250 μg/ml) f2a1 acetylation decreased as a result of coprecipitation with the enzyme. Electrophoretic analysis of the acetylated fractions indicated that multiple sites were probably acetylated on f3, f2a2, and f2a1, whereas only one residue was reactive in f1 and f2b.Interconversion of AT I and AT II has been demonstrated and it was shown that [Formula: see text]. AT I contained mainly histones f2a2 and f3, and was converted to AT II by treatment with high salt and gel filtration chromatography. After hypotonic lysis of nuclei and DEAE chromatographic analysis of extracts obtained by washing the nuclear pellet with 0.3 M sodium citrate of NaCl, most of the acetylating activity in these extracts was identified as AT II. Thus the greater amount of AT I observed initially in (NH4)2SO4 extracts was probably a consequence of the sonication step used in this extraction procedure.

Polychlorinated biphenyls and related compound interactions with specific binding sites for thyroxine in rat liver nuclear extracts

1987 ◽  
Vol 30 (1) ◽  
pp. 79-86 ◽  
Author(s):  
J. McKinney ◽  
R Fannin ◽  
S. Jordan ◽  
K. Chae ◽  
U. Rickenbacher ◽  
...  
Keyword(s):  
Polychlorinated Biphenyls ◽  
Rat Liver ◽  
Related Compound ◽  
Binding Sites ◽  
Specific Binding ◽  
Specific Binding Sites ◽  
Nuclear Extracts
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