Site-specific antibodies to the DNA-binding domain of estrogen receptor distinguish this protein from the 3H-estradiol-binding protein in pancreas.
The estradiol-binding protein (EBP) in extracts of rat and rabbit pancreata was characterized by sucrose density gradient analysis, immunoaffinity adsorption and Western blot (immunoblot) analysis using polyclonal antibodies raised against EBP. Rat pancreatic extracts labeled with 3H-estradiol contained a readily resolvable peak of steroid-binding activity that sedimented as a 4S complex on sucrose density gradients in the presence or absence of 0.4 M KCl. Estrogen receptor (ER) from calf uterine cytosols sedimented as a 4S complex on gradients containing 0.4 M KCl and as an 8S entity on gradients without KCl. Incubation of cytosol fractions from rat pancreas and calf uterus with benzoyl-DL-arginyl-p-nitroanilide (BAN) increased specific binding of 3H-estradiol to EBP but not to ER. Furthermore, two distinct site-specific antibodies to the DNA-binding domain of ER caused a marked increase in sedimentation rate of 3H-estradiol-labeled ER while normal rabbit serum and antibodies against EBP were ineffective in this regard. These data suggest that a significant portion, if not all, of the DNA-binding domain of ER is absent from EBP. Examination of the amino acid sequence of the DNA-binding domain of ER revealed a region of 10 amino acids that is significantly homologous to somatostatin, a tetradecapeptide that is a co-ligand in the binding of 3H-estradiol to EBP. Based on this observation, a possible mode of action of EBP in pancreatic acinar cells is proposed.
Grossman
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Department of Pharmacology, New York University Medical Ctr., N.Y. 10016.
Traish
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D008297:Male; D008969:Molecular Sequence Data; D010179:Pancreas / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D011960:Receptors, Estrogen / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D012738:Sex Hormone-Binding Globulin / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D014316:Tritium; D014599:Uterus / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D000939:Epitopes; D046911:Macromolecular Substances; D011960:Receptors, Estrogen; D012738:Sex Hormone-Binding Globulin; D014316:Tritium; D004958:Estradiol; D004247:DNA