Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity
The authors have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with (/sup 3/H)tamoxifen aziridine ((/sup 3/H)TAZ) were treated with trypsin (T), ..cap alpha..-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the M/sub r/ 66,000 ER. Immunoblot detection with the primate-specific antibody D75P3..gamma.. revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments were no longer immunoreactive. In contrast, use of the antibody H222SP..gamma.. revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest inn studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.
- Research Organization:
- Univ. of Illinois, Urbana
- OSTI ID:
- 5561778
- Journal Information:
- Biochemistry; (United States), Vol. 26:8
- Country of Publication:
- United States
- Language:
- English
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RECEPTORS
ENZYMATIC HYDROLYSIS
MOLECULAR STRUCTURE
AZIDO COMPOUNDS
CHEMICAL BONDS
CHYMOTRYPSIN
ELECTROPHORESIS
ESTROGENS
MAMMARY GLANDS
MAN
MONOCLONAL ANTIBODIES
PEPTIDE HYDROLASES
RATS
TRITIUM COMPOUNDS
TRYPSIN
TUMOR CELLS
UTERUS
ANIMAL CELLS
ANIMALS
ANTIBODIES
BODY
CHEMICAL REACTIONS
DECOMPOSITION
ENZYMES
FEMALE GENITALS
GLANDS
HORMONES
HYDROLASES
HYDROLYSIS
LABELLED COMPOUNDS
LYSIS
MAMMALS
MEMBRANE PROTEINS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PRIMATES
PROTEINS
RODENTS
SERINE PROTEINASES
SOLVOLYSIS
STEROID HORMONES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques