Covalent stabilization of the nontransformed chick oviduct cytosol progesterone receptor by chemical cross-linking.
The nontransformed forms of the chick oviduct cytosol progesterone receptor of sedimentation coefficient approximately 8 S (8S-PR) are heterooligomers including one hormone binding molecule, either B, approximately 110,000, or A, approximately 79,000, and two non-hormone binding subunits recently identified as heat-shock protein Mr approximately 90,000 (hsp 90) [Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., & Baulieu, E. E. (1984) Biochemistry 23, 6016-6023]. In the crude cytosol, bisimidates reacted under mild conditions and gave rise to complexes, binding progesterone and reacting with BF4, an anti-hsp 90 monoclonal antibody. These complexes have a sedimentation coefficient of 8.4 S and Rs of 8.1 nm in the presence of 0.4 M KCl and in the absence of molybdate ions, i.e., in conditions that would transform non-cross-linked 8S-PR to Rs approximately 5 nm forms of approximately 4-S sedimentation coefficient. All bisimidates tested, of an effective reagent length between 0.73 and 1.09 nm, gave comparable results in the cytosol prepared with or without molybdate ions, confirming that the latter were not responsible for the formation of the cross-linked 8S complexes. It was found that the dimethyl pimelimidate cross-linked 8S-PR was more resistant to inactivating conditions, urea, or heat treatment than the non-cross-linked 8S-PR. The 8S-PR cross-linked in the cytosol was purified by affinity chromatography in the absence of molybdate ions. After purification, it also reacted with the monoclonal antibody BF4 and had the same Rs (8.0 nm), sedimentation coefficient (approximately 8.5 S), and thus Mr (approximately 290,000) as the original cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
Arànyi
P
P
Inserm U 33, Université Paris-Sud, Bicêtre, France.
Radanyi
C
C
Renoir
M
M
Devin
J
J
Baulieu
E E
EE
D000818:Animals; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010057:Oviducts / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000302:isolation & purification / Q000378:metabolism*
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011980:Receptors, Progesterone