Purification and characterization of a macromolecular-translocation inhibitor III of activated glucocorticoid-receptor-complex binding to nuclei from rat liver.
Macromolecular-translocation inhibitors (MTI) of binding of the activated glucocorticoid-receptor complex (GRC) to nuclei from rat liver are separated into at least three components (MTI-I-III) by DEAE-cellulose column chromatography [Okamoto, K., Isohashi, F., Horiuchi, M. & Sakamoto, Y. (1982) Biochem. Biophys. Res. Commun. 108, 1655-1660]. In this study, we have purified MTI-III from the livers of adrenalectomized rats to apparent homogeneity, as determined by SDS/PAGE. The purification procedure consisted of DEAE-cellulose chromatography, acid treatment and sequential chromatographies using gel filtration, S-Sepharose and Mono S columns. The purified protein had a molecular mass of approximately 69 kDa, as estimated by SDS/PAGE, and the molecular mass of the inhibitor was approximately 68 kDa, as estimated by gel filtration. Thus, MTI-III exerts its inhibitory activity as a monomer. The sedimentation coefficient of MTI-III was approximately 3.7 S. Purified MTI-III was fairly stable at 4 degrees C but at higher temperatures, especially above 25 degrees C, it was rapidly inactivated. Under low-salt conditions, MTI-III was associated with activated GRC (4.2 S) and the resulting complex was detected on sucrose density gradients as a larger species (6.8 S). Initial treatment of nuclei or DNA-cellulose with MTI-III did not alter their abilities to bind activated GRC. These results indicate that MTI-III acts through an interaction with GRC.
Liu
G
G
Department of Oncology, Osaka University Medical School, Japan.
Okamoto
K
K
Isohashi
F
F
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism*; D002498:Centrifugation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000276:immunology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013696:Temperature
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011506:Proteins; D011965:Receptors, Glucocorticoid; C027048:receptor macromolecular translocation inhibitor