Immunochemical characterization of the ATP-stimulated glucocorticoid-receptor-translocation promoter from various organs of rat.
We previously found a novel endogenous factor in rat liver cytosol, named ATP-stimulated glucocorticoid-receptor-translocation promoter (ASTP), that increased the binding of activated glucocorticoid-receptor to nuclei in the presence of ATP. In this work, we immunized rabbits with the purified ASTP protein and characterized the antibodies with regard to titer, cross-reactivity and specificity. An IgG fraction from sera of the immunized rabbits contained specific antibodies to ASTP. The anti-ASTP IgG could precipitate the ASTP protein without the activity. Immunoblot analysis revealed a major band of 48 kDa in rat liver cytosol that migrated to the same position as the purified ASTP protein by SDS-PAGE, and an additional minor band of about 50 kDa. Monospecific antibodies purified from the IgG fraction using the antigen (the purified 48-kDa ASTP protein) immobilized on a polyvinylidene difluoride membrane also reacted with both the 48-kDa ASTP protein and the 50-kDa protein in rat liver cytosol, suggesting that this 50-kDa protein is immunologically related to the 48-kDa ASTP protein. Densitometric quantification of immunoblots demonstrated that the rat kidney cytosol contained ASTP protein at a concentration of about 20% of that of liver cytosol. Other tissues such as brain, skeletal muscle, heart, and lung, contained neither the ASTP protein nor the activity.
Okamoto
K
K
Department of Biochemistry, St. Marianna University School of Medicine, Kanagawa.
Liu
G
G
Yu
W G
WG
Isohashi
F
F
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D000918:Antibody Specificity; D002352:Carrier Proteins; D002499:Centrifugation, Density Gradient; D003429:Cross Reactions; D003600:Cytosol / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000032:analysis*; D005991:Glycerol Kinase; D015151:Immunoblotting; D007120:Immunochemistry; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D009928:Organ Specificity / Q000502:physiology; D051381:Rats
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; C485945:Gk protein, rat; D005991:Glycerol Kinase