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.
Department of Biochemistry, St. Marianna University School of Medicine, Kanagawa.
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Okamoto
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Liu
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WG
Yu
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Isohashi
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