RESUMO
The 90-kDa heat shock protein (hsp90) is a well conserved, abundant cytosolic protein believed to be a "chaperone" of most steroid receptors. We have recently demonstrated that hsp90 has an ATP-binding site and autophosphorylating activity (Csermely, P., and Kahn, C. R. (1991) J. Biol. Chem. 266, 4943-4950). Circular dichroism analysis of highly purified hsp90 from rat liver shows that ATP induces an increase of beta-pleated sheet content of hsp90. Vanadate, molybdate, and heat treatment at 56 degrees C induce a similar change in the circular dichroism spectrum. Fourier transformed infrared spectroscopy reveals an ATP-induced increase in the interchain interactions of the 90-kDa heat shock protein due to an increase in its beta-pleated sheet content. In further studies we found that ATP: 1) decreases the tryptophan fluorescence of hsp90 by 11.6 +/- 1.9%; 2) increases the hydrophobic character of the protein as determined by its distribution between an aqueous phase and phenyl-Sepharose; and 3) renders hsp90 less susceptible to tryptic digestion. Our results suggest that hsp90 undergoes an "open-->closed" conformational change after the addition of ATP, analogous in many respects to the similar changes of the DnaK protein, the immunoglobulin heavy chain binding protein (BiP/GRP78), and hsp70. The ATP-induced conformational change of hsp90 may be important in regulating its association with steroid receptors and other cellular proteins.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas de Choque Térmico/química , Fígado/química , Animais , Dicroísmo Circular , Análise de Fourier , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Masculino , Molibdênio/farmacologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espectrofotometria Infravermelho , Tripsina/metabolismo , Triptofano/química , Vanadatos/farmacologiaRESUMO
1. The metabolism of 14C-flumecinol (3-trifluoromethyl-alpha-ethyl-benzhydrol) was studied in volunteers after a single oral dose of 100 mg (11.1 MBq; 300 microCi). Radioactivity excreted in urine was 78.8 +/- 6.0% of dose and in faeces was 12.0 +/- 5.3% dose in 120 h. 2. Unchanged flumecinol was not excreted in urine, but was present in faeces unconjugated (1.2% dose) and as conjugates of glucuronic and sulphuric acids (10.8% dose). 3. Enzymic hydrolysis showed that all urinary metabolites were conjugated with glucuronic and/or sulphuric acids (77.8% dose). Unconjugated urinary metabolites were not found. 4. The major route of flumecinol metabolism was hydroxylation of the alkyl side chain and/or the phenyl group followed by conjugation. 5. Both the CF3-group and the skeleton of the original compound remained intact during metabolism.
