RESUMO
BiP is a member of the hsp70 family of proteins that is present in the endoplasmic reticulum where it functions as a molecular chaperone. Rapid quantitative assays have been used to study the effect of mutating BiP residue 229, located in the ATP binding site, from threonine to glycine. Although binding of ATP to the mutant BiP was not affected, the mutant protein possessed 10-20% of the wild-type BiP ATPase activity. Binding to a model peptide substrate, substance P (Brot et al. (1994) Proc. Natl. Acad. Sci. USA 91, 12120-12124), was twofold higher with mutant BiP at 4 degrees C than with wild-type BiP, and was ATP dependent. Under these conditions the substance P that was bound to mutant BiP, but not the wild-type, could be released by higher levels of ATP (5-10 microM), and the ratio of substance P released to ATP hydrolyzed was greater than 10. These results suggest that stoichiometric ATP hydrolysis is not required for release of a chaperone from its substrate.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Substância P/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Cricetinae , Primers do DNA/genética , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Hidrólise , Técnicas In Vitro , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Substância P/química , Substância P/genéticaRESUMO
BiP is a member of the Hsp70 heat shock protein family found in the lumen of the endoplasmic reticulum, that binds to a variety of proteins destined to be secreted. Substance P (SP) has been used as a model peptide to study the interaction of BiP with protein substrates. SP stimulates BiP ATPase activity and forms a stable complex with BiP that is dissociated in the presence of levels of ATP > 50 microM. At lower concentrations of ATP, the SP remains bound to BiP, and the results are consistent with the view that a BiP-ATP complex is initially formed that reacts with SP to form a ternary complex, SP-BiP-ATP. Hydrolysis of ATP in this complex yields a SP-BiP-ADP complex. An exchange of ATP with ADP bound to BiP has also been demonstrated, and the results suggest that the interactions of BiP with ATP resemble those seen with GTP-binding proteins and GTP.
