RESUMO
Human cytochrome P-450 (P-450) 1A2 expressed in Escherichia coli is readily converted into non-native cytochrome P-420 (P-420) in the presence of detergents. alpha-Naphthoflavone (ANF) has been used to prevent P-450 1A2 inactivation to P-420 during purification. However, the mechanism by which ANF modulates P-450 1A2 is not clearly understood. We observed that recombinant human P-450 1A2 prepared in the absence of ANF has an approx. 5 times higher maximum catalytic activity in the O-deethylation of 7-ethoxycoumarin than that in the presence of ANF, with the same K(m) values. The results revealed that the enzyme purified with ANF is not catalytically fully active, indicating that ANF tightly binds to the enzyme, only to be dissociated by heat denaturation. Furthermore, the inactive P-420 form of the enzyme could be reconverted to P-450 by ANF in high concentrations of detergents. The reconversion was concentration-dependent, confirming ANF-induced regeneration of active P-450 1A2. The reconversion coincided with the conformational change of the enzyme including increased alpha-helix content. The conformation of P-450 1A2 was also stabilized by ANF, resulting in an approx. 5 degrees C increase in thermal stability.
Assuntos
Benzoflavonas/química , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/metabolismo , Benzoflavonas/farmacologia , Cromatografia por Troca Iônica , Cumarínicos/metabolismo , Citocromo P-450 CYP1A2/isolamento & purificação , Detergentes/química , Detergentes/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Ligantes , Ligação Proteica/fisiologia , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
Kinetochores are the elaborate protein assemblies that attach chromosomes to spindle microtubules in mitosis and meiosis. The kinetochores of point-centromere yeast appear to represent an elementary module, which repeats a number of times in kinetochores assembled on regional centromeres. Structural analyses of the discrete protein subcomplexes that make up the budding-yeast kinetochore have begun to reveal principles of kinetochore architecture and to uncover molecular mechanisms underlying functions such as transmission of tension and establishment and maintenance of bipolar attachment. The centromeric DNA is probably wrapped into a compact organization, not only by a conserved, centromeric nucleosome, but also by interactions among various other DNA-bound kinetochore components. The rod-like, heterotetrameric Ndc80 complex, roughly 600 Å long, appears to extend from the DNA-proximal assembly to the plus end of a microtubule, to which one end of the complex is known to bind. Ongoing structural studies will clarify the roles of a number of other well-defined complexes.