RESUMO
Chemiluminescence has been observed during catalysis by Mn2(+)-activated ribulose-bisphosphate carboxylase/oxygenase from spinach. The luminescence is ribulose 1,5-bisphosphate (RuBP) and O2-dependent and is inhibited by 2-carboxyarabinitol 1,5-bisphosphate and high concentrations of bicarbonate; it is therefore ascribed to the RuBP oxygenase activity. The luminescence is inhibited by azide and enhanced in D2O and in the presence of diazabicyclooctane. The emission maximum is between 620 and 660 nm. The initial rate of light emission is second order in enzyme concentration. The data strongly suggest that singlet oxygen is produced during turnover, that the observed chemiluminescence is due to dimol emission of singlet oxygen, and that this provides a basis for a highly sensitive assay for RuBP oxygenase.
Assuntos
Manganês/farmacologia , Oxigênio/metabolismo , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Azidas/farmacologia , Medições Luminescentes , Pentosefosfatos/farmacologia , Piperazinas/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Ribulose-Bifosfato Carboxilase/antagonistas & inibidores , Oxigênio Singlete , Álcoois Açúcares/farmacologiaRESUMO
Irradiation of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach in the presence of vanadate at 4 degrees C resulted in rapid loss of carboxylase activity. The inactivation was light and vanadate dependent. When the enzyme was irradiated in the presence of the substrate ribulose 1,5-bisphosphate or an analogue such as fructose 1,6-bisphosphate, the inactivation was greatly reduced. Sodium bicarbonate and phosphate also protected against inactivation. No additional protection was observed in the presence of Mg2+ nor did Mg2+ alone protect. Carboxylase activity could be partially restored by treatment with NaBH4, and the photomodified protein could be tritiated with NaB3H4. Amino acid analysis showed that the tritium had been incorporated into serine. The data suggest that an active-site serine is photooxidized by vanadate to an aldehyde which results in activity loss. Irradiation in the presence of vanadate also resulted in cleavage in the large subunit of the enzyme which was subsequent to inactivation.
