RESUMO
The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.
Assuntos
Ferredoxinas/metabolismo , Glutamato Sintase/metabolismo , Anticorpos , Sítios de Ligação de Anticorpos , Dicroísmo Circular , Ferredoxinas/imunologia , Glutamato Sintase/imunologia , Imunodifusão , Imunoglobulina G , Cinética , Nitrito Redutases/metabolismo , Plantas/metabolismo , Conformação Proteica , Espectrofotometria/métodosRESUMO
Polyclonal antisera were prepared against ferredoxin-nitrite reductase (EC 1.7.7.1) and ferredoxin-glutamate synthase (glutamate synthase (ferredoxin); EC 1.4.7.1) from the green alga Chlamydomonas reinhardtii. The anti-glutamate synthase antibodies recognized both glutamate synthase and nitrite reductase, but inhibited only the ferredoxin-linked activity of the latter enzyme and not the activity dependent on methyl viologen. Analogously, the anti-nitrite reductase antibodies recognized glutamate synthase and nitrite reductase but the first enzyme was only poorly inhibited. Free ferredoxin protected the nitrite reductase against its inactivation by anti-glutamate synthase antibodies. These results indicate that the ferredoxin-dependent glutamate synthase and nitrite reductase from this alga share common antigenic determinants, and that these are located at the ferredoxin-binding domains.
Assuntos
Chlamydomonas/enzimologia , Glutamato Sintase/imunologia , NADH NADPH Oxirredutases/imunologia , Nitrito Redutases/imunologia , Transaminases/imunologia , Chlamydomonas/imunologia , Reações Cruzadas , Ferredoxina-Nitrito Redutase , Ferredoxinas/farmacologia , ImunodifusãoAssuntos
Antranilato Sintase/imunologia , Proteínas de Bactérias/imunologia , Escherichia coli/enzimologia , Glutamato Sintase/imunologia , Transaminases/imunologia , Antranilato Sintase/isolamento & purificação , Anticorpos , Proteínas de Bactérias/isolamento & purificação , Testes de Fixação de Complemento , Reações Cruzadas , Glutamato Sintase/isolamento & purificação , Testes de Precipitina , RadioimunoensaioRESUMO
The large and small subunits of Escherichia coli glutamate synthase were isolated. The small subunit catalyzes the NH(3)-dependent synthesis of glutamate. The large subunit exhibits glutaminase activity.