RESUMO
Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λmax = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H2O2 system, but not in sodium hypochlorite.
Assuntos
Apoproteínas/metabolismo , Luciferina de Vaga-Lumes/metabolismo , Imidazóis/metabolismo , Proteínas Luminescentes/metabolismo , Pirazinas/metabolismo , Apoproteínas/biossíntese , Apoproteínas/química , Escherichia coli/metabolismo , Luciferina de Vaga-Lumes/química , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Imidazóis/química , Medições Luminescentes , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/química , Pirazinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The cold-induced expression system in Escherichia coli is useful and we have applied this system to prepare the coelenterazine-utilizing luciferases including Renilla luciferase (RLase), a red-shifted variant of Renilla luciferase (RLase-547), the catalytic domain of Oplophorus luciferase (19kOLase) and Gaussia luciferase (GLase). The luminescence properties of the purified luciferases were characterized by using 10 kinds of C2-modified coelenterazine analogues as a substrate. The order of the maximal luminescence intensity for native coelenterazine was GLase (100%)>RLase (8.0%)>RLase-547 (0.73%)>19kOLase (0.09%) under our assay conditions. The substrate specificities of coelenterazine-utilizing luciferases for the C2-modified analogues showed significant differences, but the emission peaks catalyzed by coelenterazine-utilizing luciferases were not affected by the C2-substituted coelenterazine. These results suggest that the catalytic environment for the oxygenation process of coelenterazine and the excited species of coelenteramide might be different among coelenterazine-utilizing luciferases.
Assuntos
Copépodes/enzimologia , Luciferases de Renilla/genética , Luciferases de Renilla/isolamento & purificação , Renilla/enzimologia , Animais , Catálise , Escherichia coli , Imidazóis/química , Luciferases de Renilla/biossíntese , Luciferases de Renilla/química , Medições Luminescentes , Pirazinas/química , Especificidade por SubstratoRESUMO
Aequorin is a Ca(2+)-binding photoprotein and consists of an apoprotein (apoaequorin) and a 2-peroxide of coelenterazine. Eight new coelenterazine analogues modified at the C2-position were synthesized and incorporated into recombinant apoaequorin with O(2) to yield different semisynthetic aequorins. The luminescence properties and the sensitivity to Ca(2+) of these semisynthetic aequorins were characterized. Two semisynthetic aequorins, namely me- and cf3-aequorin, showed a slow decay of the luminescence pattern with less sensitivity to Ca(2+) and were useful for the cell-based G-protein-coupled receptor (GPCR) reporter assays.
Assuntos
Equorina/química , Substâncias Luminescentes/química , Receptores Acoplados a Proteínas G/metabolismo , Espectrometria de Fluorescência/métodos , Equorina/genética , Equorina/metabolismo , Animais , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Células CHO , Cálcio/química , Cricetinae , Cricetulus , Meia-Vida , Imidazóis/síntese química , Imidazóis/química , Cinética , Oxigênio/química , Pirazinas/síntese química , Pirazinas/química , Receptores Muscarínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
A novel synthetic method for v-coelenterazine (v-CTZ), which is a vinylene-bridged analog of native CTZ with a large red-shifted luminescence property, is described. The synthesis was achieved in a concise way through the use of three sequential cross-coupling reactions and ring-closing metathesis (RCM). A newly synthesized C2-modified trifluoromethyl analog cf3-v-CTZ showed slightly more red-shifted luminescence than v-CTZ when it was used as a substrate for Renilla luciferases.