RESUMO
Highly sensitive self-cleavable trimethyl lock quinone-luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H-dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.
Assuntos
Luciferina de Vaga-Lumes/análogos & derivados , Substâncias Luminescentes/metabolismo , NADP/metabolismo , Quinonas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Luciferina de Vaga-Lumes/análise , Luciferina de Vaga-Lumes/metabolismo , Humanos , Substâncias Luminescentes/análise , Medições Luminescentes , NADP/análise , Quinonas/análiseRESUMO
A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.
Assuntos
Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/metabolismo , Luciferases/metabolismo , Alquilação , Animais , Domínio Catalítico , Cinética , Luz , Luciferases/química , Luciferases de Vaga-Lume/genética , Luminescência , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
New highly sensitive latent bioluminescent luciferin substrates were designed and synthesized for monitoring mammalian glutathione S-transferase (GST) and Schistosoma japonicum enzyme activities.