A continuous fluorescence assay for cyclic nucleotide phosphodiesterase hydrolysis of cyclic GMP |
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| Authors: | J D Johnson J D Walters J S Mills |
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| Affiliation: | 1. Faculdade de Belas-Artes da Universidade de Lisboa, Largo da Academia Nacional de Belas-Artes, 1249-058 Lisboa, Portugal;2. Laboratório de Instrumentação, Engenharia Biomédica e Física da Radiação (LIBPhys-UNL), Departamento de Física, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte da Caparica, Portugal;3. Laboratório José de Figueiredo, Direcção Geral do Património Cultural, Rua das Janelas Verdes 37, 1249-018 Lisboa, Portugal;4. Laboratório HERCULES e Centro de Química de Évora, Universidade de Évora, Largo Marquês de Marialva 8, 7000 Évora, Portugal |
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| Abstract: | The fluorescent 2'-methylanthraniloyl derivative of cyclic GMP undergoes a 45% decrease in fluorescence when it is cleaved by brain phosphodiesterase in the presence of calmodulin. This fluorescence decrease is dependent upon calcium, calmodulin, and phosphodiesterase, and correlates well (r = 0.996) with the disappearance of substrate as monitored by high-performance liquid chromatography. The Kd values determined by this fluorescence method and HPLC suggest that cyclic GMP and its fluorescent derivative exhibit similar kinetic parameters in their hydrolysis. |
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