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Establishment of a steroid binding assay for goldfish membrane progesterone receptor (mPR) by coupling with graphene quantum dots (GQDs).
Hossain, Forhad; Hossain, Shakhawat; Jyoti, Maisum Sarwar; Omori, Yuki; Tokumoto, Toshinobu.
Afiliación
  • Hossain F; Department of Bioscience, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-Ku, Shizuoka, 422-8529, Japan.
  • Hossain S; Biological Science Course, Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka, 422-8529, Japan.
  • Jyoti MS; Department of Bioscience, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-Ku, Shizuoka, 422-8529, Japan.
  • Omori Y; Biological Science Course, Department of Science, Graduate School of Integrated Science and Technology, Shizuoka University, 836 Ohya, Suruga-Ku, Shizuoka, 422-8529, Japan.
  • Tokumoto T; Department of Bioscience, Graduate School of Science and Technology, National University Corporation Shizuoka University, Ohya 836, Suruga-Ku, Shizuoka, 422-8529, Japan. tokumoto.toshinobu@shizuoka.ac.jp.
Fish Physiol Biochem ; 50(3): 1331-1339, 2024 Jun.
Article en En | MEDLINE | ID: mdl-38329580
ABSTRACT
A homogeneous assay was developed to evaluate ligands that target the membrane progesterone receptor alpha (mPRα) of goldfish. This was achieved by employing graphene quantum dots (GQDs), a type of semiconductor nanoparticle conjugated to the goldfish mPRα. When progesterone-BSA-fluorescein isothiocyanate (P4-BSA-FITC) was combined with the other agents, fluorescence was observed through Förster resonance energy transfer (FRET). However, this fluorescence was quenched by binding between the ligand and receptor. This established method demonstrated the ligand selectivity of the mPRα protein. Then, the methylotrophic yeast Pichia pastoris was used to express the goldfish mPRα (GmPRα) protein. The recombinant purified GmPRα protein was coupled with graphene quantum dots (GQDs) to generate GQD-conjugated goldfish mPRα (GQD-GmPRα). Fluorescence at a wavelength of 520 nm was observed through FRET upon the combination of P4-BSA-FITC and subsequent activation by ultraviolet (UV) light. Adding free P4 to the reaction mixture resulted in a decrease in fluorescence intensity at a wavelength of 520 nm. The fluorescence was reduced by the administration of GmPRα ligands but not by steroids that do not interact with GmPRα. The findings indicated that the interaction between the ligand and receptor led to the formation of a complex involving GQD-GmPRα and P4-BSA-FITC. The interaction between the compounds and GQD-GmPRα was additionally validated by a binding experiment that employed the radiolabeled natural ligand [3H]-17α,20ß-dihydroxy-4-pregnen-3-one. We established a ligand-binding assay for the fish membrane progesterone receptor that is applicable for screening compounds.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Carpa Dorada / Receptores de Progesterona / Transferencia Resonante de Energía de Fluorescencia / Puntos Cuánticos / Grafito Límite: Animals Idioma: En Revista: Fish Physiol Biochem Año: 2024 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Carpa Dorada / Receptores de Progesterona / Transferencia Resonante de Energía de Fluorescencia / Puntos Cuánticos / Grafito Límite: Animals Idioma: En Revista: Fish Physiol Biochem Año: 2024 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Países Bajos