Purification and characterization of glutamate dehydrogenase from rainbow trout (Oncorhynchus mykiss) liver and molecular docking studies.
Biotechnol Appl Biochem
; 71(5): 1005-1024, 2024 Oct.
Article
em En
| MEDLINE
| ID: mdl-38689532
ABSTRACT
Glutamate dehydrogenase (GDH) participates in the energy metabolism of proteins and the synthesis of metabolites important for the organism. In this study, GDH enzyme was purified from the liver of rainbow trout (Oncorhynchus mykiss) by 2',5'-ADP Sepharose 4B affinity chromatography in one step. As a result of this purification process, GDH enzyme was purified 171-fold with 5.83 U/mg protein-specific activity. The characterization experiments presented that the storage stability of the purified GDH enzyme was determined as -80°C; optimum temperature 40°C; it was determined that the optimum ionic strength was 100 mM phosphate buffer and the optimum pH was 8.00. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and PAGE studies showed that the natural molar mass of the purified GDH enzyme was 346.74 kDa, and the molar mass of its subunits was 53.71 kDa. Km and Vmax values for substrates and coenzymes of GDH enzyme purified from rainbow trout liver were calculated, and the lowest Km value was found in NAD+ (1.86 mM) and the highest Vmax value in NH4 + (1.79 U/mL). The effects of some metal ions, vitamins, and solvents on the activity of the purified GDH enzyme were investigated and also IC50 values and inhibition types. The metal ion with the lowest IC50 value is Ag+ (8.65 ± 1.68 µM), and the vitamin is B6 (0.77 ± 0.04 mM). The binding affinities of inhibitors were investigated with molecular docking, based on the conformational state of GDH.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oncorhynchus mykiss
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Simulação de Acoplamento Molecular
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Glutamato Desidrogenase
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Fígado
Limite:
Animals
Idioma:
En
Revista:
Biotechnol Appl Biochem
/
Biotechnol. appl. biochem
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Biotechnology and applied biochemistry
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Turquia