Báo cáo khoa học: Purification and partial characterization of seven glutathione S -transferase isoforms from the clam Ruditapes decussatus

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus

Tác giả: Pascal Hoarau, Ginette Garello, Mauricette Gnassia-Barelli, Michèle Romeo and Jean-Pierre Girard

Lĩnh vực: Biochemistry, Marine Biology

Nội dung tài liệu: This study details the purification and partial characterization of seven glutathione S-transferase (GST) isoforms isolated from the clam Ruditapes decussatus. Employing a two-step affinity chromatography process using GSH-agarose and S-hexyl GSH-agarose, followed by anion exchange chromatography, seven distinct GST isoforms were successfully purified. Six of these isoforms were identified as homodimers, while one was a heterodimer composed of subunits 3 and 6. Kinetic parameters, including affinity and Vmax for GSH and 1-chloro-2,4-dinitrobenzene (CDNB), were investigated for these isoforms. Additionally, immunological properties were examined using antisera against mammalian GST classes (pi, mu, and alpha). N-terminal amino acid sequencing was performed to further elucidate the classification of these isoforms, with subunit 4 showing significant identity to conserved sequences of alpha/mu/pi GSTs from Fasciola hepatica. The research contributes to understanding the diversity and characteristics of GST isoenzymes in marine invertebrates.

Mục lục chi tiết:

• Introduction
• Materials and Methods
• Animal maintenance
• Preparation of the postmitochondrial fraction
• Enzyme assays
• Purification of GSTs
• Kinetic parameters
• SDS/PAGE
• Western blotting
• Results
• Purification of GSTs and HPLC separation of subunits
• Activities and kinetics specificities
• N-Terminal amino acid sequencing
• Discussion
• Acknowledgements
• References