PUBLICATION
Purification and Identification of the 20S Proteasome Complex from Zebrafish
- Authors
- Hasan, A.M., Jyoti, M.M.S., Rana, M.R., Rezanujjaman, M., Tokumoto, T.
- ID
- ZDB-PUB-220217-4
- Date
- 2022
- Source
- Zebrafish 19: 18-23 (Journal)
- Registered Authors
- Tokumoto, Toshinobu
- Keywords
- 20S proteasome, TOF-MS, proteome analysis, purification
- MeSH Terms
-
- Animals
- Electrophoresis, Gel, Two-Dimensional
- Peptides
- Proteasome Endopeptidase Complex*/genetics
- Proteasome Endopeptidase Complex*/isolation & purification
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Zebrafish*/metabolism
- Zebrafish Proteins*/genetics
- Zebrafish Proteins*/isolation & purification
- PubMed
- 35171713 Full text @ Zebrafish
Citation
Hasan, A.M., Jyoti, M.M.S., Rana, M.R., Rezanujjaman, M., Tokumoto, T. (2022) Purification and Identification of the 20S Proteasome Complex from Zebrafish. Zebrafish. 19:18-23.
Abstract
The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish (Danio rerio) from the whole body. The cytosolic fraction of zebrafish hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA), a well-known substrate for the proteasome, in the presence of sodium dodecyl sulfate. From the cytosolic fraction, the 20S proteasome was purified using five column chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S-300 gel, hydroxylapatite, and phenyl Sepharose. Electrophoresis and Western blot analyses showed that zebrafish 20S proteasome subunits have molecular masses ranging from 22 to 33 kDa. The subunit composition of the purified 20S proteasome was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation. Fourteen kinds of 20S subunits were found. As a special characteristic of zebrafish, two proteins of the α1 subunit were identified. In addition, the results suggested that the α8 subunit is in the 20S complex instead of the α4 subunit. In this study, we demonstrated the subunit composition of the 20S proteasome complex present in zebrafish cells.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping