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Specific phosphorylation of pig liver initiation factor eIF-2 by the N-ethylmaleimide-treated hemin-controlled
Abstract:
The specific phosphorylation of pig liver initiation factor 2(eIF-2) by the N-ethylmaleimide (NEM)-treated hemin-controlled translational inhibitor (HCI) from rabbit reticulocytes was investigated. The inhibitor phosphorylated the serine residue of the alpha subunit of eIF-2 (eIF-2 alpha) and 1 mol of phosphate was incorporated into 1 mol of eIF-2 alpha by the inhibitor on maximal phosphorylation, even when eIF-2 was pretreated with alkaline phosphatase prior to phosphorylation. The 32P-labeled eIF-2 alpha was subjected to tryptic digestion and the tryptic digest was analyzed by two-dimensional peptide mapping on a cellulose thin-layer sheet. After 94 h digestion, the autoradiograph of the peptide map showed a single 32P-labeled band with a molecular weight of approximately 1,200. These findings suggest that one specific serine residue of pig liver eIF-2 alpha was phosphorylated by the NEM-treated HCI.
Insights
This study shows that a specific inhibitor precisely phosphorylates the serine residue on pig liver eukaryotic initiation factor 2 alpha subunit. This phosphorylation occurs at a single site, even after enzyme pretreatment.
Area of Science:
- Molecular Biology
- Protein Biochemistry
Background:
- Eukaryotic initiation factor 2 (eIF-2) is crucial for protein synthesis.
- Hemin-controlled translational inhibitor (HCI) regulates translation initiation.
- Phosphorylation of eIF-2 affects its function.
Purpose of the Study:
- To investigate the specific phosphorylation site on pig liver eIF-2 by NEM-treated HCI.
- To characterize the stoichiometry and location of phosphate incorporation.
Main Methods:
- Phosphorylation assays using pig liver eIF-2 and NEM-treated rabbit reticulocyte HCI.
- Analysis of phosphorylated eIF-2 alpha subunit.
- Tryptic digestion and two-dimensional peptide mapping.
- Autoradiography to detect radiolabeled phosphate.
Main Results:
- NEM-treated HCI specifically phosphorylated the serine residue of the eIF-2 alpha subunit.
- Maximal phosphorylation incorporated 1 mole of phosphate per mole of eIF-2 alpha.
- Phosphorylation occurred even on eIF-2 pretreated with alkaline phosphatase.
- Peptide mapping identified a single phosphorylated site on the eIF-2 alpha subunit.
Conclusions:
- Pig liver eIF-2 alpha subunit is phosphorylated at a specific serine residue by NEM-treated HCI.
- The phosphorylation event is highly specific, targeting a single site.
- This finding contributes to understanding translational control mechanisms.