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Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver
S R Kimball1, A M Karinch, R C Feldhoff
1Department of Cellular and Molecular Physiology, Pennsylvania State University, College of Medicine, Milton S. Hershey Medical Center, Hershey, 17033.
Biochimica Et Biophysica Acta
|December 15, 1994
Summary
This study purified eukaryotic initiation factor (eIF)-2B and developed antibodies to quantify eIF-2B and eIF-2 levels in rat tissues. Results show the epsilon-subunit is the sole phosphorylation site, impacting eIF-2B activity.
Area of Science:
- Molecular Biology
- Protein Biochemistry
Background:
- Eukaryotic initiation factor (eIF)-2B is crucial for protein synthesis.
- Understanding eIF-2B's regulation and interactions is vital for cellular processes.
Purpose of the Study:
- To purify and characterize eukaryotic initiation factor (eIF)-2B.
- To develop antibodies for quantifying eIF-2B and eIF-2 in different tissues.
- To investigate the phosphorylation of eIF-2B subunits.
Main Methods:
- Purification of eIF-2B from rat and bovine liver.
- Characterization of subunit composition, molecular weights, and isoelectric points.
- Generation of monoclonal antibodies against eIF-2B subunits.
- Quantitation of eIF-2B and eIF-2 using antibody-based assays.
- Protein kinase assays using purified eIF-2B as substrate.
Main Results:
- Purified eIF-2B consists of five subunits with distinct molecular weights.
- Monoclonal antibodies were generated against the epsilon-subunit of eIF-2B.
- Direct quantitation revealed lower eIF-2B to eIF-2 ratios in reticulocytes compared to liver.
- Protein kinase activity in rat liver extracts specifically phosphorylated the epsilon-subunit of eIF-2B.
Conclusions:
- The ratio of eIF-2B to eIF-2 suggests potential sequestration of eIF-2B by phosphorylated eIF-2.
- The epsilon-subunit is the primary target for phosphorylation by liver protein kinases.
- This study provides the first direct quantitation of eIF-2 and eIF-2B in different tissues, offering insights into translational regulation.
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