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Heat-stable inhibitor of translation in reticulocyte lysates
Insights
A heat-stable protein contaminant (HS) was identified as the cause of translational inhibition in reticulocyte lysates. This protein phosphorylates eukaryotic initiation factor 2 alpha subunit, impacting protein synthesis.
Area of Science:
- Molecular Biology
- Biochemistry
- Protein Synthesis Regulation
Background:
- Earlier studies reported translational inhibition by cAMP-dependent protein kinase catalytic subunit (cS) preparations.
- This inhibition was attributed to a heat-stable protein contaminant (HS).
Purpose of the Study:
- To purify and characterize the heat-stable protein (HS) responsible for translational inhibition.
- To elucidate the mechanism of action of HS in protein synthesis.
Main Methods:
- Purification of HS from bovine heart.
- Analysis of HS polypeptide chain and molecular weight.
- Assay of translational inhibition kinetics and effects of eIF-2, GTP, and double-stranded RNA.
- Investigation of HS effect on heme-controlled translational inhibitor (HCI) and proinhibitor (proHCI).
Main Results:
- HS was purified to homogeneity, consisting of a single polypeptide chain (Mr ~68,000).
- HS exhibited biphasic translational inhibition kinetics, similar to hemin deficiency.
- HS induced phosphorylation of the eukaryotic initiation factor eIF-2 alpha subunit.
- Inhibition was relieved by eIF-2 or GTP, and not by double-stranded RNA.
- HS increased the ratio of heme-controlled translational inhibitor (HCI) to its proinhibitor form (proHCI).
Conclusions:
- A novel heat-stable protein (HS) is identified as a potent inhibitor of translation.
- HS acts by phosphorylating eIF-2 alpha, a key regulatory step in protein synthesis.
- The precise mode of action of HS remains to be determined.
Abstract:
Inhibition of translation in hemin-containing reticulocyte lysates by catalytic subunit (cS) preparations of cAMP-dependent protein kinase from bovine heart, reported earlier by our group, is due to a highly active heat-stable protein contaminant (HS). The specific activity for translational inhibition goes up by a factor of 10 when cS is heated for 10 min at 80 degrees C, which completely destroys histone phosphorylation activity. HS has been purified to homogeneity from bovine heart. It consists of a single polypeptide chain (Mr approximately 68,000). HS inhibits translation with biphasic kinetics similar to those of hemin deficiency and induces pronounced phosphorylation of the alpha subunit of the eukaryotic initiation factor eIF-2. The inhibition is relieved by eIF-2 or GTP but not by high concentrations of double-stranded RNA, thus ruling out involvement of the double-stranded RNA-activated inhibitor. Judged by poly(U) translation, HS has no effect on chain elongation. When added to crude preparations of the proinhibitor form (proHCI) of the heme-controlled translational inhibitor (HCI), HS appears to produce an increase of the HCI-to proHCI ratio. The mode of action of HS is as yet unknown.