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Effect of polyamines on globin synthesis in a rabbit reticulocyte polyamine-free protein synthetic system
T Ogasawara1, K Ito, K Igarashi
1Faculty of Pharmaceutical Sciences, Chiba University.
Abstract:
A polyamine-free protein synthetic system, established from components of rabbit reticulocytes, consisted of globin mRNA, salt-washed ribosomes, partially purified initiation factors, and pH 5 enzymes. Spermidine added to this system not only lowered the optimal magnesium concentration required for globin synthesis, but it also stimulated the globin synthesis 6- to 8-fold. The optimal spermidine concentration was 0.4 to 0.6 mM, a concentration similar to that in intact rabbit reticulocytes. The ratio of alpha to beta globin chains synthesized in the presence of spermidine and Mg2+ was approximately 1.0, while the ratio in the presence of only Mg2+ was approximately 1.5. Formation of methionyl puromycin was stimulated 4- to 6-fold by spermidine. This indicates that the stimulation is mainly at the level of initiation.
Insights
Spermidine significantly enhances rabbit reticulocyte protein synthesis by 6- to 8-fold, optimizing globin production. This polyamine also improves the balance of alpha and beta globin chains, primarily by boosting translation initiation.
Area of Science:
- Molecular Biology
- Protein Synthesis
- Biochemistry
Background:
- Rabbit reticulocyte lysates are a common system for studying protein synthesis.
- Polyamines, like spermidine, are known to play roles in cellular processes.
- Understanding the regulation of globin synthesis is crucial for hematology research.
Purpose of the Study:
- To investigate the effect of spermidine on a cell-free protein synthesis system.
- To determine the optimal concentration of spermidine for globin synthesis.
- To elucidate the mechanism by which spermidine influences globin chain production.
Main Methods:
- Established a polyamine-free protein synthesis system using rabbit reticulocyte components.
- Assayed globin synthesis in the presence and absence of varying spermidine concentrations.
- Measured the ratio of alpha to beta globin chains and methionyl puromycin formation.
Main Results:
- Spermidine stimulated globin synthesis 6- to 8-fold at optimal concentrations (0.4-0.6 mM).
- Spermidine lowered the optimal magnesium concentration required for globin synthesis.
- The alpha to beta globin chain ratio shifted towards 1.0 with spermidine, and initiation was stimulated 4- to 6-fold.
Conclusions:
- Spermidine significantly enhances cell-free globin synthesis in rabbit reticulocyte systems.
- The stimulatory effect of spermidine is primarily at the level of translation initiation.
- Spermidine also improves the stoichiometric balance of alpha and beta globin chain synthesis.