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Influence of the phosphorylation state on the biological activity of a low-molecular mitogen from group A
J H Ozegowski1, E Günther, S Vettermann
1Institut für Experimentelle Mikrobiologie, Friedrich-Schiller-Universität Jena (FSU), Germany.
Abstract:
A low molecular weight mitogen (LMP) from Streptococcus pyogenes strain NY 5 was successively purified by adsorption on phenylsepharose, chromatography on Resource S and Superdex G 30 and finally by affinity chromatography on antiphosphothreonine agarose. The N-terminal protein sequence of the mitogen was determined. The occurrence of phosphoamino acids was investigated by immunoassay using monoclonal antibodies. The LMP is a threonine-phosphorylated protein different of HPR protein of PTS-system, its mitogenic activity was lost after treatment with streptococcal protein phosphatase or alkaline phosphatase. The inactivated LMP was activated by phosphorylation with phosphokinase and ATP. The active LMP was also inactivated in streptococcal cultures secreting acid protein phosphatase during the phase of phosphate limitation.
Insights
A low molecular weight mitogen (LMP) from Streptococcus pyogenes is a threonine-phosphorylated protein. Its mitogenic activity depends on phosphorylation, being lost with phosphatases and regained with phosphokinase.
Area of Science:
- Microbiology
- Biochemistry
- Immunology
Background:
- Streptococcus pyogenes produces a low molecular weight mitogen (LMP).
- The role of post-translational modifications, such as phosphorylation, in bacterial mitogen activity is not fully understood.
Purpose of the Study:
- To purify and characterize the LMP from Streptococcus pyogenes.
- To investigate the role of phosphorylation in the mitogenic activity of LMP.
Main Methods:
- Multi-step protein purification including phenylsepharose, Resource S, Superdex G 30, and antiphosphothreonine agarose affinity chromatography.
- N-terminal protein sequencing.
- Immunoassay using monoclonal antibodies to detect phosphoamino acids.
- Enzymatic treatment with streptococcal protein phosphatase, alkaline phosphatase, phosphokinase, and ATP.
Main Results:
- A low molecular weight mitogen (LMP) was purified from Streptococcus pyogenes.
- The LMP was identified as a threonine-phosphorylated protein, distinct from the HPR protein of the PTS system.
- Mitogenic activity was abolished by treatment with protein phosphatases but restored by phosphorylation with phosphokinase and ATP.
- Active LMP was inactivated in Streptococcus cultures during phosphate limitation due to acid protein phosphatase secretion.
Conclusions:
- The mitogenic activity of Streptococcus pyogenes LMP is regulated by threonine phosphorylation.
- Phosphorylation is essential for LMP's mitogenic function, with dephosphorylation inactivating the protein and re-phosphorylation reactivating it.