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Limited proteolysis by subtilisin reveals structural differences between phosphorylase a and b.
The International Journal of Biochemistry
|January 1, 1983
Summary
Rabbit skeletal muscle phosphorylase a and b exhibit differential susceptibility to subtilisin proteolysis. AMP influences phosphorylase b conformation, making it more similar to phosphorylase a.
Area of Science:
- Biochemistry
- Enzymology
- Proteolysis
Background:
- Phosphorylase a and b are key enzymes in glycogen metabolism.
- Understanding their structural and functional differences is crucial.
- Limited proteolysis offers insights into enzyme conformation.
Purpose of the Study:
- To investigate the limited proteolysis of rabbit skeletal muscle phosphorylase a and b using immobilized subtilisin BPN'.
- To compare the susceptibility of phosphorylase a and b to proteolysis under specific conditions.
- To analyze the effect of AMP on the conformation of phosphorylase b.
Main Methods:
- Immobilization of subtilisin BPN' onto Sepharose 4B.
- Limited proteolysis assays of phosphorylase a and b at pH 7.0 and 30°C.
- Analysis of proteolytic fragments using SDS-gel electrophoresis.
Main Results:
- Phosphorylase a rapidly degraded, while phosphorylase b showed resistance to subtilisin.
- Distinct pH profiles observed for the digestion of phosphorylase a and b.
- SDS-gel electrophoresis revealed specific fragment sizes for both enzymes (e.g., 70,000 and 30,000 Da for phosphorylase a; 80,000 and 70,000 Da for phosphorylase b).
- AMP addition induced a conformational change in phosphorylase b, mimicking phosphorylase a in its recognition by subtilisin.
Conclusions:
- Subtilisin BPN' effectively differentiates between the conformations of phosphorylase a and b.
- AMP binding shifts phosphorylase b towards an 'a-like' conformation, altering its susceptibility to proteolysis.
- Proteolysis patterns provide valuable information on enzyme structure and allosteric regulation.