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Structural changes in phosphorylase b as revealed by proteolysis with subtilisin BPN'
Acta Biochimica Et Biophysica; Academiae Scientiarum Hungaricae
|January 1, 1984
Summary
Ligands significantly alter skeletal muscle phosphorylase b proteolysis by subtilisin. Some ligands accelerate inactivation, while others offer protection, and a few surprisingly increase enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Protein Chemistry
Background:
- Rabbit skeletal muscle phosphorylase b is a key enzyme in glycogen metabolism.
- Understanding its structural and functional dynamics is crucial for metabolic research.
- Proteolysis offers insights into enzyme structure-function relationships.
Purpose of the Study:
- To investigate the impact of various ligands on the subtilisin-mediated proteolysis of rabbit skeletal muscle phosphorylase b.
- To elucidate how ligand binding influences enzyme susceptibility to proteolytic degradation.
- To characterize the effects of specific ligands on enzyme activity post-proteolysis.
Main Methods:
- Enzyme proteolysis using Sepharose 4B bound subtilisin BPN' at pH 7.0 and 8.5.
- Monitoring enzyme activity to assess inactivation or activation.
- SDS-PAGE to analyze proteolytic products and subunit integrity.
Main Results:
- AMP and alpha-D-glucose-1-phosphate accelerated phosphorylase b inactivation, yielding 70 kDa and 30 kDa fragments.
- IMP and glycogen protected phosphorylase b from proteolytic degradation.
- D-glucose, caffeine, and D-glucose-6-phosphate treatment resulted in a ~20% increase in phosphorylase b activity (Vmax), without altering subunit size or ligand binding.
Conclusions:
- Ligand binding profoundly modulates the susceptibility of phosphorylase b to proteolysis.
- Specific ligands can either protect the enzyme, accelerate its degradation, or paradoxically enhance its catalytic activity.
- These findings highlight the complex allosteric regulation of phosphorylase b structure and function.