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Related Experiment Videos

Effect of NaF on type-1 phosphatase aggregation.

R Pesi1, E Villa-Moruzzi

  • 1Ist. di Patologia Generale, Universita di Pisa, Italy.

Biochemical and Biophysical Research Communications
|August 31, 1990
PubMed
Summary

Sodium fluoride (NaF) treatment reveals phosphorylase phosphatase exists as a 70 kDa dimer in muscle. This finding contrasts with its larger, higher molecular weight forms observed without NaF, suggesting a structural change.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Phosphorylase phosphatase plays a crucial role in glycogen metabolism.
  • The native molecular weight and quaternary structure of phosphorylase phosphatase in muscle tissue are not fully understood.

Purpose of the Study:

  • To investigate the effect of sodium fluoride (NaF) on the molecular state of phosphorylase phosphatase in muscle.
  • To determine the subunit composition and assembly of phosphorylase phosphatase.

Main Methods:

  • Preparation of muscle cytosolic and glycogen fractions.
  • Analysis of protein complexes using SDS-PAGE.
  • Treatment with sodium fluoride (NaF) and cross-linking agents.
  • Purification and fragmentation of phosphatase-1 catalytic subunit.

Main Results:

  • In the presence of 50 mM NaF, phosphorylase phosphatase formed an approximately 70 kDa complex, differing from the >250 kDa form observed without NaF.
  • Purified phosphatase-1 catalytic subunit (37 kDa) formed a 70 kDa complex with NaF, while a 33 kDa tryptic fragment did not.
  • Cross-linking of the 37 kDa subunit-NaF complex resulted in a 66 kDa band, indicating a dimeric structure of the 70 kDa complex.

Conclusions:

  • Sodium fluoride induces a conformational change or dissociation, revealing a dimeric form of phosphorylase phosphatase.
  • The 37 kDa catalytic subunit is essential for the formation of the NaF-induced dimeric complex.
  • Phosphorylase phosphatase exists as a dimer in muscle under specific conditions, influencing its functional state.

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