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Related Experiment Videos

Cholinergic synaptic vesicles contain a V-type and a P-type ATPase.

S K Yamagata1, S M Parsons

  • 1Department of Chemistry, IES, University of California, Santa Barbara 93106.

Journal of Neurochemistry
|November 1, 1989
PubMed
Summary

Cholinergic synaptic vesicles contain two types of ATPases. A V-type ATPase acts as the proton pump for acetylcholine transport, while a P-type ATPase has an unknown function.

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Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica.

Journal of neurochemistry·2000

Area of Science:

  • Neuroscience
  • Biochemistry
  • Molecular Biology

Background:

  • Cholinergic synaptic vesicles utilize ATPases for active transport.
  • Distinguishing between different ATPase types is crucial for understanding vesicle function.

Purpose of the Study:

  • To identify and characterize the specific ATPases involved in acetylcholine transport in Torpedo electric organ cholinergic synaptic vesicles.
  • To differentiate the roles of P-type and V-type ATPases in vesicle function.

Main Methods:

  • Enzyme activity assays using vanadate and N-ethylmaleimide inhibition.
  • Proteolytic protection assays.
  • Western blot analysis using specific antisera.
  • Vesicle solubilization and purification.

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Main Results:

  • Vanadate inhibited 50-85% of vesicle ATPase activity, attributed to a P-type ATPase.
  • Vanadate stimulated acetylcholine transport, indicating the P-type ATPase is not the proton pump.
  • N-ethylmaleimide sensitive ATPase activity, protected by vanadate, suggests a V-type ATPase.
  • ACh transport was inhibited by N-ethylmaleimide, confirming V-type ATPase involvement.
  • Western blots confirmed the presence of V-type ATPase subunits.
  • Both P-type and V-type ATPases copurified with synaptic vesicles.

Conclusions:

  • Torpedo cholinergic synaptic vesicles possess both a P-type ATPase of unknown function and a V-type ATPase that functions as the proton pump for acetylcholine transport.
  • Differential detergent solubilization explains previous difficulties in detecting V-type ATPase activity.