Activation of rat choline acetyltransferase by limited proteolysis

D. Wu, S. N. Ahmed, W. Lian, L. B. Hersh

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13 Scopus citations


In the past, purification of choline acetyltransferase (ChAT, EC, the enzyme responsible for the biosynthesis of the neurotransmitter acetylcholine, has yielded fragmented species of the enzyme. The nature and possible function of these forms of ChAT are not well understood. Using a bacterial expression system, recombinant rat ChAT in its active form has been purified to homogeneity. The purified enzyme was found to be activated to >25-fold when assayed at low ionic strength and >5-fold when assayed at high ionic strength by limited proteolysis with either trypsin or chymotrypsin, but not with proteinase K. The activated ChAT shows an increased K(m) for both substrates, diminished sensitivity to salt activation and a pH optimum that is shifted approximately 1 pH unit. On a denaturing SDS-polyacrylamide gel, the activated ChAT is composed of three to four polypeptides; however, it migrates as an intact 68-kDa protein species on gel filtration. In order to delineate the site of cleavage by proteolysis, the newly generated fragments have been subjected to N-terminal sequencing. By comparing cleavage sites between trypsin and chymotrypsin, the putative activation sites were identified.

Original languageEnglish
Pages (from-to)19395-19401
Number of pages7
JournalJournal of Biological Chemistry
Issue number33
StatePublished - Aug 18 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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