title: Activation of rat choline acetyltransferase by limited proteolysis.
abstract: In the past, purification of choline acetyltransferase (ChAT, EC 2.3.1.6.), 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 Km 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-k-Da 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.
authors.affiliation: Department of Biochemistry, University of Kentucky Lexington 40536-2284, USA.
authors.forename: D
authors.initials: D
authors.lastname: Wu
authors.forename: S N
authors.initials: SN
authors.lastname: Ahmed
authors.forename: W
authors.initials: W
authors.lastname: Lian
authors.forename: L B
authors.initials: LB
authors.lastname: Hersh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D004789:Enzyme Activation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002795:Choline O-Acetyltransferase; D014357:Trypsin