ACTIVATION OF RAT CHOLINE-ACETYLTRANSFERASE BY LIMITED PROTEOLYSIS

In the past, purification of choline acetyltransferase (ChAT, EC 2.3.1 .6), the enzyme responsible for the biosynthesis of the neurotransmitt er acetylcholine, has yielded fragmented species of the enzyme. The na ture and possible function of these forms of ChAT are not well underst ood, 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 a nd >5-fold when assayed at high ionic strength by limited proteolysis with either trypsin or chymotrypsin, but not with proteinase K. The ac tivated ChAT shows an increased K-m for both substrates, diminished se nsitivity to salt activation and a pH optimum that is shifted approxim ately 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 del ineate the site of cleavage by proteolysis, the newly generated fragme nts have been subjected to N-terminal sequencing, By comparing cleavag e sites between trypsin and chymotrypsin, the putative activation site s were identified.