Preparation and characterization of two isozymes of choline acetyltransferase from squid head ganglia. II. Self-association, molecular weight determinations, and studies with inactivating antisera.
The two isozymes of choline acetyltransferase (Acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) from head ganglia of Loligo pealei have been examined by polyacrylamide gel electrophoresis, gel chromatography, and equilibrium sedimentation in the ultracentrifuge. Inactivating antisera, prepared to both native and dithiothreitol-treated isozymes 1 and 2 of squid choline acetyltransferase, were used to demonstrate the immunologic identity of isozymes 1 and 2. Each isozyme appeared to contain two non-identical catalytically active subunits, with molecular weights of approx. 37 000 and 56 000. A staining method was developed to visualize choline acetyltransferase activity in acrylamide gels. The method is based on the formation of a precipitate of manganese ferrocyanide at sites where free coenzyme A is released. By this method, and by analysis of gel slices, it was found that each of the isozymes can form aggregates of several different sizes. The formation of immune precipitates with the aggregates showed the identity of the multiple bands of enzyme protein resolved on disc gel electrophoresis. Isozyme 1 was most active as a small aggregate, whereas isozyme 2 was most active as a large aggregate. Both chromatography on Sephadex G-200 and isoelectric focusing yielded a number of active species with molecular weights ranging from 35 000 to 300 000. In addition, we demonstrated the dissociation of enzyme protein in the presence of 1.0 - 10(-2) M dithiothreitol, the formation of multiple precipitin bands by aged enzyme, and the identity of the different isoelectric fractions of each of the isozymes.
D000123:Acetyltransferases / Q000378:metabolism*; D002795:Choline O-Acetyltransferase / Q000276:immunology / Q000378:metabolism*; D049832:Decapodiformes / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004589:Electrophoresis, Disc; D004591:Electrophoresis, Polyacrylamide Gel; D005295:Ferrocyanides; D005724:Ganglia / Q000201:enzymology*; D006257:Head; D007106:Immune Sera; D005779:Immunodiffusion; D007527:Isoenzymes / Q000378:metabolism; D008345:Manganese; D008970:Molecular Weight; D011487:Protein Conformation; D013194:Staining and Labeling
D005295:Ferrocyanides; D007106:Immune Sera; D007527:Isoenzymes; D008345:Manganese; D000123:Acetyltransferases; D002795:Choline O-Acetyltransferase; D004229:Dithiothreitol