Multiple isoelectric and molecular weight variants of choline acetyltransferase. Artifact or real?

Abstract

The existence of isoelectric variants of bovine brain and human placental choline acetyltransferase was confirmed by chromatofocusing. The identification of molecular weight variants (bovine brain, Mr = 68,000 and 63,000; human placental, Mr = 66,000 and 64,000) was also demonstrated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by "Western blotting" and immunochemical visualization of choline acetyltransferase with monoclonal antibodies. No correlation between the isoelectric variants and molecular weight variants could be observed; however, in the case of the bovine brain enzyme the more alkaline isoelectric variant was enriched in the higher molecular weight form of the enzyme. Treatment of the bovine brain enzyme with Staphylococcus aureus V8 protease resulted in the conversion of the Mr = 68,000 form of the enzyme to the Mr = 63,000 form. During this conversion no change in the enzyme activity was observed demonstrating that the Mr = 63,000 form of the enzyme and probably also the Mr = 68,000 form of the enzyme are active. Preparation of the bovine brain enzyme in the presence of proteolytic enzyme inhibitors yielded a new higher molecular weight form of the enzyme, Mr = 73,000, which was enzymatically active. The Mr = 73,000 form of the enzyme exhibited a single isoelectric form when analyzed by chromatofocusing. These results suggest that the Mr = 73,000 form of the enzyme represents the native form, and that other molecular weight and isoelectric variants may arise by proteolysis.