Generation of aspartate aminotransferase multiple forms by deamidation

Area of Science:

• Biochemistry
• Enzymology

Background:

• Aspartate aminotransferase (AST) exists in various subforms.
• Understanding AST subform development and activity is crucial for biochemical studies.

Purpose of the Study:

• To investigate the in vitro development of aspartate aminotransferase subforms.
• To analyze the activity and inactivation mechanisms of these developing subforms.

Main Methods:

• Thin-film isoelectric focusing followed by densitometry to track AST subform development.
• Measurement of ammonia production to assess enzymatic activity.
• Spectrophotometric analysis to characterize enzyme properties.

Main Results:

• In vitro AST subform development was observed, with more negatively charged subforms exhibiting high activity.
• Enzyme inactivation occurred through coenzyme modification, a process independent of deamidation.
• The newly formed enzyme showed distinct absorption properties (maximal at 340nm) compared to naturally inactive AST.

Conclusions:

• AST subform development in vitro involves both active formation and independent inactivation pathways.
• Coenzyme modification represents a distinct inactivation mechanism for AST, separate from deamidation.
• The in vitro generated AST subforms possess unique biochemical characteristics.