Purification and activity assays of N-terminal acetyltransferase D

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Alpha-N-terminal acetyltransferase D (NatD) modifies histones H4 and H2A, impacting gene expression.
• Characterizing NatD's function and identifying inhibitors requires robust assays.

Purpose of the Study:

• To present novel fluorescence-based and MALDI-MS assays for NatD-mediated acetylation.
• To enable detailed characterization of enzymatic kinetics and inhibitor screening for NatD.

Main Methods:

• Development and validation of a fluorescence-based assay for NatD activity.
• Implementation and application of MALDI-MS for quantifying NatD acetylation.
• Comparative analysis of the developed assays against existing methods.

Main Results:

• Detailed protocols for enzymatic kinetic characterization using both assay types.
• Demonstrated utility of the assays for assessing NatD inhibitor activity.
• Evaluation of the strengths and limitations of each assay.

Conclusions:

• The presented fluorescence and MALDI-MS assays effectively characterize NatD-mediated acetylation.
• These methods are valuable tools for biochemical studies of NatD and N-terminal acetylation.
• The assays offer broader applicability for evaluating other N-terminal acetyltransferases and their inhibitors.