Assays for mechanistic investigations of protein/histone acetyltransferases |
| |
Authors: | Berndsen Christopher E Denu John M |
| |
Affiliation: | Department of Biomolecular Chemistry, University of Wisconsin-Madison, 1300 University Avenue Madison, WI 53706-1532, USA. |
| |
Abstract: | Protein/histone acetyltransferases (PATs/HATs) have been implicated in a number of cellular functions including gene regulation, DNA synthesis, and repair. This paper reviews methods that can be used to quantitatively determine the activity and ultimately the catalytic/kinetic mechanism of PAT/HATs in vitro. Two methods will be described in detail. The first method is a filter-binding assay that measures the transfer of radiolabeled acetate from acetyl-CoA to protein. The second method is a continuous, spectroscopic, enzyme-coupled assay that links the PAT/HAT reaction to the reduction of NAD+ by pyruvate or alpha-ketoglutarate dehydrogenase. Both methods are highly applicable in determining steady-state reaction rates, and obtaining the kinetic constants Vmax, Km, and V/K from substrate saturation curves. We describe a new application of the filter-binding assay to determine the kinetic parameters for HATs using low concentrations of nucleosomal substrates. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|