期刊
METHODS
卷 36, 期 4, 页码 321-331出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2005.03.002
关键词
histone; acetyltransferase; coupled assay; acetyl-CoA; enzyme mechanism
资金
- NIGMS NIH HHS [GM065386] Funding Source: Medline
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 V-max, K-m, 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. (c) 2005 Elsevier Inc. All rights reserved.
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