Assays for mechanistic investigations of protein/histone acetyltransferases.
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.
Berndsen
Christopher E
CE
Department of Biomolecular Chemistry, University of Wisconsin-Madison, 1300 University Avenue Madison, WI 53706-1532, USA.
Denu
John M
JM
D000105:Acetyl Coenzyme A / Q000737:chemistry; D000123:Acetyltransferases / Q000737:chemistry* / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D002384:Catalysis; D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry; D019411:Clinical Laboratory Techniques; D005374:Filtration; D051548:Histone Acetyltransferases; D006801:Humans; D007700:Kinetics; D008851:Micropore Filters; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D013053:Spectrophotometry; D014316:Tritium / Q000737:chemistry
D009243:NAD; D014316:Tritium; D000105:Acetyl Coenzyme A; D002482:Cellulose; C008881:phosphocellulose; D000123:Acetyltransferases; D051548:Histone Acetyltransferases