Enzymatic assays for NAD-dependent deacetylase activities.
The NAD-dependent deacetylases are a new class of enzymes responsible for the removal of acetyl groups from lysines on proteins. Instead of water, the NAD-dependent deacetylases use a highly reactive ADP-ribose intermediate as a recipient for the acetyl group. The products of the reaction are nicotinamide, acetyl-ADP-ribose, and a deacetylated substrate. Many assays have been developed for the measurement of NAD-dependent deacetylase activity. In this review we present assays based on each of the two reactions catalyzed by these enzymes, deacetylation and NAD hydrolysis. First we describe methods for the production of acetylated protein and peptide substrates for use in deacetylation reactions. Then we describe four methods for assaying deacetylation, three of which directly measure the loss of acetyl groups from a protein or peptide substrate, and one that measures acetate production. We also describe two indirect methods for following enzyme activity, NAD hydrolysis and a novel NAD-nicotinamide exchange reaction. Finally, a quantitative method using a monoacetylated peptide as a substrate and HPLC to measure products is described.
Landry
Joseph
J
Program in Genetics, Stony Brook University, Stony Brook, NY 11794-5215, USA.
Sternglanz
Rolf
R
D000107:Acetylation; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006655:Histone Deacetylases / Q000302:isolation & purification / Q000378:metabolism; D006657:Histones / Q000302:isolation & purification / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D037761:Sirtuins / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D006657:Histones; D007202:Indicators and Reagents; D010455:Peptides; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D037761:Sirtuins; D006655:Histone Deacetylases