Assay of mono ADP-ribosyltransferase activity by using guanylhydrazones.
Guanylhydrazones of p-nitrobenzaldehyde and methylglyoxal serve as acceptors of ADP-ribosyl groups for the reactions catalyzed by cholera toxin. The absorption spectrum of the ADP-ribosylated p-nitrobenzylidine aminoguanidine is similar to that of a 1:1 mixture of ADP-ribose and p-nitrobenzylidine aminoguanidine. Results from fast atom bombardment mass spectrometry prove that the product is mono-ADP-ribosylated. ADP-ribosylation lowers the pKa of the p-nitrobenzylidine aminoguanidine by 0.7-0.8 pH unit. Assay methods are developed for measuring the ADP-ribosyltransferase reaction by following the rate of disappearance of p-nitrobenzylidine aminoguanidine by high-performance liquid chromatography or spectrophotometrically by monitoring the absorbance increase at 370 nm accompanying ADP-ribosylation of p-nitrobenzylidine aminoguanidine. The high-performance liquid chromatographic system can be utilized to measure ADP-ribosyltransferase activity in animal tissues. By using this procedure, the presence and quantitation of an ADP-ribosyltransferase in a homogenate of rabbit skeletal muscle is reported.
D036002:ADP Ribose Transferases; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006146:Guanidines / Q000138:chemical synthesis; D013058:Mass Spectrometry; D009132:Muscles / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000032:analysis*; D011065:Poly(ADP-ribose) Polymerases; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; C039996:4-nitrobenzylidene aminoguanidine; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases