Mono(adenosine diphosphate ribosyl) transferase in Xenopus tissues. Direct demonstration by a zymographic localization in sodium dodecyl sulfate--polyacrylamide gels.
A semiquantitative method to measure mono(adenosine diphosphate ribosyl) transferase activity [mADPRT] in tissue extracts is described. After electrophoretic separation in sodium dodecyl sulfate (SDS)--polyacrylamide gels, renatured enzymatic activity is demonstrated in situ by incubation of the slab gels with radiolabeled NAD+ and histones. Precipitation of the radiolabeled product in the gel allows localization of the enzyme by autoradiography. This method is suitable for two-dimensional gel electrophoresis, whereby proteins are electrofocused in the presence of 9 M urea and subsequently subjected to electrophoresis in SDS. A single major band showing mADPRT activity of Mr approximately 30 Kda was observed in all crude extracts of Xenopus tissues examined. Accumulation of acid-insoluble radiolabeled products was dependent on added histones and was specifically inhibited by agmatine. The ADPRT activity of cholera toxin A fragment could also be demonstrated by this technique. Reducing agents stimulated the activity of cholera toxin A fragment while depressing that of Xenopus mADPRT.
D000818:Animals; D002772:Cholera Toxin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D009132:Muscles / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011065:Poly(ADP-ribose) Polymerases; D012967:Sodium Dodecyl Sulfate; D014982:Xenopus laevis
D012967:Sodium Dodecyl Sulfate; D002772:Cholera Toxin; D011065:Poly(ADP-ribose) Polymerases; D009713:Nucleotidyltransferases