XB-ART-29814
Anal Biochem
1984 Mar 01;1372:287-96. doi: 10.1016/0003-2697(84)90087-3.
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Mono(adenosine diphosphate ribosyl) transferase in Xenopus tissues. Direct demonstration by a zymographic localization in sodium dodecyl sulfate--polyacrylamide gels.
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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.
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Species referenced: Xenopus laevis
Genes referenced: parp1