Mono(adenosine diphosphate ribosyl) transferase in Xenopus tissues. Direct demonstration by a zymographic localization in sodium dodecyl sulfate--polyacrylamide gels |
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Authors: | F Godeau D Belin S S Koide |
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Institution: | The Population Council, The Rockefeller University, New York, New York 10021 USA |
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Abstract: | 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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