Fluorescent assay of cell-permeable C3 transferase activity

C3 transferase has been extensively used in the investigation of Rho signaling. It acts as a Rho antagonist by covalently attaching an ADP-ribose group to RhoA, B, and C proteins. Cell-permeable C3 fusion proteins have been developed so that lower doses of C3 can be used. We describe a simple and fast fluorescence-based assay to evaluate the enzymatic activity of cell-permeable C3 proteins purified from Escherichia coli. The assay measures glycohydrolase (GH) activity of C3 that cleaves NAD+ into ADP-ribose and nicotinamide. Results from the GH activity correlate with other tests carried out in tissue culture cells such as neurite outgrowth or ADP ribosylation of RhoA. This method provides reliable measurements of the activity of permeable C3 proteins or other C3-related proteins.