ADP-ribosylation of Proteins by Pertussis Toxin

ADP-ribosylation of tfie samples is carried out in glass tubes (approximate volume 5 ml) in case the reaction has to be followed by precipitation with organic solvents. Therefore, the markings on the tubes should withstand solvents. Each tube contains 10 (xl of preactivated toxin (100 ng PT/tube) and 20 [il of membrane suspension. The reaction is readily started by addition of 30 1 of the P-NAD -containing buffer, resulting in a total volume of 60 (il per tube. ADP-ribosylation of the thoroughly vortexed sample is conducted for 30 min at 30°C or tor 20 min at 37°C while the tubes are agitated con-tinously. 

We stop the reaction after not more than 45 min. If desired, isoform identification of labeled proteins may be done by immunoprecipita-tion employing specific antisera. First, the membranes are pelleted in 

Extraction of G proteins from membranes usually enhances ADP-ribosylation. In addition, small amounts of detergents like SDS, CHAPS or Lubrol PX facilitate the toxin s enzymatic activity (see above). For detection and identification of G proteins during G protein purification procedures, aliquots are also subjected to PT-catalyzed ADP-ribosylation for detection and identification (Rosenthal 

Ten xl of G-protein-containing samples is mixed with 10 [il of preactivated toxin (see section 4.3.1 100 ng PT/tube) and started by addition of another 10 il of reaction buffer (total volume 30fil). The assay is conducted as outlined in section 4.3.3. After termination of the reaction samples may be quantified by filtration assay as described by Sternweis and Pang (1990). 

The continous support of Gunter Schultz is greatly appreciated. Many thanks to my colleagues tor valuable discussions. The author s own research reported here was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 366, and Fonds der Chemischen Industrie. 

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