Differential Glucosylation by Toxin

Glucosylation of Rho at Thr-37 prevents subsequent C3-catalyzed ADP-ribosylation of Rho at Asn-41 (Just efal., 1994 Just etal., 1995a). Therefore, C3-catalyzed ADP-ribosylation can be used to quantify the amount of glucosylation of Rho by comparing ADP-ribosylation in lysates from control cells and toxin B-treated cells (Fig. 2). However, only Rho but not Rac and Cdc42 are ADP-ribosylated. 

40 nl of cell lysate or membranous fraction (for preparation see Section 15.3.1) plus 10 [xM of pP]NAD (0.3 (xCi) plus 10 mM thymidine is incubated with 1 [xg/ml of C3 (total volume 50n.l) for 30 min at 37°C. Thymidine blocks the poly(ADP-ribose)-polymerase (approx. 120 kDa), thereby preventing consumption of NAD which is essential for quantitative ADP-ribosylation of Rho. 

The ADP-ribosylation reaction is terminated by addition of 10 1 Laemmli sample buffer or of 1 ml of trichlororacetic acid (20 %, w/v). 

The proteins are separated by 12.5 % SDS-gel electrophoresis, and the labeled proteins are analyzed using a phosphorimager system. The exposure time of the gels varies from 1 h up to 4 h. For autoradiography, the exposure time is 12 to 24 h. 

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