ENHANCEMENT OF CLOSTRIDIUM-BOTULINUM C3-CATALYZED ADP-RIBOSYLATION OFRECOMBINANT RHOA BY SODIUM DODECYL-SULFATE

The influence of sodium dodecyl sulfate (SDS) on ADP-ribosylation by C lostridium botulinum C3 exoenzyme (C3) was studied. SDS increased the ADP-ribosylation of recombinant rhoA and human platelet cytosolic prot eins maximally at 0.01 % whereas higher concentrations of the detergen t (>0.01 %) inhibited the ADP-ribosylation. In contrast, ADP-ribosylat ion of human platelet membranes and of recombinant rhoB was inhibited by the detergent. The K(m) for NAD of the ADP-ribosylation of rhoA was decreased by SDS from about 10 to 0.6 muM. Whereas in the absence of SDS, the C3-induced ADP-ribosylation of recombinant rhoA is not affect ed by the amphiphilic wasp venom mastoparan, in the presence of SDS (0 .01 %) mastoparan (100 muM) inhibited the ADP-ribosylation. C3-associa ted NAD-glycohydrolase activity was maximally and half-maximally inhib ited by 0.1 and 0.013 % SDS, respectively. Inhibition of NAD-glycohydr olase activity was reversed by diluting out SDS indicating that C3 was not irreversibly denatured by SDS treatment. SDS (0.01 %) completely inhibited the [H-3]GTP binding of rhoA whereas the release of previous ly bound nucleotide was not affected. The data indicate that changes i n the lipophilicity of rhoA protein largely affect its ability to serv e as a substrate for C3-like ADP-ribosyltransferases.