IDENTIFICATION OF SITES FOR ALKYLATION BY N-ETHYLMALEIMIDE AND PERTUSSIS TOXIN-CATALYZED ADP-RIBOSYLATION ON GTP-BINDING PROTEINS

An alpha-beta-gamma-trimeric GTP-binding protein (G(o)) serving as the substrate of pertussis toxin-(IAP) catalyzed ADP-ribosylation was purified from rat brain membranes. The constituent alpha-subunit (alpha-o) was alkylated with N-ethylmaleimide (NEM), and the functionally important sulfhydryl groups were investigated. There were at least two cysteine residues highly reactive to NEM on the GDP-bound form of alpha-o. These alkylations resulted in loss of its ability to be ADP-ribosylated by IAP and to associate with beta-gamma, but leaving the GTP-binding site of alpha-o intact. The reacted cysteine residues were identified by the sequencing of tryptic fragments of alpha-o. One of the alkylation sites was Cys-351, which was four amino acid residues away from the carboxyl-terminus of the molecule. The Cys-351 was proven to be also a site for IAP-catalyzed ADP-ribosylation. Possible roles of cysteine residues on the alpha-subunit of G(o) are discussed in the functions of the signal transducing protein.