Identification of sites for alkylation by N‐ethylmaleimide and pertussis toxin‐catalyzed ADP‐ribosylation on GTP‐binding proteins

Abstract

An αβγ‐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 α‐subunit (α_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 α_o. These alkylations resulted in loss of its ability to be ADP‐ribosylated by IAP and to associate with βγ, but leaving the GTP‐binding site of α_o intact. The reacted cysteine residues were identified by the sequencing of tryptic fragments of α_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 α‐subunit of G_o are discussed in the functions of the signal transducing protein.