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

[摘要]

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.

[学科分类] 生物化学/生物物理

[关键词] Alkylation;ADP-ribosylation;GTP-binding protein;Islet-activating protein;Go;a GTP-binding protein of unknown function purified from brain tissues;IAP;islet-activating protein or pertussis toxin;GTPγS;guanosine 5 '-(3-O-thio)triphosphate;NEM;N-ethylmaleimide;DTT;dithiothreitol