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Identification of sites for alkylation by N ‐ethylmaleimide and pertussis toxin‐catalyzed ADP‐ribosylation on GTP‐binding proteins
Author(s) -
Hoshino Shin-ichi,
Kikkawa Satoshi,
Takahashi Katsunobu,
Itoh Hiroshi,
Kaziro Yoshito,
Kawasaki Hiroshi,
Suzuki Koichi,
Katada Toshiaki,
Ui Michio
Publication year - 1990
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(90)80548-w
Subject(s) - adp ribosylation , n ethylmaleimide , pertussis toxin , gtp' , cysteine , g alpha subunit , chemistry , g protein , biochemistry , protein subunit , binding site , alkylation , palmitoylation , stereochemistry , enzyme , receptor , catalysis , nad+ kinase , gene
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.