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 (Go) 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 Go are discussed in the functions of the signal transducing protein.
Hoshino
S
S
Department of Physiological Chemistry, University of Tokyo, Japan.
Kikkawa
S
S
Takahashi
K
K
Itoh
H
H
Kaziro
Y
Y
Kawasaki
H
H
Suzuki
K
K
Katada
T
T
Ui
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide / Q000378:metabolism* / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D037342:Pertussis Toxin; D051381:Rats; D014357:Trypsin; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D014357:Trypsin; D019204:GTP-Binding Proteins; D005033:Ethylmaleimide