Reactive sulfhydryl groups of alpha 39, a guanine nucleotide-binding protein from brain. Location and function.
The guanine nucleotide-binding proteins which mediate hormonal inhibition of adenylate cyclase as well as hormonal regulation of other membrane functions are alpha, beta, and gamma heterotrimers which are structurally homologous to each other. In brain, the predominant guanine nucleotide-binding component is a 39-kDa protein whose physiological role is as yet unknown. We have used N-ethylmaleimide to define functionally important sulfhydryl groups on alpha 39. Three cysteine residues in the molecule are reactive in unliganded alpha 39. Alkylation of two of these is reduced when guanosine 5'-(3'-O-thio)triphosphate (GTP gamma S) is bound. We have isolated and sequenced tryptic peptides containing the three reactive cysteines. The octapeptide containing the GTP gamma S-insensitive cysteine is at a position equivalent to amino acids 106-113 of the transducin alpha subunit (Lochrie, M. A., Hurley, J. B., and Simon, M. I. (1985) Science 228, 96-99). However, the equivalent peptide in transducin does not contain a cysteine residue. Alkylation of this cysteine blocks ADP-ribosylation of cysteine 351 by pertussis toxin. However, alkylation does not prevent association of alpha with the beta X gamma subunits nor does it inhibit GTPase activity. The two GTP gamma S-sensitive cysteines are at positions equivalent to cysteines 139 and 286 of the transducin alpha subunit. Alkylation of these residues inhibits GTPase activity. Neither of these GTP gamma S-sensitive cysteines are in those regions of alpha 39 which are highly homologous to the GTP-binding site of elongation factor Tu (Jurnak, F. (1985) Science 230, 32-36). However, both are present in the brain 41-kDa guanine nucleotide-binding protein and in the two transducins. The conservation of these cysteine residues suggests that they are important for the function of the subunits.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine; D005033:Ethylmaleimide; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors; D019204:GTP-Binding Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D014357:Trypsin
D010446:Peptide Fragments; D000246:Adenosine Diphosphate Ribose; D014357:Trypsin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D003545:Cysteine; D005033:Ethylmaleimide