Winslow J W, Bradley J D, Smith J A, Neer E J
J Biol Chem. 1987 Apr 5;262(10):4501-7.
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.
介导激素对腺苷酸环化酶的抑制作用以及对其他膜功能的激素调节作用的鸟嘌呤核苷酸结合蛋白是α、β和γ异源三聚体，它们在结构上彼此同源。在脑中，主要的鸟嘌呤核苷酸结合成分是一种39 kDa的蛋白质，其生理作用尚不清楚。我们使用N-乙基马来酰亚胺来确定α39上功能重要的巯基。该分子中的三个半胱氨酸残基在未结合配体的α39中具有反应性。当结合鸟苷5'-(3'-O-硫代)三磷酸(GTPγS)时，其中两个的烷基化作用减弱。我们已经分离并测序了含有这三个反应性半胱氨酸的胰蛋白酶肽段。含有对GTPγS不敏感的半胱氨酸的八肽位于与转导素α亚基的氨基酸106 - 113相当的位置(Lochrie, M. A., Hurley, J. B., and Simon, M. I. (1985) Science 228, 96 - 99)。然而，转导素中的等效肽段不包含半胱氨酸残基。该半胱氨酸的烷基化作用会阻断百日咳毒素对半胱氨酸351的ADP核糖基化作用。然而，烷基化作用并不阻止α与βXγ亚基的结合，也不抑制GTP酶活性。这两个对GTPγS敏感的半胱氨酸位于与转导素α亚基的半胱氨酸139和286相当的位置。这些残基的烷基化作用会抑制GTP酶活性。这两个对GTPγS敏感的半胱氨酸都不在α39中与延伸因子Tu的GTP结合位点高度同源的区域(Jurnak, F. (1985) Science 230, 32 - 36)。然而，两者都存在于脑41 kDa的鸟嘌呤核苷酸结合蛋白和两种转导素中。这些半胱氨酸残基的保守性表明它们对亚基的功能很重要。
