使用特异性抗体对脑中鸟嘌呤核苷酸结合蛋白Go进行定量分析。
Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain.
作者信息
Gierschik P, Milligan G, Pines M, Goldsmith P, Codina J, Klee W, Spiegel A
出版信息
Proc Natl Acad Sci U S A. 1986 Apr;83(7):2258-62. doi: 10.1073/pnas.83.7.2258.
We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the alpha subunit (39 kDa) of a G protein of unknown function, termed Go, as well as with the beta subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the alpha subunits of the stimulatory (Gs) or inhibitory (Gi) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affinity-purified antiserum showed that RV3 specifically recognizes the Go alpha subunit and the beta subunit in crude brain membranes. Using RV3, we found approximately equal amounts of Go in brain membranes from frog, chicken, rat, cow, and man. Quantitative immunoblotting gave Go alpha subunit/ beta subunit ratios approximately equal to 1 in cerebral cortex, raising the possibility that free Go alpha subunit (unassociated with beta subunit) may exist in brain. The concentration of Go alpha subunit in cortex is about 5 times that of Gi alpha subunit. The results show that Go is an immunochemically distinct, highly conserved protein distributed throughout the brain, with particularly high concentrations in forebrain.
我们用从牛脑中纯化的鸟嘌呤核苷酸结合蛋白（G蛋白）免疫兔子，获得了一种抗血清RV3，它能与一种功能未知的G蛋白Go的α亚基（39 kDa）特异性反应，也能与所有G蛋白共有的β亚基（35 kDa）反应。RV3与与腺苷酸环化酶相关的刺激性（Gs）或抑制性（Gi）G蛋白的α亚基均无交叉反应，也与视杆外段G蛋白转导蛋白无交叉反应。用粗制和亲和纯化抗血清进行的免疫印迹表明，RV3能在粗制脑膜中特异性识别Goα亚基和β亚基。利用RV3，我们发现青蛙、鸡、大鼠、牛和人的脑膜中Go的含量大致相等。定量免疫印迹显示，大脑皮层中Goα亚基/β亚基的比例约为1，这增加了大脑中可能存在游离Goα亚基（未与β亚基结合）的可能性。皮层中Goα亚基的浓度约为Giα亚基的5倍。结果表明，Go是一种免疫化学性质独特、高度保守的蛋白，分布于整个大脑，在前脑中浓度尤其高。
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