Huff R M, Neer E J
J Biol Chem. 1986 Jan 25;261(3):1105-10.
Bovine cerebral cortex contains two major substrates for ADP-ribosylation by pertussis toxin: a 39-kDa protein, alpha 39, and a 41-kDa protein, alpha 41 (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229). Both of these proteins bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with a similar affinity (Kd = 30 +/- 10 nM for alpha 39, Kd = 32 +/- 14 nM for alpha 41). Both proteins associate with a beta X gamma subunit made up of a 36-kDa beta component and a 6-kDa gamma component. We have previously shown that the beta X gamma unit is required for pertussis toxin-catalyzed ADP-ribosylation (Neer et al. (1984)). By measuring the amount of beta X gamma required for maximal incorporation of ADP-ribose, we now find that the EC50 for beta X gamma in this reaction is 3 +/- 1 times lower for alpha 41 than for alpha 39. ADP-ribosylation by pertussis toxin does not prevent dissociation of alpha 41 X beta X gamma or alpha 39 X beta X gamma by GTP gamma S. GTP gamma S decreases the sedimentation coefficient of ADP-ribosylated alpha 41 from 4.2 S to 3.0 S and the sedimentation coefficient of ADP-ribosylated alpha 39 from 4.3 S to 2.9 S. The conclusion that GTP gamma S dissociates both ADP-ribosylated heterotrimers was confirmed by the observation that GTP gamma S blocks precipitation of ADP-ribosylated alpha 39 or alpha 41 by anti-beta antibody. Neither alpha 41 X beta X gamma nor alpha 39 X beta X gamma is dissociated by GTP whether or not the proteins are ADP-ribosylated. The observation that alpha 41 more readily associates with beta X gamma than does alpha 39 may explain our earlier observation that alpha 41 is more readily ADP-ribosylated than alpha 39. In most intact membranes, only a 41-kDa ADP-ribosylated protein is seen. However, alpha 39 is also present in most tissues since we can detect it with anti-alpha 39 antibody. The functional consequences of pertussis toxin treatment may depend on whether one or both proteins are ADP-ribosylated. This in turn may depend on the ratio of alpha 41 and alpha 39 to beta X gamma in a given tissue.
牛大脑皮层含有百日咳毒素进行ADP-核糖基化修饰的两种主要底物：一种39 kDa的蛋白质，α39，和一种41 kDa的蛋白质，α41（尼尔，E.J.，洛克，J.M.，和沃尔夫，L.G.（1984年）《生物化学杂志》259，14222 - 14229）。这两种蛋白质以相似的亲和力结合鸟苷5'-（3 - O - 硫代）三磷酸（GTPγS）（α39的Kd = 30 ± 10 nM，α41的Kd = 32 ± 14 nM）。这两种蛋白质都与由一个36 kDa的β组分和一个6 kDa的γ组分组成的βXγ亚基结合。我们之前已经表明，βXγ亚基是百日咳毒素催化的ADP-核糖基化所必需的（尼尔等人（1984年））。通过测量ADP-核糖最大掺入量所需的βXγ的量，我们现在发现，在该反应中，α41对βXγ的EC50比α39低3 ± 1倍。百日咳毒素介导的ADP-核糖基化并不阻止GTPγS使α41XβXγ或α39XβXγ解离。GTPγS使ADP-核糖基化的α41的沉降系数从4.2 S降至3.0 S，使ADP-核糖基化的α39的沉降系数从4.3 S降至2.9 S。GTPγS使两种ADP-核糖基化的异源三聚体都解离这一结论通过以下观察得到证实：GTPγS阻止抗β抗体沉淀ADP-核糖基化的α39或α41。无论蛋白质是否被ADP-核糖基化，GTP都不会使α41XβXγ或α39XβXγ解离。α41比α39更容易与βXγ结合这一观察结果可能解释了我们之前的观察：α41比α39更容易被ADP-核糖基化。在大多数完整的膜中，只能看到一种41 kDa的ADP-核糖基化蛋白质。然而，α39也存在于大多数组织中，因为我们可以用抗α39抗体检测到它。百日咳毒素处理的功能后果可能取决于一种还是两种蛋白质被ADP-核糖基化。这反过来可能取决于给定组织中α41和α39与βXγ的比例。
