MECHANISM OF CHOLERA TOXIN ACTION - COVALENT MODIFICATION OF GUANYL NUCLEOTIDE-BINDING PROTEIN OF ADENYLATE-CYCLASE SYSTEM
MECHANISM OF CHOLERA TOXIN ACTION - COVALENT MODIFICATION OF GUANYL NUCLEOTIDE-BINDING PROTEIN OF ADENYLATE-CYCLASE SYSTEM
复制标题
DOI：
10.1073/pnas.75.6.2669
复制
发表时间：
1978-01-01
影响因子：
11.1
通讯作者：
PFEUFFER, T
中科院分区：
文献类型：
--
作者：
CASSEL, D;PFEUFFER, T
用霍乱毒素和NAD+处理鸽子红细胞膜增强了GTP刺激并抑制了腺苷酸环化酶[ATP焦磷酸裂解酶（环化），EC 4.6.1.1]的F-活化。在AMP部分中存在用32 P标记的NAD+的情况下，毒素催化放射性共价结合到MW为200，000、86，000和42，000的膜蛋白中。用Lubrol PX提取毒素处理的膜，然后在GTP-琼脂糖柱上进行亲和层析，导致42，000 MW标记蛋白质的200倍纯化，并使其与其他标记蛋白质完全分离。从毒素处理的膜含有纯化的GTP结合组分的馏分赋予增强的GTP刺激的腺苷酸环化酶从未处理的膜溶解的活性。同样地，从未处理的膜的GTP结合馏分添加到从毒素处理的膜溶解的酶恢复F-刺激的腺苷酸环化酶。毒素诱导的腺苷酸环化酶修饰和放射性掺入42，000 MW蛋白质中，在pH 6.1下与毒素和烟酰胺孵育后部分逆转。霍乱毒素通过催化带有鸟苷酸调节位点的42，000 MW组分的ADP-核糖基化而明显影响腺苷酸环化酶系统。
Treatment of pigeon erythrocyte membranes with cholera toxin and NAD+ enhanced the GTP stimulation and suppressed the F- activation of the adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. In the presence of NAD+ labeled with 32P in the AMP moiety the toxin catalyzed the covalent incorporation of radioactivity into membrane proteins with MW of 200,000, 86,000 and 42,000. Extraction of toxin-treated membranes with Lubrol PX followed by affinity chromatography on a GTP-Sepharose column resulted in a 200-fold purification of the 42,000 MW labeled protein and in its complete separation from the other labeled proteins. The fraction containing the purified GTP-binding component from toxin-treated membranes conferred an enhanced GTP-stimulated activity on adenylate cyclase solubilized from nontreated membranes. Likewise, the addition of GTP-binding fraction from nontreated membranes to an enzyme solubilized from toxin-treated membranes restored F- stimulation of the adenylate cyclase. The toxin-induced modification of adenylate cyclase and the incorporation of radioactivity into the 42,000 MW protein were partially reversed upon incubation with toxin and nicotinamide at pH 6.1. Cholera toxin apparently affects the adenylate cyclase system by catalyzing an ADP-ribosylation of the 42,000 MW component bearing the guanyl nucleotide regulatory site.