Evidence for the involvement of a pertussis toxin-insensitive G-protein in egg activation of the frog, Xenopus laevis.
Evidence for the involvement of a pertussis toxin-insensitive G-protein in egg activation of the frog, Xenopus laevis.
复制标题
对百日咳毒素不敏感的 G 蛋白参与非洲爪蟾卵激活的证据。
DOI：
10.1016/0012-1606(91)90072-b
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发表时间：
1991
影响因子：
2.7
通讯作者：
Jaffe,LA
中科院分区：
文献类型：
--
作者：
Kline,D;Kopf,GS;Muncy,LF;Jaffe,LA
青蛙卵在受精时的激活反应包括从细胞内储存的钙的释放和钙依赖性氯离子通道的打开，从而产生受精潜力。为了研究鸟嘌呤核苷酸结合蛋白（G 蛋白）的存在及其在非洲爪蟾卵中启动这些事件的作用，我们测定了百日咳和霍乱毒素底物，并应用了 G 蛋白的激活剂和抑制剂。百日咳毒素催化aMr40,000成分的[32P]ADP核糖基化，但没有证明霍乱毒素底物。注射 ⩾25 pmole 鸟苷-5′-O-(3-硫代三磷酸) (GTP-γ-S)（一种 G 蛋白激活剂）会产生模拟受精电位的膜电位变化，并引起皮质颗粒胞吐作用和皮质收缩。注射高达 600 pmole 鸟苷 3′:5′-环状单磷酸盐或 9 nmole 鸟苷-5′-(β-γ-imido)三磷酸盐不会激活卵。对 GTP-γ-S 注射的膜电位响应显示出与受精电位相同的峰值和氯依赖性，尽管 GTP-γ-S 响应的持续时间稍长。如果通过预先注射钙螯合剂 BAPTA 来阻止钙升高，GTP-γ-S 不会激活卵子。注射高达 200 ng 的霍乱毒素并不能激活卵。然而，通过将 1 nM 血清素注射到注射了血清素 1c 受体（通过 G 蛋白起作用的受体类成员）的特定 mRNA 的鸡蛋中，鸡蛋被激活。在大约 80-90% 的毒素底物被 ADP 核糖基化的实验条件下，百日咳毒素不会抑制对精子或血清素的卵子激活。这些结果支持这样的假设：精子在受精时通过对百日咳和霍乱毒素不敏感的 G 蛋白激活爪蟾卵。
Activation responses of the frog egg at fertilization include the release of calcium from intracellular stores and the opening of calcium-dependent chloride channels, which produce the fertilization potential. To investigate the presence of guanine nucleotide-binding proteins (G-proteins), and their role in initiation of these events in the egg of the frogXenopus laevis, we assayed for pertussis and cholera toxin substrates, and applied activators and inhibitors of G-proteins. Pertussis toxin catalyzed the [32P]ADP ribosylation of aMr40,000 component, but no cholera toxin substrates were demonstrated. Injection of ⩾25 pmole of guanosine-5′-O-(3-thiotriphosphate) (GTP-γ-S), an activator of G-proteins, produced a change in membrane potential that mimicked the fertilization potential and also caused cortical granule exocytosis and cortical contraction. Injections of up to 600 pmole of guanosine 3′:5′-cyclic monophosphate or 9 nmole of guanosine-5′-(β-γ-imido)triphosphate did not activate eggs. The membrane potential response to GTP-γ-S injection showed the same peak and chloride dependence as the fertilization potential, although the duration of the GTP-γ-S response was somewhat greater. GTP-γ-S did not activate eggs if the calcium rise was prevented by prior injection of the calcium chelator BAPTA. Injection of up to 200 ng of cholera toxin did not activate eggs. However, eggs were activated by applying 1 nMserotonin to eggs that had been injected with a specific mRNA for the serotonin 1c receptor, a member of the class of receptors that act by way of G-proteins. Egg activation in response to either sperm or serotonin was not inhibited by pertussis toxin, under experimental conditions where ∼80–90% of the toxin substrate was ADP-ribosylated. These results support the hypothesis that sperm activateXenopuseggs at fertilization by way of a pertussis and cholera toxin-insensitive G-protein.