Schmalzing G, Kröner S
Max-Planck-Institut für Biophysik, Frankfurt, Federal Republic of Germany.
Biochem J. 1990 Aug 1;269(3):757-66. doi: 10.1042/bj2690757.
As demonstrated previously, digitonin-permeabilized Xenopus oocytes have a large internal pool of sodium pumps which are inaccessible to cytosolic ouabain [Schmalzing, Kröner & Passow (1989) Biochem. J. 260, 395-399]. Access to internal ouabain-binding sites required permeabilization of inner membranes with SDS. In the present study, micromolar free Ca2+ was found to stimulate ouabain binding in the digitonin-permeabilized cells (K0.5 0.5 microM-Ca2+, h 1.9, average of seven experiments) without disrupting intracellular membranes. Sustained incubation at 9 microM-Ca2+ was as effective as SDS in inducing access to the ouabain-binding sites of the internal sodium pumps. Omission of either Mg2+ or ATP completely abolished the Ca2+ effect. Half-maximal stimulation by Ca2+ required approx. 0.4 mM-MgATP. Of a variety of nucleotides tested, none was as effective as ATP (rank order ATP greater than ADP greater than ATP[S] (adenosine 5'-[gamma-thio]triphosphate) greater than CTP greater than UTP greater than ITP = XTP greater than GTP). Pi, AMP, cyclic AMP, cyclic GMP, GTP[S] (guanosine 5'-[gamma-thio]triphosphate) and a stable ATP analogue p[NH]ppA (adenosine 5'-[beta gamma-imido]triphosphate), were ineffective. The metalloendoproteinase inhibitor carbobenzoxy-Gly-Phe-amide reduced the Ca2+ effect by some 50%. Inhibitors of chymotrypsin and the Ca2+ proteinase calpain had no effect. Ca2+ ionophores (A23187 and ionomycin) and the polycations neomycin and polymixin B blocked the Ca2+ response entirely. Neomycin also abolished a Ca2(+)-independent stimulation of ouabain binding by the wasp venom mastoparan. The requirements for increasing the accessibility of ouabain-binding sites are remarkably similar to those for exocytosis in secretory cells, suggesting that oocytes and eggs possess a Ca2(+)-regulated pathway for the plasma membrane insertion of sodium pumps.
如先前所示，洋地黄皂苷通透的非洲爪蟾卵母细胞有大量位于内部的钠泵池，胞质中的哇巴因无法接触到这些钠泵[施马尔青、克罗纳与帕索（1989年）《生物化学杂志》260卷，395 - 399页]。要接触内部的哇巴因结合位点，需要用十二烷基硫酸钠使内膜通透。在本研究中，发现微摩尔浓度的游离Ca²⁺能刺激洋地黄皂苷通透细胞中的哇巴因结合（半数效应浓度0.5微摩尔/升Ca²⁺，h值1.9，七次实验的平均值），且不会破坏细胞内膜。在9微摩尔/升Ca²⁺下持续孵育在诱导接触内部钠泵的哇巴因结合位点方面与十二烷基硫酸钠同样有效。去除Mg²⁺或ATP会完全消除Ca²⁺的作用。Ca²⁺产生半数最大刺激效应大约需要0.4毫摩尔/升的MgATP。在测试的多种核苷酸中，没有一种与ATP一样有效（效力顺序为ATP＞ADP＞ATP[S]（腺苷5'-[γ-硫代]三磷酸）＞CTP＞UTP＞ITP = XTP＞GTP）。磷酸、AMP、环AMP、环GMP、GTP[S]（鸟苷5'-[γ-硫代]三磷酸）和一种稳定的ATP类似物p[NH]ppA（腺苷5'-[βγ-亚氨基]三磷酸）均无效。金属内蛋白酶抑制剂苄氧羰基-甘氨酰-苯丙氨酸酰胺使Ca²⁺的作用降低了约50%。胰凝乳蛋白酶抑制剂和Ca²⁺蛋白酶钙蛋白酶没有作用。Ca²⁺离子载体（A23187和离子霉素）以及多阳离子新霉素和多粘菌素B完全阻断了Ca²⁺反应。新霉素还消除了黄蜂毒液mastoparan对哇巴因结合的不依赖Ca²⁺的刺激作用。增加哇巴因结合位点可及性的条件与分泌细胞中胞吐作用的条件非常相似，这表明卵母细胞和卵细胞拥有一条受Ca²⁺调节的钠泵插入质膜的途径。
