Clegg J S, Jackson S A, Liang P, MacRae T H
Bodega Marine Laboratory, University of California (Davis), Bodega Bay, California 94923, USA.
Exp Cell Res. 1995 Jul;219(1):1-7. doi: 10.1006/excr.1995.1197.
Embryos of the crustacean Artemia franciscana survive continuous anoxia for periods of years, during which their metabolism comes to a reversible stand-still. A question of some interest concerns the maintenance of cellular integrity in the absence of biosynthesis and an ongoing energy metabolism. The present paper continues previous work on an abundant protein (p26) that undergoes extensive intracellular translocation during aerobic-anoxic transitions, exhibits several characteristics of stress proteins, and might be involved in metabolic regulation during aerobic-anoxic transitions. Since it has been established that intracellular pH (pHi) plays a major role in aerobic-anoxic transitions in this system we examined the pH-dependence of nuclear-cytoplasmic translocations of p26. In unincubated and aerobic-incubated embryos (pHi > or = 7.9) p26 was located in the "soluble" fraction, whereas in anoxic embryos (pH about 6.3) roughly 50% was translocated into the nucleus as shown by immunocolloidal gold electron microscopy. These nuclear translocations also took place in vitro, simply by manipulating buffer pH in a physiologically appropriate fashion. Immunostaining of Western blots prepared after two-dimensional electrophoresis revealed several isoforms of native p26. The isoelectric point of the major isoform was 7.10 +/- 0.05, a value close to the pH at which p26 translocation into the nucleus was first initiated in vitro. 31P-NMR measurements indicated that pHi was maintained at acidic levels (about 6.3) during prolonged anoxia. We also found that pHi of hydrated (0 degree C) but otherwise unincubated embryos was alkaline, allowing for rapid resumption of metabolism under permissive conditions. The significance of these pH-dependent translocations of p26 is discussed.
卤虫（Artemia franciscana）的胚胎能够在持续缺氧的环境中存活数年，在此期间它们的新陈代谢会进入可逆的停滞状态。一个颇受关注的问题是，在缺乏生物合成和持续能量代谢的情况下，细胞完整性是如何维持的。本文延续了之前对一种丰富蛋白质（p26）的研究工作，该蛋白质在有氧 - 缺氧转变过程中会发生广泛的细胞内转运，表现出应激蛋白的几个特征，并且可能参与有氧 - 缺氧转变过程中的代谢调节。由于已经确定细胞内pH（pHi）在该系统的有氧 - 缺氧转变中起主要作用，我们研究了p26核 - 质转运的pH依赖性。在未孵育和有氧孵育的胚胎中（pHi≥7.9），p26位于“可溶性”部分，而在缺氧胚胎中（pH约为6.3），通过免疫胶体金电子显微镜观察显示，约50%的p26转运到了细胞核中。这些核转运在体外也会发生，只需以生理合适的方式调节缓冲液pH即可。二维电泳后制备的Western印迹免疫染色显示了天然p26的几种同工型。主要同工型的等电点为7.10±0.05，该值接近体外p26首次开始转运到细胞核时的pH。31P - NMR测量表明，在长时间缺氧期间，pHi维持在酸性水平（约6.3）。我们还发现，水合（0℃）但未孵育的胚胎的pHi呈碱性，这使得在允许的条件下代谢能够迅速恢复。本文讨论了p26这些pH依赖性转运的意义。
