Joseph S K, Rice H L, Nicchitta C V
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Biochim Biophys Acta. 1988 Nov 22;945(2):185-94. doi: 10.1016/0005-2736(88)90481-6.
Guanosine triphosphate (GTP) can release Ca2+ and enhance responses to D-myo-inositol 1,4,5-trisphosphate (IP3) in crude liver microsomes in the presence of polyethylene glycol (PEG) (Dawson et al. (1986) Biochem. J. 234, 311-315). The mechanism of these responses has been further investigated. GTP gamma S which antagonizes the actions of GTP on microsomes, does not promote Ca2+ re-uptake when added after the completion of GTP-mediated Ca2+ release. However, the effects of GTP could be reversed by washing or dilution of the microsomes. Addition of PEG to the incubation medium promoted the aggregation of microsomes. Electron microscopy provided no evidence for the fusion of microsomal vesicles in the presence or absence of GTP. In the presence of PEG, GTP produced an alteration of the permeability properties of the microsomal membrane as indicated by increased leakage of an intraluminal esterase, a reduction in the mean buoyant density of the vesicles, and a decrease in the latency of mannose 6-phosphate hydrolysis. All three effects developed relatively slowly, whereas the effects of GTP on Ca2+ fluxes occurred more rapidly (complete within 15 min). A low permeability to mannose 6-phosphate was restored upon washing away the GTP. These results suggest that non-specific permeability changes may underly the effects of GTP on Ca2+ release and that, under certain conditions, GTP can reversibly modulate the permeability of a transmembrane 'pore' in microsomal membranes that can pass ions and macromolecules. The possibility that such a pore serves to link IP3-sensitive vesicles with other Ca2+-containing compartments is discussed.
在聚乙二醇（PEG）存在的情况下，三磷酸鸟苷（GTP）可在粗制肝微粒体中释放Ca2+并增强对D-肌醇1,4,5-三磷酸（IP3）的反应（道森等人，（1986年）《生物化学杂志》234卷，311 - 315页）。对这些反应的机制进行了进一步研究。GTPγS可拮抗GTP对微粒体的作用，在GTP介导的Ca2+释放完成后添加时，它不会促进Ca2+的再摄取。然而，通过洗涤或稀释微粒体可逆转GTP的作用。向孵育培养基中添加PEG可促进微粒体的聚集。电子显微镜观察未发现无论有无GTP存在时微粒体小泡融合的证据。在PEG存在的情况下，GTP使微粒体膜的通透性发生改变，表现为腔内酯酶泄漏增加、小泡平均浮力密度降低以及甘露糖6 - 磷酸水解延迟时间缩短。所有这三种效应发展相对缓慢，而GTP对Ca2+通量的影响发生得更快（15分钟内完成）。洗去GTP后，对甘露糖6 - 磷酸的低通透性得以恢复。这些结果表明，非特异性通透性变化可能是GTP对Ca2+释放作用的基础，并且在某些条件下，GTP可可逆地调节微粒体膜中可通透离子和大分子的跨膜“孔”的通透性。讨论了这种孔可能用于将IP3敏感小泡与其他含Ca2+区室相连的可能性。
