Mullaney J M, Chueh S H, Ghosh T K, Gill D L
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
J Biol Chem. 1987 Oct 5;262(28):13865-72.
The GTP-activated Ca2+ release process we recently described (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464) was revealed in the preceding report to operate via a mechanism likely to be induced by close membrane association but which appears not to involve membrane fusion (Chueh, S. H., Mullaney, J. M., Ghosh, T. K., Zachary, A. L., and Gill, D. L. (1987) J. Biol. Chem. 262, 13857-13864). To determine more about the GTP-activated Ca2+ translocation process, effects of GTP on cells loaded with Ca-oxalate were investigated. Using permeabilized cells of both the N1E-115 neuroblastoma and DDT1MF-2 smooth muscle cell lines, 10 microM GTP activates a profound uptake of Ca2+ in the presence of oxalate, as opposed to release observed without oxalate. GTP stimulation of Ca2+ uptake was observed at oxalate concentrations (2 mM) only slightly augmenting Ca2+ uptake without GTP; with 8 mM oxalate (which alone induces linear Ca2+ accumulation) GTP still increases the rate of uptake. GTP-activated uptake in the presence of oxalate is completely reversed by 1 mM vanadate. 3% polyethylene glycol enhances the effect of GTP although GTP-activated uptake is still observed without polyethylene glycol. The Km for GTP for activation of Ca2+ uptake is 0.9 microM. Uptake is not activated by guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp); however, GTP gamma S (but not GppNHp) completely blocks the action of GTP. GDP gives a delayed uptake response which is blocked by ADP, indicating its action arises from conversion to GTP. In the presence of ADP, GDP blocks the action of GTP; guanosine 5'-O-(2-thio)diphosphate, which does not activate uptake, also blocks the action of GTP. These data reveal almost exact correlation between parameters affecting GTP-activated uptake and release, strongly suggesting the same process mediates both events. To explain the opposite effects of GTP in the absence and presence of oxalate, it is proposed that GTP activates a transmembrane conveyance of Ca2+ between oxalate-permeable and -impermeable compartments.
我们最近描述的GTP激活的Ca2+释放过程（吉尔，D.L.，上田，T.，朱，S.H.，和诺埃尔，M.W.（1986年）《自然》320，461 - 464），在前一份报告中显示其通过一种可能由紧密的膜结合诱导但似乎不涉及膜融合的机制起作用（朱，S.H.，穆拉尼，J.M.，戈什，T.K.，扎卡里，A.L.，和吉尔，D.L.（1987年）《生物化学杂志》262，13857 - 13864）。为了更多地了解GTP激活的Ca2+转运过程，研究了GTP对加载草酸钙的细胞的影响。使用N1E - 115神经母细胞瘤和DDT1MF - 2平滑肌细胞系的通透细胞，10微摩尔GTP在有草酸盐存在的情况下激活Ca2+的大量摄取，这与无草酸盐时观察到的释放相反。在草酸盐浓度（2毫摩尔）仅略微增加无GTP时的Ca2+摄取的情况下观察到GTP对Ca2+摄取的刺激作用；对于8毫摩尔草酸盐（其单独诱导线性Ca2+积累），GTP仍增加摄取速率。草酸盐存在时GTP激活的摄取被1毫摩尔钒酸盐完全逆转。3%聚乙二醇增强了GTP的作用，尽管在没有聚乙二醇的情况下仍观察到GTP激活的摄取。激活Ca2+摄取的GTP的Km为0.9微摩尔。鸟苷5'-O-(3 - 硫代)三磷酸（GTPγS）或鸟苷5'-(β,γ - 亚氨基)三磷酸（GppNHp）不激活摄取；然而，GTPγS（但不是GppNHp）完全阻断GTP的作用。GDP产生延迟的摄取反应，该反应被ADP阻断，表明其作用源于转化为GTP。在有ADP存在的情况下，GDP阻断GTP的作用；鸟苷5'-O-(2 - 硫代)二磷酸不激活摄取，也阻断GTP的作用。这些数据揭示了影响GTP激活的摄取和释放的参数之间几乎完全的相关性，强烈表明相同的过程介导这两个事件。为了解释GTP在无草酸盐和有草酸盐存在时的相反作用，有人提出GTP激活Ca2+在草酸盐可通透和不可通透的区室之间的跨膜转运。
