Partial characterization of mitochondrial G proteins in adrenal cells.
Partial characterization of mitochondrial G proteins in adrenal cells.
复制标题
肾上腺细胞线粒体 G 蛋白的部分表征。
DOI：
10.1016/s0005-2736(99)00185-6
复制
发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Hall,PF
中科院分区：
文献类型：
--
作者：
Sleer,LS;Hall,PF
在牛肾上腺皮质线粒体膜上发现了四种低分子量G蛋白。蛋白质1、2和4的分子质量分别为28、27、26和24 kDa，等电点分别为8.1、5.6和6.3。蛋白质3的等电点为5.0-6.1。用12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离并转移到硝基纤维素上，通过与[α-32P]鸟苷三磷酸结合鉴定蛋白质。未标记的竞争核苷磷酸配体对鸟苷三磷酸(GTP)和鸟苷二磷酸(GDP)显示出特异性，与鸟苷一磷酸的结合很少，与腺苷磷酸没有检测到结合。结合强度不到10%，GDP和GTP超额100倍，表现出相同的结合强度。三磷酸胞苷和三磷酸尿苷对结合的抑制作用约为1000倍。10%镁离子可促进四种蛋白质与GTP的结合。镁离子对蛋白质1、2和3的影响基本相同，而蛋白质4在浓度为-lt；10−3M时对镁的敏感性较低。经蔗糖密度梯度超声分离的线粒体膜显示，所有四种蛋白质都存在于接触点。在内膜和外膜中存在较低浓度的蛋白质(每毫克蛋白质表达)，这表明这些膜中有少量是目前制备的接触点的一部分，或者蛋白质存在于接触点中，在内膜和外膜中的程度要小得多。有人建议研究这些蛋白在胆固醇从外线粒体膜向内线粒体膜运输中的可能作用。
Four low molecular mass G proteins have been identified in mitochondrial membranes from bovine adrenal cortex. These proteins (referred to as proteins 1 to 4) showed molecular masses of 28, 27, 26 and 24 kDa with isoelectric points (pI) of 8.1, 5.6, and 6.3 respectively for proteins 1, 2 and 4. Protein 3 was shown to be heterogeneous, with isoelectric points of 5.0–6.1. Proteins were identified by binding of [α-32P]guanosine triphosphate (GTP) after separation by 12% SDS-polyacrylamide gel electrophoresis and transfer to nitrocellulose. Competitive binding by unlabelled competing nucleoside phosphate ligands showed specificity for guanosine triphosphate (GTP) and guanosine diphosphate (GDP) with little binding of guanosine monophosphate and no detectable binding with adenosine nucleoside phosphates. Binding was less than 10% with 100-fold excess GDP and GTP which showed equal intensities of binding. Inhibition of binding by 1000-fold cytidine triphosphate and uridine triphosphate was approx. 10%. Magnesium (Mg2+) stimulated binding of GTP by all four proteins. The effect of Mg2+was essentially the same for proteins 1, 2 and 3, while protein 4 was less sensitive to Mg2+at concentrations <10−3M. Centrifugation of sonicated mitochondrial membranes through sucrose density gradients showed the presence of all four proteins in contact points. The presence of lower concentrations (expressed per mg protein) of the proteins in inner and outer membranes suggests that either small amounts of these membranes are part of contact points as presently prepared or that the proteins occur in contact points and to a much smaller extent in inner and outer membranes. It is proposed to examine a possible role for these proteins in transport of cholesterol from outer to inner mitochondrial membranes.