Redistribution of ADP-ribosylation factor during stimulation of permeabilized cells with GTP analogues.
Redistribution of ADP-ribosylation factor during stimulation of permeabilized cells with GTP analogues.
复制标题
用 GTP 类似物刺激透化细胞期间 ADP-核糖基化因子的重新分布。
DOI：
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发表时间：
1991
影响因子：
4.1
通讯作者：
Claes B. Wollheim
中科院分区：
文献类型：
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作者：
Romano Regazzi;Susanne Ullrich;Richard A. KAHNt;Claes B. Wollheim
低分子量GTP结合蛋白的ras家族进行了分析，[32 P]GTP结合后，PAGE和转移到硝酸纤维素膜。通过这种技术，在RINm 5 F细胞的胞浆和微粒体部分中检测到了几种20-30 kDa范围内的GTP结合蛋白。其中之一，显示约20 kDa的表观分子量和6.7的pI，主要是胞质和被证明是ADP-核糖基化因子（ARF）通过使用特定的抗体。当透化的RINm 5 F细胞与稳定的GTP类似物鸟苷5 '-[γ-硫代]三磷酸（GTP[S]）和鸟苷5'-[β-γ-亚氨基]三磷酸（p[NH]ppG）孵育时，在含有高尔基体和质膜标记物的部分中ARF的量增加，但在含有分泌颗粒、线粒体和溶酶体的部分中不增加。GTP、GDP及其β-硫代类似物没有增强ARF与膜的结合，smg 25/rab 3和rho以及通过[32 P]GTP结合检测到的所有其他小GTP结合蛋白在这些条件下没有重新分布。由于GTP[S]刺激这些细胞中的胰岛素分泌，我们还检查了ARF易位与胰岛素分泌之间的关系。这两种现象都是由GTP[S]引起的，其EC 50（中位有效浓度）约为10 μ M。p[NH]ppG在诱导胰岛素分泌方面与GTP[S]等效（EC 50约10 μ M），但需要更高的浓度（约500 μ M）来实现相同的最大ARF再分布。这些结果表明：（1）ARF在膜结合和游离/松散结合形式之间循环，由结合的鸟嘌呤核苷酸种类决定;（2）ARF单独似乎不调节胰岛素分泌细胞的胞吐作用。
Low-molecular-mass GTP-binding proteins of the ras family were analysed by [32P]GTP binding after PAGE and transfer to nitrocellulose membranes. By this technique, several GTP-binding proteins in the 20-30 kDa range were detected in both cytosolic and microsomal fractions of RINm5F cells. One of these, displaying an apparent molecular mass of about 20 kDa and a pI of 6.7, was mainly cytosolic and was shown to be the ADP-ribosylation factor (ARF) by using specific antibodies. When permeabilized RINm5F cells were incubated with the stable GTP analogues guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) the amount of ARF increased in a fraction containing both Golgi and plasma-membrane markers, but not in the fraction containing secretory granules, mitochondria and lysosomes. GTP, GDP and its beta-thio analogue did not enhance ARF binding to membranes, smg25/rab3 and rho, as well as all the other small GTP-binding proteins detected by the [32P]GTP binding, did not redistribute under these conditions. As GTP[S] stimulates insulin secretion in these cells, we also examined the relationship between ARF translocation and insulin secretion. Both phenomena were elicited by GTP[S] with an EC50 (median effective concentration) of about 10 microM. p[NH]ppG was equipotent with GTP[S] in inducing insulin secretion (EC50 about 10 microM), but higher concentrations (about 500 microns) were required to achieve the same maximal ARF redistribution. These results suggest that: (1) ARF is subject to cycling between a membrane-associated and a free/loosely attached form, determined by the species of bound guanine nucleotide; (2) ARF alone does not seem to regulate exocytosis in insulin-secreting cells.