Cell-surface biotinylation of GLUT4 using bis-mannose photolabels
Cell-surface biotinylation of GLUT4 using bis-mannose photolabels
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DOI：
10.1042/bj3301209
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发表时间：
1998-03-15
影响因子：
4.1
通讯作者：
Holman, GD
中科院分区：
文献类型：
--
作者：
Koumanov, F;Yang, J;Holman, GD
已经开发了新的细胞不渗透性双甘露糖光标记，其中生物素基团通过聚乙氧基sgacer（Bio-ATB-BMPA）或额外的己酸间隔基（Bio-LC-ATB-BMPA）连接到4-（1-azi-2，2，2-trifluoroethyl）-苯甲酰基-1，3-bis（D-mannos-4-yloxy）-2-丙胺（ATB-BMPA）。对于Bio-ATB-BMPA和Bio-LC-ATB-BMPA，胰岛素刺激的大鼠脂肪细胞中葡萄糖转运活性抑制的半最大抑制常数K值分别为359 +/- 10和273 +/- 28 μ M。这些值与先前报告的非生物素化化合物ATB-BMPA的值相似。紫外线照射诱导交联的生物素化的光标记大鼠脂肪细胞，生物素化的葡萄糖转运蛋白亚型4（GLUT 4）可以检测到非放射性和放射性的方法，利用与链霉亲和素的相互作用。从1-2 μ g脂肪细胞膜，沉淀到磁性链霉亲和素珠上的生物素化GLUT 4，可以使用电致发光测定方法进行灵敏和定量检测。这利用了在ORIGEN分析仪中在电极处激发时产生电致发光信号的经硒标记的抗GLUT 4抗体。或者，表面生物素化的GLUT 4可以很容易地，但不太敏感，检测链霉亲和素琼脂糖沉淀，通过传统的GLUT 4蛋白质印迹分析。使用非放射性方法获得的数据与使用生物素化探针的氚化版本的数据相比是有利的。当使用电致发光或蛋白质印迹检测方法时，脂肪细胞的胰岛素处理使来自表面生物素化的GLUT 4的信号水平分别增加了约10倍或约20倍，并且这些信号被细胞松弛素B阻断。
New cell-impermeant bis-mannose photolabels have been developed with biotinyl groups attached to 4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1,3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA) by either a polyethoxy sgacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal inhibition constants, K, values, for inhibition of glucose transport activity in insulin-stimulated rat adipocytes were determined to be 359 +/- 10 and 273 +/- 28 mu M for Bio-ATB-BMPA and Bio-LC-ATB-BMPA, respectively. These values are similar to those previously reported for the non-biotinylated compound ATB-BMPA. Following UV-irradiation-induced cross-linking of the biotinylated photolabels to rat adipocytes, the biotinylated glucose transporter isoform 4 (GLUT4) could be detected by non-radioactive and radioactive methods that utilized the interaction with streptavidin. Biotinylated GLUT4 from 1-2 mu g of adipose cell membranes, precipitated onto magnetic streptavidin beads, could be sensitively and quantitatively detected using an electrochemiluminescent assay method. This utilized a ruthenium-tagged anti-GLUT4 antibody that on excitation at an electrode generated an electrochemiluminescent signal in an ORIGEN analyser. Alternatively, surface-biotinylated GLUT4 could be easily, but less sensitively, detected in streptavidin agarose precipitates which were analysed by conventional GLUT4 Western blotting. Data obtained using the non-radioactive methods compared favourably with those using tritiated versions of the biotinylated probes. Insulin treatment of adipocytes increased the levels of signals from surface biotinylated GLUT4 by similar to 10-fold or similar to 20-fold, respectively, when the electrochemiluminescent or the Western blot detection methods were used and these signals were blocked by cytochalasin B.