Koumanov F, Yang J, Jones A E, Hatanaka Y, Holman G D
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1209-15. doi: 10.1042/bj3301209.
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 spacer (Bio-ATB-BMPA) or an additional hexanoic acid spacer (Bio-LC-ATB-BMPA). The half-maximal inhibition constants, Ki values, for inhibition of glucose transport activity in insulin-stimulated rat adipocytes were determined to be 359+/-10 and 273+/-28 microM 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 microg 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 approximately 10-fold or approximately 20-fold, respectively, when the electrochemiluminescent or the Western blot detection methods were used and these signals were blocked by cytochalasin B.
新的细胞非渗透性双甘露糖光标记物已被开发出来，其生物素基团通过聚乙氧基间隔物（Bio-ATB-BMPA）或额外的己酸间隔物（Bio-LC-ATB-BMPA）连接到4-(1-叠氮基-2,2,2-三氟乙基)-苯甲酰基-1,3-双（d-甘露糖-4-氧基）-2-丙胺（ATB-BMPA）上。在胰岛素刺激的大鼠脂肪细胞中，抑制葡萄糖转运活性的半数最大抑制常数Ki值，Bio-ATB-BMPA和Bio-LC-ATB-BMPA分别为359±10和273±28微摩尔。这些值与先前报道的非生物素化化合物ATB-BMPA的值相似。在紫外线照射诱导生物素化光标记物与大鼠脂肪细胞交联后，利用与链霉亲和素的相互作用，可通过非放射性和放射性方法检测生物素化的葡萄糖转运蛋白异构体4（GLUT4）。从1-2微克脂肪细胞膜沉淀到磁性链霉亲和素珠上的生物素化GLUT4，可使用电化学发光测定法进行灵敏和定量检测。该方法使用了钌标记的抗GLUT4抗体，在电极激发下，在ORIGEN分析仪中产生电化学发光信号。或者，表面生物素化的GLUT4在链霉亲和素琼脂糖沉淀物中很容易检测到，但灵敏度较低，通过传统的GLUT4蛋白质免疫印迹法进行分析。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获得的数据相比具有优势。当使用电化学发光或蛋白质免疫印迹检测方法时，胰岛素处理脂肪细胞分别使表面生物素化GLUT4的信号水平增加约10倍或约20倍，并且这些信号被细胞松弛素B阻断。
