Paul P, Bordmann A, Rosenfelder G, Towbin H
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basle, Switzerland.
Anal Biochem. 1992 Aug 1;204(2):265-72. doi: 10.1016/0003-2697(92)90237-2.
We assessed inhibitors of glycosylation by simultaneous determination of [14C]Gal incorporated into glycosphingolipids and glycoproteins as well as of [3H]Leu incorporated into proteins of intact cells. After metabolic labeling in 96-well plates in the presence or absence of a test substance, cells were collected on glass-fiber filters. The lipid components were extracted from the filter and radioactivities of both extract and filter determined. The reliability of the procedure was tested with different drugs. Using the glucocerebroside synthetase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP; 5 microM), glycolipid biosynthesis was shown to be reduced to 50% in the murine T-cell EL-4 6.1 line, whereas glycosylation of proteins was not disturbed. With 0.5 microM tunicamycin, the glycosylation of proteins was 50% of that in the control. The procedure was also able to detect various specific effects: the inhibition of protein glycosylation with D-glucosamine and castanospermine, the inhibition of glycosphingolipid biosynthesis with L-cycloserine, and a slight enhancement of glycosphingolipid biosynthesis with conduritol B epoxide and castanospermine. Within a series of N-acyl homologs of PDMP the inhibitory potency increased with chain length. In contrast, these homologs were equipotent by enzymatic in vitro assays.
我们通过同时测定掺入糖鞘脂和糖蛋白中的[14C]半乳糖以及掺入完整细胞蛋白质中的[3H]亮氨酸来评估糖基化抑制剂。在存在或不存在测试物质的情况下，在96孔板中进行代谢标记后，将细胞收集在玻璃纤维滤器上。从滤器中提取脂质成分，并测定提取物和滤器的放射性。用不同的药物测试了该程序的可靠性。使用葡萄糖脑苷脂合成酶抑制剂1-苯基-2-癸酰氨基-3-吗啉代-1-丙醇（PDMP；5 microM），在小鼠T细胞EL-4 6.1系中，糖脂生物合成显示减少至50%，而蛋白质糖基化未受干扰。用0.5 microM衣霉素处理时，蛋白质糖基化是对照的50%。该程序还能够检测到各种特定效应：D-葡萄糖胺和栗精胺对蛋白质糖基化的抑制，L-环丝氨酸对糖鞘脂生物合成的抑制，以及环缩醛醇B环氧化物和栗精胺对糖鞘脂生物合成的轻微增强。在一系列PDMP的N-酰基同系物中，抑制效力随链长增加。相比之下，这些同系物在体外酶促试验中具有同等效力。
