TOPOLOGY OF GLUCOSYLCERAMIDE SYNTHESIS IN GOLGI MEMBRANES FROM PORCINE SUBMAXILLARY-GLANDS
TOPOLOGY OF GLUCOSYLCERAMIDE SYNTHESIS IN GOLGI MEMBRANES FROM PORCINE SUBMAXILLARY-GLANDS
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DOI：
10.1016/0005-2736(86)90285-3
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发表时间：
1986-06-13
期刊：
影响因子：
--
通讯作者：
GOT, R
中科院分区：
文献类型：
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作者：
COSTE, H;MARTEL, MB;GOT, R
在源自高尔基体的猪颌下腺的密封和“右侧朝外”囊泡中研究了神经酰胺葡萄糖基转移酶和从头合成的葡萄糖神经酰胺的拓扑结构。链霉蛋白酶处理不会引起管腔糖蛋白半乳糖基转移酶活性的任何破坏，在蛋白酶与高尔基体蛋白的比例为1:100时，将神经酰胺葡糖基转移酶抑制到50%以上。相同浓度的胰蛋白酶虽然不会使管腔半乳糖基转移酶失活，但会导致神经酰胺糖基转移酶活性完全丧失。膜不可渗透的化合物DID不引起半乳糖基转移酶的任何抑制，但抑制神经酰胺葡糖基转移酶(在80μM DIDS时减少70％)。因此，神经酰胺葡萄糖基转移酶可从高尔基体囊泡的细胞质侧进入。通过葡萄糖神经酰胺酶在完整和破坏的囊泡上水解该化合物的能力来研究新合成的葡萄糖神经酰胺的方向。在 3 小时的孵育过程中获得了相同的水解百分比（分别为 36% 和 30%），表明葡萄糖神经酰胺根本没有受到外部水解的保护。链霉蛋白酶处理的囊泡显示葡萄糖神经酰胺酶水解增加（高达 45%），这表明葡萄糖神经酰胺可能是隐秘的。所有这些结果表明，神经酰胺葡萄糖基转移酶以及相关的葡萄糖神经酰胺在猪颌下腺的高尔基体囊泡中以细胞质为导向。
The topology of ceramide glucosyltransferase and de novo synthesized glucosylceramide was studied in sealed and ''right-side-out'' vesicles of porcine submaxillary glands derived from Golgi apparatus. Pronase treatment which did not cause any breakdown of the luminal glycoprotein galactosyltransferase activity, inhibited the ceramide glucosyltransferase to more than 50% at a ratio proteinase to Golgi protein 1:100. Trypsin at the same concentration, while producing no inactivation of luminal galactosyltransferase, caused a complete loss of ceramide glycosyltransferase activity. The membrane-impermeable compound, DIDs, which did not cause any inhibition of the galactosyltransferase, inhibited the ceramide glucosyltransferase (70% reduction at 80 .mu.M DIDS). Thus, the enzyme ceramide glucosyltransferase is accessible from the cytoplasmic side of the Golgi vesicles. The orientation of the newly synthesized glucosylceramide is studied by the ability of the enzyme glucosylceramidase to hydrolyse this compound both on intact and on disrupted vesicles. The same percentage (respectively, 36 and 30%) of hydrolysis was obtained during an incubation of 3 h, showing that glucosylceramide is not at all protected from external hydrolysis. Pronase-treated vesicles revealed an increase in glucosylceramidase hydrolysis (up to 45%), which indicates that glucosylceramide may be cryptic. All these results indicate that the ceramide glucosyltransferase, as well as related glucosylceramide, are cytoplasmically oriented in Golgi vesicles from porcine submaxillary glands.