title: Topology of glucosylceramide synthesis in Golgi membranes from porcine submaxillary glands.
abstract: 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 glucosyltransferase activity. The membrane-impermeable compound, DIDS, which did not cause any inhibition of the galactosyltransferase, inhibited the ceramide glucosyltransferase (70% reduction at 80 microM 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 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.
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002554:Cerebrosides / Q000096:biosynthesis*; D003593:Cytoplasm / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D005964:Glucosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D066298:In Vitro Techniques; D013363:Submandibular Gland / Q000378:metabolism / Q000648:ultrastructure; D013552:Swine; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D002554:Cerebrosides; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D005700:Galactosyltransferases; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D014532:Uridine Diphosphate Glucose