Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose: ceramide galactosyltransferase (CGalT), it has been stably expressed in CGalT-negative Chinese hamster ovary (CHO) cells. After fractionation of transfected cells, CHO-CGT, on sucrose gradients, the activity resides at the density of endoplasmic reticulum and not of Golgi. A lipid chromatogram from CHO-CGT cells revealed two new iodine-staining spots identified as GalCer, since they comigrate with GalCer standards, can be metabolically labelled with [3H]galactose, are recognized by anti-GalCer antibodies, and are resistant to alkaline hydrolysis. A third [3H]galactose lipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing ceramides. Remarkably, endogenous GalCer of transfected cells contains exclusively non-hydroxy fatty acids: fast atom bombardment and collision-induced dissociation mass spectrometric analysis revealed mainly C16:0 in the lower GalCer band on TLC and mainly C22:0 and C24:0 in the upper band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depending on their local availability. Thus, CGalT alone may be responsible for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyldiglyceride in myelin.
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July 1996
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Research Article|
July 15 1996
Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase
Petra van der BIJL;
Petra van der BIJL
*Department of Cell Biology, Faculty of Medicine, Institute of Biomembranes, The Netherlands
†Department of Basic Sciences, Division of Biochemistry, Faculty of Veterinary Medicine, Institute of Biomembranes, The Netherlands
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Ger J. STROUS;
Ger J. STROUS
*Department of Cell Biology, Faculty of Medicine, Institute of Biomembranes, The Netherlands
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Matthijs LOPES-CARDOZO;
Matthijs LOPES-CARDOZO
†Department of Basic Sciences, Division of Biochemistry, Faculty of Veterinary Medicine, Institute of Biomembranes, The Netherlands
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Jane THOMAS-OATES;
Jane THOMAS-OATES
‡Department of Mass Spectrometry, Faculty of Chemistry, Utrecht University, Utrecht, The Netherlands
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Gerrit van MEER
Gerrit van MEER
§
*Department of Cell Biology, Faculty of Medicine, Institute of Biomembranes, The Netherlands
§To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
January 02 1996
Revision Received:
March 11 1996
Accepted:
March 19 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (2): 589–597.
Article history
Received:
January 02 1996
Revision Received:
March 11 1996
Accepted:
March 19 1996
Citation
Petra van der BIJL, Ger J. STROUS, Matthijs LOPES-CARDOZO, Jane THOMAS-OATES, Gerrit van MEER; Synthesis of non-hydroxy-galactosylceramides and galactosyldiglycerides by hydroxy-ceramide galactosyltransferase. Biochem J 15 July 1996; 317 (2): 589–597. doi: https://doi.org/10.1042/bj3170589
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