Inhibition of Glycolipid Biosynthesis

Adriana E. Manzi1

1 University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.10B
DOI:  10.1002/0471142727.mb1710bs32
Online Posting Date:  May, 2001
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Abstract

Adequate inhibition of glycolipid biosynthesis allows the study of their biological functions. The method presented in this unit employs a synthetic analog of ceramide, PDMP (1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol), that inhibits glycolipid biosynthesis in cultured cells. Optimum conditions for inhibition of glycolipid biosynthesis are determined, glycolipids extracted from cultures grown with and without inhibitor, and the patterns of glycolipids analyzed by HPTLC. Detection is achieved using colorimetric reactions, or by monitoring radioactivity when cells have been metabolically radiolabeled.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1:

  Materials
  • Adherent cell culture
  • recipe10 mM PDMP in ethanol (see recipe)
  • Culture medium appropriate for cell line, with or without serum
  • Ethanol
  • Radiolabeled precursors (unit 17.4; for metabolic labeling)
  • PBS ( appendix 22)
  • 2:1, 1:1, and 1:2 (v/v) chloroform/methanol
  • 10:10:1 (v/v/v) chloroform/methanol/water
  • HPTLC standards: mixture of gangliosides or neutral lipids from bovine brain (Sigma or Accurate Chemical) or mixture of neutral glycolipids (Accurate Chemical)
  • recipeOrcinol/sulfuric acid reagent (see recipe)
  • recipeResorcinol/hydrochloric acid reagent (see recipe)
  • Screw‐cap glass tube, sterile
  • Conical glass tubes (10‐ or 50‐ml, depending on volume of pellet to be extracted)
  • Sonicator bath
  • Nitrogen stream
  • Rotary or shaker evaporator
  • Silica‐gel HPTLC plates
  • Enhance spray (DuPont NEN; for metabolic labeling)
  • Glass spray unit
  • Oven adjustable up to 140°C
  • Additional reagents and equipment for HPTLC (unit 9.7) and for metabolic radiolabeling (unit 17.4) and autoradiography ( appendix 3A; both optional)
NOTE: All incubations should be carried out under the conditions normally used for the cell line to be analyzed.
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Figures

Videos

Literature Cited

Literature Cited
   Datta, S.C. and Radin, N.S. 1986. Glucosylceramide and level of glucosidase‐stimulating proteins. Lipids 21:702‐709.
   Hospattankar, A.V. and Radin, N.S. 1982. Changes in liver lipids after administration of 2‐decanoylamino‐3‐morpholinopropiophenone and chlorpromazine. Lipids 17:538‐543.
   Inokuchi, J.I. and Radin, N.S. 1987. Preparation of the active isomer of 1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol, inhibitor of murine glucocerebroside synthetase. J. Lipid Res. 28:565‐571.
   Inokuchi, J.I., Mason, I., and Radin, N.S. 1987. Antitumor activity via inhibition of glycosphingolipid biosynthesis. Cancer Ltrs. 38:23‐30.
   Inokuchi, J., Momosaki, K., Shimeno, H., Nagamatsu, A., and Radin, N.S. 1989. Effects of D‐threo‐PDMP, an inhibitor of glucosylceramide synthetase, on expression of cell surface glycolipid antigen and binding to adhesive proteins by B16 melanoma cells. J. Cell. Physiol. 141:573‐583.
   Radin, N.S. and Inokuchi, J. 1991. Use of PDMP for the study of glycosphingolipid functions. Trends Glycos. Glycotech. 3:200‐213.
Key Reference
   Radin and Inokuchi, 1991. See above.
  Excellent review, providing very useful references for comparing conditions used for different systems along with specific protocols for studying the different biological roles affected by inhibition of glycolipid biosynthesis.
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