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The ceramide structure of GM1 ganglioside differently affects its recovery in low-density membrane fractions prepared from HL-60 cells with or without triton-X100

Abstract

Gangliosides are characteristically enriched in various membrane domains that can be isolated as low density membrane fraction insoluble in detergents (detergent-resistant membranes, DRMs) or obtained after homogenization and sonication in 0.5 M sodium carbonate (low-density membranes, LDMs). We assessed the effect of the ceramide structure of four [3H]-labeled GM1 ganglioside molecular species (GM1s) taken up by HL-60 cells on their occurrence in LDMs, and compared it with our previous observations for DRMs. All GM1s contained C18 sphingosine, which was acetylated in GM1(18:1/2) or acylated with C14, C18 or C18:1 fatty acids (Fas)

Abbreviations

cPMs:

crude plasma membranes

CT:

cholera toxin

DRMs:

detergent-resistant membranes

Fa:

fatty acid

GM1 ganglioside:

Galβ3GalNAcβ4 (Neu5Acα3)Galβ4GlcCer (GM1s are abbreviated according to Palestini et al. [39] as follows: GM1(18:1/2), GM1 with N-acetylated C18 sphingosine; GM1(18:1/14), GM1 with myristic acid-acylated C18 sphingosine; GM1(18:1/18), GM1 with stearic acid-acylated C18 sphingosine; and GM1(18:1/18:1), GM1 with oleic acid-acylated C18 sphingosine)

LDMs:

low-density membrane fraction

ld :

liquid disordered

lo :

liquid ordered

medium H:

RPMI 1640 medium containing 10 mM Hepes buffer, pH 7.3, and 5 μg/ml insulin and transferrin

PBS-G:

PBS containing 0.1% gelatin

sodium carbonate buffer:

a solution consisting of 2.5 mM Tris, 500 mM Na2CO3, 5 mM NaCl, 2.5 mM EDTA, pH 11.0, 2 mM Pefabloc SC and chymostatin, leupeptin, antipain, and pepstatin, each at 5 μg/ml

TX:

Triton X-100

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Correspondence to Tadeusz Pacuszka.

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Keywords

  • Ceramide
  • Gangliosides
  • GM1
  • Membrane domains
  • Myristic acid
  • Sonication