Solubilization of a membrane-associated protein from rat nervous system tissues which binds anionic glycolipids and phospholipids

Tracy K. White, Ronald L. Schnaar

Research output: Contribution to journalArticlepeer-review

Abstract

An anionic glycolipid and phospholipid binding protein was characterized in detergent-solubilized rat brain synaptosomes using a synthetic, polyvalent radiolabeled ganglioside-protein conjugate as radioligand. Gangliosides are prominent cell surface glycoconjugates in vertebrate brain, where they may function in membrane protein regulation or in cell-cell recognition. The neoganglioprotein (GT1b)13BSA was radioiodinated and used to probe solubilized synaptosomal proteins for ganglioside binding activity using a receptor-ligand precipitation assay. Binding data revealed a high affinity (KD = 1 nM), saturable (Bmax = 173 pmol/mg protein) binding activity that was proteinase sensitive, calcium independent and maximal at neutral pH. Size exclusion chromatography of the synaptosomal (GT1b)13BSA binding activity indicated a Mr of ≈ 28 kDa. Binding activity with similar characteristics was solubilized from other rat tissues, with activity from sciatic nerve = muscle > synaptosomes > central nervous system myelin = liver. Gangliosides added as mixed detergent-lipid micelles inhibited (GT1b)13BSA binding: GT1b, GD1a and GD1b were the most effective inhibitors (IC50 ≈ 200 nM), while GM1 and GM3 were 5-fold less effective. In addition, the sphingolipids sulfatide and sialylneolactotetraosylceramide were effective inhibitors, with IC50 values of 300 nM and 200 nM, respectively. The neutral sphingolipid GA1 did not block (GT1b)13BSA binding. Phosphatidylcholine and phosphatidylethanolamine were non-inhibitory, however phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol inhibited half-maximally at 200-300 nM. Inhibition by both gangliosides and anionic phospholipids was competitive with (GT1b)13BSA. We conclude that a binding protein for anionic glycolipids and phospholipids is distributed on nerve and muscle membranes.

Original languageEnglish (US)
Pages (from-to)218-226
Number of pages9
JournalBBA - Biomembranes
Volume1196
Issue number2
DOIs
StatePublished - Dec 30 1994

Keywords

  • Brain membrane
  • Carbohydrate receptor
  • Ganglioside
  • Lipid binding

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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