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

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 (G_T1b)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 (K_D = 1 nM), saturable (B_max = 173 pmol/mg protein) binding activity that was proteinase sensitive, calcium independent and maximal at neutral pH. Size exclusion chromatography of the synaptosomal (G_T1b)₁₃BSA binding activity indicated a M_r 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 (G_T1b)₁₃BSA binding: G_T1b, G_D1a and G_D1b were the most effective inhibitors (IC₅₀ ≈ 200 nM), while G_M1 and G_M3 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 G_A1 did not block (G_T1b)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 (G_T1b)₁₃BSA. We conclude that a binding protein for anionic glycolipids and phospholipids is distributed on nerve and muscle membranes.