Interaction of G_M1 Ganglioside with Bovine Serum Albumin Formation and Isolation of Multiple Complexes

Abstract

The binding of ganglioside C r M l to bovine serum albumin has been studied by using absorption and fluorescence properties of the protein chromophores. Differences in the ultraviolet absorption spectrum and in fluorescence quenching, as well as a marked shift of the wavelength at the fluorescence maximum provide information about the binding of this ganglioside to albumin. Ultracentrifugal studies showed that there are two forms of the GM1-protein complexes which differ markedly in their molecular weight. These two forms have been separated on this basis, by a chromatographic sieving procedure, and designated as complexes I and IT. Both complexes are characterized by a G M~ : protein ratio of one ganglioside micelle per albumin polypeptide chain. Complex I1 polymerizes slowly and irreversibly to a dimer, complex I. These results have been correlated with the optical studies in order to draw limited inferences as to the environment of the binding sites on the native protein. The interaction between G M M I micelles and albumin is mostly hydrophobic and the two complexes are actually mixed ganglioside-protein micelles. At submicellar concentrations of ganglioside a binding of ganglioside GMI to albumin also occurs. This process is due, however, to an aspecific, reversible adhesion of GMl inolecules on the albumin surface with no apparent perturbation of the albumin' structure.In the past few years, it has become increasingly evident that gangliosides, glycosphingolipids containing sialic acid [l], contribute to several properties of natural cell membranes. With their hydrophilic oligosaccharide moieties protruding out of the membrane, gangliosides are well suited to play a role in the transmission of biochemical sigiiak across the membrane and into the cell. The biochemical information, generated by ligand-ganglioside interaction at the cell surface, forins an intrinsic part of a variety of events, such as the cellular processes activated by the bacterial toxins of Vihrio cholercte [2] and C'lostridium tctnizi [3], by lectins [4,5], by hormones [6]. by interferon (71 and by cell-cell recognition [8,9]. The carbohydrate pattern of the oligosaccharide portion of the gangliosidcs seems to determine the specificity of the Ahhreviation. Ganglioside GMI, galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminosyl)-ga~actosy~g~ucosy~c~ramide. ['13)-Gu,, tritium-labelled GM,.Enzyme. Galactose oxidase (EC 1.1.3.9); Vihrio cholcwe neuraminidase (EC 3.2.1.18).ganglioside-protein interaction. Moreover, proteins that can cross-link gangliosides on the cell surface may cause redistribution of these gangliosides into segregated regions [lo, 111, which results in a modification of membrane fluidity. Apparently a higher membrane fluidity due to ganglioside aggregation is a characteristic of the structuring and stability of functional synaptic membranes [12,13]. This different localization of gangliosides in the lipid matrix will expose proteins inside the aggregate to a different environment than that surrounding proteins outside tli...

Sphingolipid Uptake by Cultured Cells

“…Cell Feeding with [1][2][3] H]Sphingosine- [1-3 H]Sphingosine dissolved in methanol was transferred into a sterile glass tube and dried under a nitrogen stream; the residue was then dissolved in an appropriate volume of pre-warmed (37°C) 2% FCS-EMEM (fibroblasts) or cellconditioned medium (granule cells) or 10% FCS-DMEM (neuroblastoma cells) to obtain a final 3 ϫ 10 Ϫ8 M concentration. After a 2-h incubation (pulse), the medium was removed, and cells were washed with culture medium and incubated for 48 h with culture medium not containing radioactive precursors in the presence of 10% FCS.…”

“…Some data suggest that under this condition the amount of gangliosides taken up by the cells is much lower, but the rate of the catabolic process higher (11). This is probably because of the formation of stable complexes between gangliosides and the proteins and lipoproteins of FCS (3)(4)(5) that reduce the number of free monomers available for a direct interaction with the cell membranes but allows endocytosis of sphingolipid aggregates (12). Sphingomyelin is also taken up by neuronal cells and largely catabolized (13).…”

“…The critical aggregative concentration of sphingolipids is in the nano-or picomolar range (1,2), thus the amount of free monomers in solution is low. Nevertheless, the quantity of free monomers should increase when a favorable equilibrium leads to the formation of stable lipoprotein complexes (3)(4)(5). This process should also occur in living organisms, where significant amounts of sphingolipids are detectable in serum and cerebrospinal fluid from normal and pathological subjects (6 -8), as components of serum lipoproteins or associated with albumin (4).…”

“…In this paper we performed the following: 1) the release of all 1 The abbreviations used are: FCS, fetal calf serum; Neu5Ac, Nacetylneuraminic acid; Cer, ceramide; N-acylsphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy-octadecene; [1-3 H]sphingosine, (2S,3R,4E)-2-amino-1,3-dihydroxy- [1-3 H]octadecene; [1][2][3] H]sphinganine, (2S,3R)-2-amino-1,3-dihydroxy- [1-3 H]octadecane; PE, phosphatidylethanolamine; SM, sphingomyelin; BME, basal modified Eagle's medium; DMEM, Dulbecco's modified Eagle's medium; EMEM, Eagle's minimum essential medium; HPTLC, high performance thin layer chromatography; GlcCer, ␤-Glc-(1-1)-Cer; LacCer, ␤-Gal-(1-4)-␤-Glc-(1-1)-Cer; GM3, II classes of sphingolipids from both normal and tumor cells in culture, 2) the association of sphingolipids released by cells with proteins and lipoproteins of FCS present in the culture medium, and 3) the fate of sphingolipids released by the cells and taken up by the same or different cells. Our results suggest that the endogenous and specific sphingolipid content of plasma membrane cannot be changed in vivo by the incorporation into cells of circulating sphingolipids, and suggest that the sphingolipids that are taken up by the cells are largely and rapidly catabolized.…”

Determination of Gangliosides in Human Cerebrospinal Fluid by High-Performance Thin-Layer Chromatography and Direct Densitometry

“…The procedure is based on the assumption that CSF gangliosides are associated with proteins. Albumin is known to strongly bind to gangliosides (14). Moreover, a rapid procedure of CSF volume reduction causes an abrupt increase in ganglioside concentration, thereby triggering the formation of micelles of higher molecular mass (2).…”