g(m1)-ganglioside and sodium-borohydride

Liposomes were used to determine whether gangliosides containing certain structurally defined analogues of sialic acid could inhibit activation of the alternative pathway of human C. Gangliosides containing sialic acid residues with modifications in the N-acetyl group, carboxyl group, or polyhydroxylated tail were either isolated from natural sources or prepared by chemical modification of the native sialic acid structure. Sialic acid lost more than 90% of its inhibitory activity after removal of just the C9 carbon from the polyhydroxylated tail. Sialic acid was also unable to inhibit activation after converting the carboxyl group to a hydroxymethyl group. Galactose oxidase/NaB3H4 treatment of liposomes containing gangliosides with native or modified sialic acid residues confirmed that neither modification altered the amount of gangliosides exposed at the liposome surface. Changing the N-linked acetyl group to a glycolyl group had no effect on the inhibitory activity of sialic acid. These data further define the structural features of sialic acid that are important in regulation of alternative pathway activation. Both the C9 carbon of the polyhydroxylated tail and the carboxyl group are essential for this function; whereas, the N-linked acetyl group may be modified without loss of activity.

Topics: Binding, Competitive; Borohydrides; Carbohydrate Conformation; Complement Activation; Complement C3b Inactivator Proteins; Complement Pathway, Alternative; G(M1) Ganglioside; Humans; Hydroxylation; Liposomes; N-Acetylneuraminic Acid; Sialic Acids; Structure-Activity Relationship

Among lipids, gangliosides can be selectively stained with Schiff's reagent if the oxidizing agent (sodium metaperiodate) is sufficiently dilute to exclude all but the readily oxidized sialic acid sugars. A borohydride-periodate-Schiff (BhPS) sequence is recommended as a reliable method, convenient to perform, for the detection of the intraneuronal lipid accumulations in the ganglioside storage disorders.

Topics: Borohydrides; Brain Chemistry; G(M1) Ganglioside; Gangliosides; Gangliosidoses; Histocytochemistry; Humans; Oxidation-Reduction; Periodic Acid-Schiff Reaction; Spinal Cord; Tay-Sachs Disease

The ganglioside II3NeuAc-GgOse4Cer and other glycosphingolipids can be radiolabeled to high specific activity by the galactose oxidase-NaB3H4 procedure, by purifying the oxidized compounds prior to reductive labeling. The oxidized products are separated from nonoxidized compounds and detergents (Triton X-100 and sodium taurocholate) present during the enzymatic oxidation. Since the oxidized derivatives are separated, the final specific activity depends solely upon the specific activity of the NaB3H4 and the reduction conditions.

Topics: Animals; Borohydrides; Cattle; Chromatography, Gel; G(M1) Ganglioside; Galactose Oxidase; Gangliosides; Glycosphingolipids; Humans; Isotope Labeling; Oxidation-Reduction; Tritium

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

Topics: Animals; Borohydrides; Cell Line; Cholera Toxin; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; G(M1) Ganglioside; Galactose Oxidase; Gangliosides; Glycoproteins; Immunosorbent Techniques; Mice; Mice, Inbred BALB C; Receptors, Cell Surface; Receptors, Immunologic