g(m1)-ganglioside and lucifer-yellow

A fluorescent derivative of ganglioside GM1 was prepared by oxidation of the sialic acid residue with sodium periodate and reaction of the resulting aldehyde with Lucifer yellow CH. The biological activity of the fluorescent derivative was compared with that of native GM1 using GM1-deficient rat glioma C6 cells. When the cells were exposed to either native or fluorescent GM1, their ability to bind 125I-labeled cholera toxin was increased to a similar extent. This increase in binding was directly proportional to the amount of ganglioside added to the medium. The affinity of the toxin for cells treated with either native or fluorescent GM1 also was similar. More importantly, the fluorescent GM1 was as effective as native GM1 in enhancing the responsiveness of the cells to cholera toxin. Thus, the ganglioside-treated cells exhibited a 9-fold increase in toxin-stimulated cyclic AMP production over cells not exposed to GM1. There was a similar increase in iodotoxin binding and toxin-stimulated cyclic AMP accumulation in cells treated with other GM1 derivatives containing rhodaminyl or dinitrophenyl groups. On the basis of these results, it is clear that these modified gangliosides retain the ability to function as receptors for cholera toxin. Consequently, fluorescent gangliosides are likely to be useful as probes for investigating the dynamics and function of these membrane components.

Topics: Animals; Cell Line; Cholera Toxin; Chromatography, Thin Layer; Cyclic AMP; Fluorescent Dyes; G(M1) Ganglioside; Glioma; Isoquinolines; Kinetics; Rats; Receptors, Cell Surface; Receptors, Immunologic

Fluorescent derivatives of gangliosides were prepared by oxidizing the sialyl residues to aldehydes and reacting them with fluorescent hydrazides. When rhodaminyl gangliosides were incubated with lymphocytes, the cells incorporated them in a time- and temperature-dependent manner. Initially, the gangliosides were evenly distributed on the cell surface but were redistributed into patches and caps by antirhodamine antibodies. When the cells were then stained with a second antibody or protein A labeled with fluorescein, the fluorescein stain revealed the coincident movement of both the gangliosides and the antirhodamine antibodies. When the cells were treated with both rhodamine and Lucifer yellow CH-labeled gangliosides, the antirhodamine antibodies induced patching and capping of both fluorescent gangliosides but had no effect on cells incubated only with Lucifer yellow CH-labeled gangliosides. In addition, capping was observed on cells exposed to cholera toxin, antitoxin antibodies, and rhodamine-labeled protein A, indirectly showing the redistribution of endogenous ganglioside GM1, the cholera toxin receptor. By incorporating Lucifer yellow CH-labeled GM1 into the cells and inducing capping as above, we were able to demonstrate directly the coordinate redistribution of the fluorescent GM1 and the toxin. When the lymphocytes were stained first with Lucifer yellow CH-labeled exogenous ganglioside GM3, which is not a toxin receptor, there was co-capping of endogenous GM1 (rhodamine) and exogenous GM3 (Lucifer yellow CH). These results suggest that gangliosides may self-associate in the plasma membrane which may explain the basis for ganglioside redistribution and capping.

Topics: Animals; Antibodies; Cholera Toxin; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Hydrazines; Isoquinolines; Mice; Rats; Rats, Inbred Lew; Rhodamines; T-Lymphocytes; Thiocyanates