Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.
Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.
D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D001894:Borohydrides / Q000494:pharmacology; D002471:Cell Transformation, Neoplastic; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005692:Galactose Oxidase; D005732:Gangliosides / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010504:Periodic Acid; D014734:Vibrio cholerae
D001427:Bacterial Toxins; D001894:Borohydrides; D005732:Gangliosides; D010504:Periodic Acid; D005692:Galactose Oxidase