Preparation of affinity-purified,biotinylated tetanus toxin,and characterization and localization of cell surface binding sites on nerve growth factor-treated PC12 cells |
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Authors: | Ko Fujita Dr Gordon Guroff Ephraim Yavin Gerthrud Goping Richard Orenberg Philip Lazarovici |
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Institution: | (1) Section on Growth Factors, National Institute of Child Health and Human Development, National Institutes of Health, 20892 Bethesda, Maryland;(2) Department of Neurobiology, The Weizmann Institute of Science, 26100 Rehovot, Israel;(3) Laboratory of Cell Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 20892 Bethesda, Maryland;(4) National Institute of Child Health and Human Development, National Institutes of Health, Building 6, Room 130, 20892 Bethesda, MD |
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Abstract: | Biotinylated derivatives of tetanus toxin were prepared and isolated by chromatofocusing and ganglioside-affinity chromatography. Biotinylation was monitored by the appearance of a 210,00 dalton complex upon SDS-polyacrylamide gel electrophoresis in the presence of avidin, and by selective binding to an avidin-Sepharose gel. At molar biotin:toxin ratios from 11 to 201 only biotinylated derivatives with low toxicity were obtained; these derivatives, however, retained 60–80% of their specific binding affinity for brain synaptosomes. A biotinylated tetanus toxin derivative purified by ganglioside-affinity chromatography was used to identify and localize tetanus toxin binding sites on PC12 cells. Electron microscopic analysis with streptavidin-gold revealed very low levels of tetanus toxin binding sites on the surface of untreated cells, and the appearance of such binding sites during the second week of nerve growth factor-induced differentiation. Examination of micrographs of the differentiated cells indicated that the tetanus toxin binding sites sites are concentrated on the neurites, with relatively few appearing on the cell bodies. Cognate studies using125I-labeled, affinity-purified tetanus toxin revealed an increase in PC12 binding capacity from about 0.07 nmol/mg protein in untreated cells to 0.8 nmoles/mg protein in cells treated for 14 days with nerve growth factor. Cells treated in suspension for 2–3 weeks with nerve growth factor do not express tetanus toxin binding sites; upon plating, these cells required one week for the appearance of binding sites, although neurites grew much more rapidly from these primed cells. The high binding capacity of these tetanus toxin sites, as well as their sensitivity to neuraminidase, is indicative of a polysialoganglioside structure. The advantages of biotinylated tetanus toxin derivatives are discussed and the significance of nerve growth factor-differentiated PC12 cells grown as monolayers as a model for the study of the development, localization, and function of neuraminidase-sensitive tetanus toxin binding sites is presented.Abbreviations PBS
phosphate-buffered saline
- STS
sucrose-Tris-serum solution
- NGF
nerve growth factor
- C
collagen
- PL
polylysine
- BBG
bovine brain ganglioside mixture
- GM1
gafactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide
- GD1a
N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide
- GT1a
N-aceylneuraminyl]-galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide
- GD1b
galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl-N-acetylneuraminyl]-galactosylglucosyl ceramide
- GT1b
N-acetylneuraminyl]-galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl-N-acetylneuraminyl] galactosylglucosyl ceramide
- NANA
N-acetylneuraminic acid |
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Keywords: | Biotinylated tetanus toxin streptavidin-gold nerve growth factor (NGF) PC12 |
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