Oligosaccharide composition of the neurotoxin responsive Na/sup +/ channel and the requirement of sialic acid for activity
The neurotoxin responsive Na/sup +/ channel was purified to homogeneity in an 18% yield from a clonal cell line of mouse neuroblastoma, N-18, metabolically labeled with L-(/sup 3/H)fucose. The Na/sup +/ channel, a glycoprotein, M/sub r/=200,000 (gradient 7-14% PAGE) was digested with Pronase and the glycopeptides were characterized by serial lectin affinity chromatography. greater than 90% of the oligosaccharides contained sialic acid and 18% were biantennary, 39% were triantennary and 30% tetraantennary. The glycoprotein was reconstituted into artificial phospholipid vesicles and /sup 86/Rb flux was stimulated (65%) by 200 ..mu..M veratridine and 1.2 ..mu..g of scorpion venom and was inhibited (95%) by 5 ..mu..M tetrodotoxin. The requirement of sialic acid for Na/sup +/ channel activity was demonstrated since neuraminidase (0.01 U) treatment of the reconstituted glycoprotein eliminated the response of /sup 86/Rb flux to the stimulating neurotoxins. In other experiments, treatment of N-18 cells with 10 ..mu..M swainsonine, an inhibitor of glycoprotein processing, altered the oligosaccharide composition of the Na/sup +/ channel. When the abnormally glycosylated Na/sup +/ channel was reconstituted into artificial phospholipid vesicles, /sup 86/Rb flux in response to neurotoxins was impaired. Thus, glycosylation of the polypeptide with oligosaccharides of specific composition and structure is essential for expression of the biological activity of the neurotoxin responsive Na/sup +/ channel.
- Research Organization:
- Univ. of Pennsylvania Medical School, Philadelphia
- OSTI ID:
- 7108639
- Report Number(s):
- CONF-8606151-; TRN: 86-039057
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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GLUCOPROTEINS
CHEMICAL COMPOSITION
PURIFICATION
O-GLYCOSYL HYDROLASES
ENZYME ACTIVITY
CHROMATOGRAPHY
MICE
OLIGOSACCHARIDES
PHOSPHOLIPIDS
RUBIDIUM 86
SIALIC ACID
SODIUM COMPOUNDS
TOXINS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TUMOR CELLS
ALKALI METAL COMPOUNDS
ALKALI METAL ISOTOPES
ANIMAL CELLS
ANIMALS
ANTIGENS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOHYDRATES
DAYS LIVING RADIOISOTOPES
ENZYMES
ESTERS
GLYCOSYL HYDROLASES
HYDROLASES
INTERMEDIATE MASS NUCLEI
ISOMERIC TRANSITION ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LIPIDS
MAMMALS
MATERIALS
MINUTES LIVING RADIOISOTOPES
MONOSACCHARIDES
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
PROTEINS
RADIOISOTOPES
RODENTS
RUBIDIUM ISOTOPES
SACCHARIDES
SEPARATION PROCESSES
TOXIC MATERIALS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques