Characterization of the binding and internalization of tetanus toxin in a neuroblastoma hybrid cell line.

Abstract

Tetanus toxin is known to bind neuronal tissue selectively. To study the interactions of this potent neurotoxin in an intact cell system, the binding of 125I-tetanus toxin was characterized in a neuroblastoma retina hybrid cell line, N18-RE-105. The binding of 125I-tetanus toxin to membranes prepared from N18-RE-105 cells showed many similarities to the interactions of 125I-toxin with rat synaptic membranes. The binding was decreased with increasing temperature, ionic strength, and pH. 125I-Toxin bound to membranes with high affinity: KD = 0.62 +/- 0.05 nM; Bmax = 196 +/- 45 pmol/mg protein. Quantitative thin-layer chromatography and acid-degradation analysis revealed that N18-RE-105 cells contained polysialogangliosides GD1a and GT1b in high concentrations. An assay was developed to quantitate surface-bound and internalized 125I-tetanus toxin by exploiting the observation that surface-bound 125I-toxin is susceptible to pronase digestion. When cells were incubated with 125I-tetanus toxin at 0 degree C, all of the bound 125I-toxin could be degraded with pronase. In contrast, when the incubations were performed at 37 degrees C, within 10 min about 50% of the total cell-associated 125I-toxin was pronase-resistant. Temperature pulse experiments demonstrated that 125I-tetanus toxin that was bound to cells at 0 degree C rapidly disappeared from the surface when the cells were warmed to 37 degrees C, as revealed by the appearance of pronase-resistant radioactivity. This internalization was sensitive to metabolic inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

• Authors: G C Staub, K M Walton, R L Schnaar, T Nichols, R Baichwal, K Sandberg, T B Rogers
• Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience (J Neurosci) Vol. 6 Issue 5 Pg. 1443-51 (May 1986) ISSN: 0270-6474 [Print] United States
• PMID: 3711989 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Gangliosides
• Membrane Proteins
• Oligomycins
• Receptors, Cholinergic
• Tetanus Toxin
• tetanus toxin receptor
• Rotenone
• Pronase

Topics

• Animals
• Binding, Competitive
• Cell Line
• Chromatography, Thin Layer
• Drug Resistance
• Gangliosides (analysis)
• Hybrid Cells (analysis, metabolism)
• Membrane Proteins
• Mice
• Neuroblastoma (analysis, metabolism, pathology)
• Oligomycins (pharmacology)
• Pronase (pharmacology)
• Rats
• Receptors, Cholinergic (metabolism)
• Rotenone (pharmacology)
• Temperature
• Tetanus Toxin (antagonists & inhibitors, metabolism)