Characterization of the binding and internalization of tetanus toxin in a neuroblastoma hybrid cell line.
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)
G C
GC
Staub
K M
KM
Walton
R L
RL
Schnaar
T
T
Nichols
R
R
Baichwal
K
K
Sandberg
T B
TB
Rogers
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002855:Chromatography, Thin Layer; D004351:Drug Resistance; D005732:Gangliosides / Q000032:analysis; D006822:Hybrid Cells / Q000032:analysis / Q000378:metabolism*; D008565:Membrane Proteins; D051379:Mice; D009447:Neuroblastoma / Q000032:analysis / Q000378:metabolism* / Q000473:pathology; D009840:Oligomycins / Q000494:pharmacology; D011402:Pronase / Q000494:pharmacology; D051381:Rats; D011950:Receptors, Cholinergic / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D013696:Temperature; D013744:Tetanus Toxin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005732:Gangliosides; D008565:Membrane Proteins; D009840:Oligomycins; D011950:Receptors, Cholinergic; D013744:Tetanus Toxin; C028728:tetanus toxin receptor; D012402:Rotenone; D011402:Pronase