Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component.
The pharmacokinetic interaction of an affinity-purified 125I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 X 10(-9) M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 degrees C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 degrees C but not at 37 degrees C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. Trypsin and chymotrypsin inhibited binding between 55% and 68% while bacterial protease abolished it by 91-95%. The effect was species-specific as it was not seen in rat or bovine synaptosomes. Collagenase and hyaluronidase had little or no inhibitory effect when applied to synaptosomes (27% and 9%) but inhibited binding to synaptic vesicles by 56% and 49%, respectively. Phospholipases A2 and C caused 42-43% inhibition of binding in vesicles and less than 22% in synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D006168:Guinea Pigs; D006867:Hydrolases / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins; D009439:Neuraminidase; D009498:Neurotoxins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism; D014357:Trypsin
D008565:Membrane Proteins; D009498:Neurotoxins; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D017981:Receptors, Neurotransmitter; C028728:tetanus toxin receptor; D006867:Hydrolases; D009439:Neuraminidase; D014357:Trypsin