Molecular characterization, reconstitution, and "transport-specific fractionation" of the saxitoxin binding protein/Na+ gate of mammalian brain.
The saxitoxin (STX) binding protein has been solubilized by sodium cholate, both from axolemma and from synaptosomal membranes of mammalian brain. On the basis of agarose gel filtration and sedimentation properties in H2O and 2H2O, the solubilized particle has the following molecular properties: Stokes radius, 120 A; partial specific volume, 0.85 cm3/g; mass, 1,020,000 daltons; frictional ratio f/fo, 1.6. The solubilized STX binding protein was incorporated into unilamellar (approximately 550-A) artificial phosphatidylcholine vesicles. Based on the expectation that the STX binding protein contains functional monovalent cation gating activity ("action potential Na+ gate") that can be activated by veratridine and inhibited by tetrodotoxin, a strategy was devised for partial purification of the reconstituted sodium gate/STX binding protein by "transport-specific fractionation." When the entire vesicle population was preloaded with 0.4 M cesium ion, addition of veratridine allowed Cs+ efflux from specifically those vesicles containing the ion gate; the concomitant reduction in intravesicular density permitted the ion gate/STX binding protein to be fractionated on density gradients. These observations demonstrate functional reconstitution and partial (30- to 50-fold) purification of the STX binding protein/Na+ gate of mammalian brain.
D029845:Amphibian Proteins; D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000302:isolation & purification; D002417:Cattle; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification; D009441:Neurilemma / Q000032:analysis; D012530:Saxitoxin / Q000302:isolation & purification*; D013574:Synaptosomes / Q000032:analysis; D013779:Tetrodotoxin / Q000494:pharmacology; D014701:Veratridine / Q000494:pharmacology
D029845:Amphibian Proteins; D002352:Carrier Proteins; D007473:Ion Channels; D008081:Liposomes; D008565:Membrane Proteins; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; D013779:Tetrodotoxin; D014701:Veratridine