Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid.
The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including alpha-(2----8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis alpha-(2----8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated binding and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3200 pmol of [3H]TTX-binding sites/mg of protein and a single polypeptide of approximately 285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. We further describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.
James
W M
WM
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Emerick
M C
MC
Agnew
W S
WS
D000818:Animals; D002846:Chromatography, Affinity; D004557:Electric Organ / Q000378:metabolism*; D004563:Electrochemistry; D004593:Electrophorus / Q000378:metabolism; D007075:Immunoglobulin M; D019158:N-Acetylneuraminic Acid; D012116:Resins, Plant; D012794:Sialic Acids / Q000276:immunology; D015222:Sodium Channels / Q000032:analysis*; D013779:Tetrodotoxin
D007075:Immunoglobulin M; D012116:Resins, Plant; D012794:Sialic Acids; D015222:Sodium Channels; D013779:Tetrodotoxin; D019158:N-Acetylneuraminic Acid