Affinity Purification of the Voltage-Sensitive Sodium Channel from Electroplax with Resins Selective for Sialic Acid

William M. James, Mark C. Emerick, William S. Agnew

Research output: Contribution to journalArticlepeer-review

Abstract

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including '-(2→8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Umax 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 α-(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 ~ 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.

Original languageEnglish (US)
Pages (from-to)6001-6009
Number of pages9
JournalBiochemistry
Volume28
Issue number14
DOIs
StatePublished - Jul 1 1989

ASJC Scopus subject areas

  • Biochemistry

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