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Title: Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

Abstract

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}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{yields}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 3,200 pmol of ({sup 3}H)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. The authors describe a cation-dependent stabilization with millimolarmore » levels of monovalent and micromolar levels of divalent species.« less

Authors:
; ;  [1]
  1. Yale Univ. School of medicine, New Haven, CT (USA)
Publication Date:
OSTI Identifier:
5060259
Resource Type:
Journal Article
Journal Name:
Biochemistry; (USA)
Additional Journal Information:
Journal Volume: 28:14; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; RECEPTORS; PURIFICATION; RESINS; PERFORMANCE; CELL MEMBRANES; DOSE-RESPONSE RELATIONSHIPS; EEL; ELECTROPHORESIS; GLYCOPROTEINS; IMMUNOGLOBULINS; LECTINS; SIALIC ACID; SODIUM COMPOUNDS; TRITIUM COMPOUNDS; ALKALI METAL COMPOUNDS; ANIMALS; AQUATIC ORGANISMS; CARBOHYDRATES; CELL CONSTITUENTS; FISHES; GLOBULINS; HYDROGEN COMPOUNDS; MEMBRANE PROTEINS; MEMBRANES; MONOSACCHARIDES; ORGANIC COMPOUNDS; ORGANIC POLYMERS; PETROCHEMICALS; PETROLEUM PRODUCTS; POLYMERS; PROTEINS; SACCHARIDES; VERTEBRATES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

James, W M, Emerick, M C, and Agnew, W S. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid. United States: N. p., 1989. Web. doi:10.1021/bi00440a042.
James, W M, Emerick, M C, & Agnew, W S. Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid. United States. https://doi.org/10.1021/bi00440a042
James, W M, Emerick, M C, and Agnew, W S. Tue . "Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid". United States. https://doi.org/10.1021/bi00440a042.
@article{osti_5060259,
title = {Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid},
author = {James, W M and Emerick, M C and Agnew, W S},
abstractNote = {The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}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{yields}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 3,200 pmol of ({sup 3}H)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. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.},
doi = {10.1021/bi00440a042},
url = {https://www.osti.gov/biblio/5060259}, journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 28:14,
place = {United States},
year = {1989},
month = {7}
}