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

Journal Article · Tue Jul 11 04:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00440a042· OSTI ID:5060259

James, W M; Emerick, M C; Agnew, W S ^[1]

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1. Yale Univ. School of medicine, New Haven, CT (USA)

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.

• OSTI ID: 5060259
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 28:14; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALI METAL COMPOUNDS
ANIMALS
AQUATIC ORGANISMS
CARBOHYDRATES
CELL CONSTITUENTS
CELL MEMBRANES
DOSE-RESPONSE RELATIONSHIPS
EEL
ELECTROPHORESIS
FISHES
GLOBULINS
GLYCOPROTEINS
HYDROGEN COMPOUNDS
IMMUNOGLOBULINS
LECTINS
MEMBRANE PROTEINS
MEMBRANES
MONOSACCHARIDES
ORGANIC COMPOUNDS
ORGANIC POLYMERS
PERFORMANCE
PETROCHEMICALS
PETROLEUM PRODUCTS
POLYMERS
PROTEINS
PURIFICATION
RECEPTORS
RESINS
SACCHARIDES
SIALIC ACID
SODIUM COMPOUNDS
TRITIUM COMPOUNDS
VERTEBRATES