Pharmacological and biochemical properties of saxiphilin, a soluble saxitoxin-binding protein from the bullfrog ( Rana catesbeiana)

Abstract

Supernatant fractions of various tissues and plasma from the North American bullfrog, Rana catesbeiana, specifically bind saxitoxin with high affinity. Binding of [ 3H]saxitoxin to bullfrog plasma follows single-site behavior with an equilibrium dissociation constant of K d = 0.16±0.03 nM at 0°C and a maximum binding capacity of 380±60 pmole/ml plasma. High-affinity binding of [ 3H]saxitoxin is chemically specific since it is unaffected by tetrodotoxin and a variety of cationic peptides, amino acids and drugs. The structure-activity dependence of binding to this site was investigated with eight different natural and synthetic derivatives of saxitoxin. Substitution of the carbamoyl side chain or the C-12 β-hydroxyl group of saxitoxin with a hydrogen atom had little effect on binding affinity, but addition of a hydroxyl group at the N-1 position decreased the binding affinity from 430- to 710-fold in three different molecular pairs. High performance size exclusion chromatography of supernatant from bullfrog skeletal muscle showed that the [ 3H]saxitoxin-binding component migrates with an apparent molecular weight of M r = 74,000±8000 or a Stokes radius of 35±2 A ̊ . The [ 3H]saxitoxin-binding protein in skeletal muscle extract or plasma is retained on a cation-exchange column at pH 6.0, suggesting that the protein contains a region of exposed basic residues. Column isoelectric focusing of a sample from plasma indicated that the protein has a basic isoelectric point near pH = 10.7. These pharmacological and biochemical properties imply that this soluble saxitoxin-binding activity is associated with a novel protein named saxiphilin that is structurally distinct from known subtypes of functional sodium channel proteins which are the biological target of saxitoxin's paralyzing effect.