Pharmacological and biochemical properties of saxiphilin, a soluble saxitoxin-binding protein from the bullfrog (Rana catesbeiana).
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 Kd = 0.16 +/- 0.03 nM at 0 degrees 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 beta-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 Mr = 74,000 +/- 8000 or a Stokes radius of 35 +/- 2A. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Mahar
J
J
Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510.
Lukács
G L
GL
Li
Y
Y
Hall
S
S
Moczydlowski
E
E
D029845:Amphibian Proteins; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D007525:Isoelectric Focusing; D009132:Muscles / Q000378:metabolism; D011892:Rana catesbeiana; D012530:Saxitoxin / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship
D029845:Amphibian Proteins; D002352:Carrier Proteins; D015222:Sodium Channels; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin