Purification and partial sequencing of saxiphilin, a saxitoxin-binding protein from the bullfrog, reveals homology to transferrin.
Plasma from the bullfrog, Rana catesbeiana, contains a soluble component of unknown function that specifically binds the neurotoxin, [3H]saxitoxin, with a Kd of approximately 0.2 nM. Saxiphilin, the protein responsible for this activity, was purified approximately 440-fold from bullfrog plasma by column chromatography on heparin-Sepharose followed by chromatofocusing. The purified saxiphilin preparation exhibits a binding capacity of 9.6 nmol/mg protein and a Kd of 0.32 nM for [3H]saxitoxin. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a predominant band migrating with an apparent Mr of approximately 89,000 which is similar to the expected size of saxiphilin previously estimated by nondenaturing size exclusion chromatography. Amino-terminal sequencing of the approximately 89-kDa protein and sequencing of four different tryptic peptide fragments revealed that each of the partial saxiphilin sequences can be aligned by homology with members of the transferrin protein family with sequence identity as high as 69%. The available sequence corresponding to conserved residues that comprise part of the two Fe3+ binding sites in lacto-transferrin show several substitutions in saxiphilin, suggesting that saxiphilin is not an Fe(3+)-binding protein. Saxiphilin appears to be a monomeric approximately 89-kDa protein that is evolutionarily related to transferrin but which binds saxitoxin instead of Fe3+.
Li
Y
Y
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Moczydlowski
E
E
D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011892:Rana catesbeiana; D012530:Saxitoxin / Q000378:metabolism*; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014168:Transferrin / Q000235:genetics*; D014357:Trypsin
D029845:Amphibian Proteins; D002352:Carrier Proteins; D014168:Transferrin; C027525:saxitoxin-binding protein, Rana catesbeiana; D012530:Saxitoxin; D014357:Trypsin