Biochemical and immunochemical comparison of saxiphilin and transferrin, two structurally related plasma proteins from Rana catesbeiana

Y Li, L Llewellyn and E Moczydlowski

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.

Saxiphilin is a approximately 90-kDa protein in bullfrog plasma that binds the neurotoxin saxitoxin (STX) with high affinity (Kd, approximately 0.2 nM). The relationship between saxiphilin and transferrin was examined because partial sequencing of saxiphilin previously revealed an unexpected homology to members of the transferrin family of Fe(3+)-binding proteins. Transferrin was purified from bullfrog plasma and shown to be distinct from saxiphilin on the basis of its size (approximately 78 kDa), chromatographic behavior, visible absorption spectrum, and ligand-binding properties. High affinity binding of [3H]STX was found to be a distinctive property of saxiphilin that was not exhibited by transferrins from various species of animals. Conversely, under conditions appropriate for transferrins, purified saxiphilin did not bind 55Fe3+, implying that it is not involved in iron metabolism. Polyclonal antibodies raised against native saxiphilin precipitated [3H]STX-binding activity from whole bullfrog plasma. On immunoblots such antibodies recognized the denatured saxiphilin protein but only weakly labeled bullfrog transferrin. In an enzyme-linked immunosorbent assay using native proteins, antisaxiphilin antibodies weakly cross-reacted with transferrin from bullfrog and a number of other species. Likewise, antibodies against human transferrin cross-reacted with saxiphilin in a similar immunosorbent assay. These results lead to the conclusion that saxiphilin is not bullfrog transferrin but is structurally related to the transferrin family. As a novel member of the transferrin superfamily, saxiphilin may help to uncover new functions mediated by this class of proteins.

Volume 44, Issue 4, pp. 742-748, 10/01/1993
Copyright © 1993 by American Society for Pharmacology and Experimental Therapeutics

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