Identification of tyrosyl residue present in the high-affinity saccharide-binding site of ricin D.

Abstract

We analyzed [125I]iodotyrosyl residues in the B-chain of ricin D iodinated at pH 7.0 and 0°C for 60min in the presence and absence of lactose. Peptides containing such [125I]iodotyrosyl residues were isolated from the subtilisin digest of iodinated B-chains by gel filtration on Sephadex G-25 followed by paper chromatography and paper electrophoresis. Their locations in the primary structure were assigned based on their amino acid composition. In the absence of lactose, five tyrosyl residues were susceptible to iodonation, but their reactivities varied; Tyr-248 seemed to be the most reactive, followed by Tyr-148, Tyr-78 (or Tyr125), and Tyr-67; Tyr-176 was the least reactive. In the presence of lactose, however, Tyr-248 was completely protected from iodination, and the reactivity of Tyr-67 increased more than two-fold; that of Tyr-78 (or Tyr-125) decreased by about half. From these and earlier results, we concluded that Tyr-248 exists in the high-affinity saccharide-binding site of the B-chain. Tyr-67 and Tyr-78 (or Tyr-125) may exist next to each other near the low-affinity saccharide-binding site of the B-chain, and their iodination may cause a loss of lactosebinding ability.