Vicia graminea lectin binding to human M and N erythrocytes

Abstract

The mode of binding of Vicia graminea 125I-labelled lectin to human M and N erythrocytes at 4°C has been investigated. The labelled lectin retained the full activity of native lectin. Lectin association at 4°C was characterized by a t 1 2 of 3 to 5 min, reaching steady-state within 15 min. Incubation of cells for 15 min at 4°C with increasing concentrations of Vicia graminea 125I-labelled lectin showed that saturation binding occurred. Scatchard analysis of equilibrium data determined over a wide range of lectin concentrations yielded a curvilinear plot with an upward concave slope; this representation indicated that there was not a single homogeneous class of noninteracting binding sites. This result could indicate two or more independent classes of binding sites or one class of interacting sites exhibiting negative cooperativity. Since unlabelled lectin, which at the concentration used, rapidly binds to available receptors, did not affect the dissociation rate of the labelled lectin and since identical Scatchard plots were found using native and formaldehyde-fixed erythrocytes we conclude that there are two classes of independent Vicia graminea binding sites on human erythrocytes. Computer analysis of the Scatchard plots gave high- and low-affinity constant (7.07±1.1) · 10 7 M −1 and (0.2±0.01) · 10 7 M −1, respectively, for N erythrocytes and (1.13±0.18) · 10 7 M −1 and (0.24±0.01) · 10 7 M −1, respectively for the M cells. N erythrocytes were estimated to have 0.085 · 10 5 high-affinity and 2.1 · 10 5 low-affinity sites and M erythrocytes, 0.011 · 10 5 high affinity and 0.13 · 10 5 low-affinity sites. N cells therefore have 10-times as many sites as M cells. Studies of the dissociation of 125I-labelled lectin from N and M cells in the presence of unlabelled lectin gave dissociation rate constants of 51 · 10 −4 s −1 and 1.97 · 10 −4 s −1 for the high- and low-affinity sites of N cells and 13 · 10 −4 s −1 and 1.6 · 10 −4 s −1 for the high- and low-affinitym sites of M cells, indicating that the binding of Vicia graminea lectin to human erythrocytes is reversible.