Crosslinking of lectins and receptors in membranes with hetero-bifunctional crosslinking reagents

Abstract

125I-Con A was reacted with the imidoester group of methyl 4-azidobenzoimidate and isolated from excess reagents on a Sephadex G-25 column. The hetero-bifunctional crosslinking reagent has two dissimilar functional groups, imidoester and arylazide, instead of two identical functional groups found in conventional homo-bifunctional crosslinking reagents. The two functional groups react under different conditions and it is possible to react one first and to activate the other later. Here the reagents were attached to the lectins by the imidoester reactions, and the arylazides now attached to the lectins were intended for the later use to crosslink the lectins and receptors. Human erythrocyte ghosts were incubated with the activated lectins, unbound lectins removed, and irradiated with UV to photolyze arylazides, thus crosslinking the lectins to the receptors in membranes. Upon the solubilization and subsequent electrophoresis of the sample, a new band appeared on the gel. 125I-Con A was detected in band A, the new band, and band 3, one of the membrane glycoproteins, diminished in parallel to the appearance of band A. The concurrent appearance of band A accompanied by 125I-Con A and decrease of band 3 were significantly reduced, when ghosts were incubated with the activated lectins but were not irradiated, ghosts were incubated with nonactivated 125I-Con A and irradiated, or ghosts were incubated with the activated concanavalin A in the presence of alpha-methylmannoside, the lectin inhibitor, and irradiated. The inhibitor removed a significant amount of the activated lectins bound to membranes, but failed to do so the activated lectins crosslinked into band A.