Membrane binding sites for the human blood group H-type 2 determinant, an inducer of laminarinase activity in protoplasts of Rubus fruticosus L.

Abstract

The human blood-group determinants H-type 1 (α-l-Fuc-(1 → 2)-β-d-Gal-(1 → 3)β-d-GlcNAc), or type 2 (α-l-Fuc(1 → 2)-β-d-Gal(1 → 4)-β-d-GlcNAc) and their mono- and disaccharidic precursors, have been reported to induce d-glycanase (laminarinase) activity in Rubus cells (Y. Liénart et al. 1990, Plant Science 68, 197–202) and protoplasts (Y. Liénart et al. 1991, Plant Science 77, 41–45). Using immunoadsorbent H-type 1 as a matrix for the affinity purification of membrane proteins, and the H-type 2 trisaccharide neoglycoprotein as ligand in kinetic-dependent enzyme-linked-immunosorbent assay for measuring binding, we were able to show that Rubus microsomes contain high-affinity binding sites for the laminarinase inducers. The N-acetyl glucosamine (GlcNAc) eluate was found to contain a saturable, high affinity binding activity for GlcNAc, compatible with the presence of a single class of binding sites K_d = 2 nM, B_max = 400 pmol · (mg protein)⁻¹. In contrast, the Scatchard plot of proteins in the lactose eluate was nonlinear. In competition studies, the precursors of H-type 1 (GlcNAc-OCH₃, β-d-Gal-(1 → 3)-β-d-GlcNAc-OCH₃) or of H-type 2 (GlcNAc, N-acetyl lactosamine) trisaccharides inhibited the binding of the proteins in the GlcNAc eluate by H-type 2 neoglycoprotein with respective IC₅₀ values of 0.6, 0.6 or 2, 0.4 nM. These data, and the binding of the H-type 2 trisaccharide by a protein of M_r 260 kDa in a ligand-blot process, are indicative of the general properties exhibited by receptors.