title: 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 Kd = 2 nM, Bmax = 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 Mr 260 kDa in a ligand-blot process, are indicative of the general properties exhibited by receptors.
authors.affiliation: Centre de Recherches sur les Macromolécules Végétales, C.N.R.S., 53, F-38041, Grenoble Cedex, France.
authors.forename: Y
authors.initials: Y
authors.lastname: Liénart
authors.forename: R
authors.initials: R
authors.lastname: Dubois-Dauphin
authors.forename: C
authors.initials: C
authors.lastname: Gautier
authors.forename: M
authors.initials: M
authors.lastname: Khitri
authors.forename: H
authors.initials: H
authors.lastname: Driguez