Membrane binding sites for the human blood group H-type 2 determinant, an inducer of laminarinase activity in protoplasts ofRubus fruticosusL.
✍ Scribed by Y. Liénart; R. Dubois-Dauphin; C. Gautier; M. Khitri; H. Driguez
- Publisher
- Springer-Verlag
- Year
- 1992
- Tongue
- English
- Weight
- 973 KB
- Volume
- 188
- Category
- Article
- ISSN
- 0032-0935
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✦ Synopsis
The human blood-group determinants H-type 1 (~-L-Fuc-(1-~2)-fl-D-Gal-(1-~3)fl-D-GlcNAc), or type 2 (~-L-Fuc(l~2)-fl-D-Gal(1 ~4)-fl-o-GlcNAc) and their mono-and disaccharidic precursors, have been reported to induce D-glycanase (laminarinase) activity in Rubus cells (Y. Li6nart et al. 1990, Plant Science 68, 197-202) and protoplasts (Y. Li6nart 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, highaffinity binding activity for GlcNAc, compatible with the presence of a single class of binding sites (Kd = 2 nM, Bma x : 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, fl-D-Gal-(1 ~3)-fl-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 IC5o 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.