Purification and partial characterization of a beta-1,3-glucan-binding-protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus.
A receptor for the 100 kDa beta-1,3-glucan-binding protein [Duvic, B. and Söderhäll, K. (1990) J. Biol. Chem. 265, 9327-9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE-cellulose chromatography of detergent-solubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide-gel electrophoresis and was composed of two non-covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide-gel electrophoresis or two-dimensional electrophoresis. The receptor could only bind the beta-1,3-glucan-binding protein if this protein had previously reacted with a beta-1,3-glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin-treated beta-1,3-glucan-binding protein to its receptor was a saturable process and binding data indicated a single high-affinity-binding site with a Kd of 0.35 +/- 0.15 microM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin-treated beta-1,3-glucan-binding protein. Laminarin, as well as oligosaccharides such as D-glucose, sialic acid, N-acetyl glucosamine or methyl-alpha-D-mannoside, could not affect the binding of the beta-1,3-glucan-binding protein to its receptor.
Duvic
B
B
Department of Physiological Botany, University of Uppsala, Sweden.
Söderhäll
K
K
D000818:Animals; D003400:Astacoidea; D001667:Binding, Competitive; D001773:Blood Cells / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D037102:Lectins; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism
D002352:Carrier Proteins; D037102:Lectins; D011956:Receptors, Cell Surface; C037937:glucan-binding proteins