Affinity purification and affinity characterization of carbohydrate-binding proteins in bovine kidney.
Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney by two-step affinity chromatography on fetuin and heparin columns and subsequent anion-exchange high-performance liquid chromatography. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the purified fraction gave two protein bands corresponding to proteins of relative molecular mass 33,000 (p33) and 41,000 (p41), respectively. Although the proteins had no haemogglutinating activities towards human and rabbit erythrocytes, their carbohydrate-binding activity was examined by a newly developed method using horseradish peroxidase (HRP) and/or biotin-labelled glycoconjugates as affinity probes. They could bind in a Ca(2+)-dependent manner to labelled fetuin and heparin in a specific and dose-dependent manner by solid-phase assay after immobilization on plastic plate surface. Inhibition assay of the binding revealed that N-acetylneuraminic acid is the most potent inhibitor of the proteins among the monosaccharides tested. Fucoidin and heparan sulphate most strongly inhibited the binding of the proteins to labelled heparin. Direct binding assay to acidic glycolipids prepared from bovine kidney showed that the proteins react with the ganglioside fraction but not with sulphatide [Gal(3-SO4) beta 1-1Cer]. These results indicated that the purified proteins have a significant affinity to charged oligosaccharides linking to glycoproteins, glycolipids and charged polysaccharides in a Ca(2+)-dependent manner.
Kojima
K
K
Department of Chemistry, Faculty of Science, Ochanomizu University, Tokyo, Japan.
Ogawa
H K
HK
Seno
N
N
Matsumoto
I
I
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects; D006028:Glycosphingolipids / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000737:chemistry*; D008970:Molecular Weight; D011485:Protein Binding; D011817:Rabbits; D011956:Receptors, Cell Surface
D000596:Amino Acids; D002352:Carrier Proteins; D006028:Glycosphingolipids; D011956:Receptors, Cell Surface; C055213:saccharide-binding proteins