Purification and characterization of multiple species (isolectins) of a slime mold lectin implicated in intercellular adhesion.
An improved purification procedure for the carbohydrate-binding proteins (lectins) of cohesive Polysphondylium pallidum cells has been devised. The procedure uses extraction of cells with lactose-containing buffer followed by ammonium sulfate precipitation and affinity chromatography of the redissolved precipitate on a column of acid-treated Sepharose 6B. All hemagglutination activity is adsorbed to the column and recoveries are about 70% of the activity of the starting cell lysate. Sodium dodecyl sulfate-gel electrophoresis of the protein obtained with this procedure resolved three subunits with molecular weights of 26,500 (A), 26,000 (B), and 25,000 (C). Three species are resolved by isoelectric focusing with apparent pI values of 6.4 (I), 7.3 (II), and 7.5 (III) which contain Subunits A, B, and C in the following ratios: I, B:C at 2:1; II, A:B at 2:1, and III, A:B at 1:2. All three isoforms agglutinate rabbit and human type O erythrocytes and are thus isolectins. Isoforms II and III are separated from Isoform I by galactose-gradient elution of the Sepharose 6B column. Isoforms II and III aggregate extensively (nonamers and multiples thereof), but reduction with 2-mercaptoethanol reverses this process yielding a single species of Mr = 73,000 (trimer). Isoform I exists as trimers and hexamers and reduction has no effect on this distribution. Amino acid compositions and tryptic peptide maps of S-[14C]carboxymethyl-isolectins indicate that Subunits A and B are very similar and may represent the same peptide chain, while Subunit C is a peptide quite distinct from A and B.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates; D002448:Cell Adhesion; D006386:Hemagglutination Tests; D006595:Hexosamines / Q000032:analysis; D006801:Humans; D037102:Lectins / Q000302:isolation & purification; D008970:Molecular Weight; D009235:Myxomycetes / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D000509:alpha-Fetoproteins
D000596:Amino Acids; D002241:Carbohydrates; D006595:Hexosamines; D037102:Lectins; D010446:Peptide Fragments; D000509:alpha-Fetoproteins