Studies on perivitelline fluid of horseshoe crab embryo. II. Purification of agglutinin-binding substance from the perivitelline fluid of Tachypleus gigas embryo.
Three glycoproteins with potent agglutinin-binding activity have been isolated from the perivitelline fluid of Tachypleus gigas, horseshoe crab, embryo. In the native form, these agglutinin-binding substances were highly aggregated. After being dissociated in 10 M urea, these proteins were fractionated by gel-filtration on a Fractogel TSK (Toyopearl) HW-60 in Tris-NaCl-CaCl2 (0.05 M Tris-HCl, pH 7.5, containing 0.5 M NaCl and 0.1 M CaCl2) containing 10 M urea. The proteins thus obtained were designated as ABS-I, -II, and -III in the order of elution and have apparent molecular weights of 25,000 (ABS-II) and 10,000 (ABS-III) as judged by both gel-filtration on Fractogel TSK (Toyopearl) HW-60 in 10 M urea and sodium dodecyl sulfate-gel electrophoresis; the molecular weight of ABS-I could not be estimated in the two systems since it was too high. ABS-I, -II, and -III, of which only ABS-I is water-soluble, inhibit one hemagglutination unit of activity with minimum quantities of 0.5 micrograms/ml, 7.8 micrograms/ml, and 1.0 micrograms/ml, respectively. They were found to be glycoproteins in which 6.6% of the dry weight (ABS-I), 4.2% of the dry weight (ABS-II), and 7.5% of the dry weight (ABS-III) were carbohydrate. The dry weight ratio of hexosamines in these substances is 3:1:2 (ABS-I: ABS-II: ABS-III), and that of sialic acid is also 3:1:2. Amino acid analyses of these proteins indicated that they have high contents of aspartic acid, glutamic acid, and glycine in common.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification*; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification*; D006384:Hemagglutination; D006385:Hemagglutination Inhibition Tests; D006388:Hemagglutinins; D006737:Horseshoe Crabs / Q000196:embryology*; D008970:Molecular Weight; D012794:Sialic Acids / Q000032:analysis; D014817:Vitelline Membrane / Q000276:immunology
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D006023:Glycoproteins; D006388:Hemagglutinins; D012794:Sialic Acids