Physical and chemical characterization of the major lactose-blockable lectin activity from fetal calf skeletal muscle.
The lactose-blockable lectin activity from fetal calf skeletal muscle has been purified to apparent homogeneity. The purification entails differential centrifugation, ammonium sulfate precipitation, asialofetuin affinity chromatography with a lactose gradient and ion-exchange chromatography on DEAE-cellulose. In the last step, the activity is resolved into a major and minor species, designated ion-exchange-purified lectins I and II, respectively. Both lectin activities are reversibly inhibited by lactose and appear as single bands with identical mobilities on SDS-polyacrylamide gel electrophoresis. Lectin II was not obtained in sufficient quantities for further characterization. Lectin I is characterized by a functional requirement for reducing agents and sensitivity to N-ethylmaleimide, which suggests a role for an essential thiol in its activity. Subunit molecular weight determinations by SDS-polyacrylamide gel electrophoresis (12 000 +/- 1 000) and by gel filtration in 6 M guanidine . HCl (13 000 +/- 1 000), when compared with that obtained under native conditions on Bio-Rad P-60 gels (27 000 +/- 2 000), suggest a true Mr of 25 000 +/- 3 000 for the dimeric molecule. Amino acid composition data, when fitted to this molecular weight, lead to the tentative conclusion that the intact dimer is composed of two very similar but compositionally non-identical chains, designated by alpha and beta. While the only detectable N-terminal amino acid is tryptophan, the isoelectric focusing pattern of lectin I supports this heterodimeric structure. In addition, a lactose-sensitive hemagglutinating activity which can be separated from the lactose-blockade activity by affinity chromatography was also observed.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D005033:Ethylmaleimide / Q000494:pharmacology; D006386:Hemagglutination Tests; D007785:Lactose / Q000494:pharmacology*; D037102:Lectins / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis / Q000196:embryology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D037102:Lectins; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D007785:Lactose; D005033:Ethylmaleimide