Isolation and physicochemical characterization of electrolectin, a beta-D-galactoside binding lectin from the electric organ of Electrophorus electricus.
Electrolectin, a beta-D-galactoside binding lectin, has been isolated from the electric organ of the electric eel Electrophorus electricus. Electrolectin is purified 1000-fold with a yield of 10 mg/kg of tissue by steps including low speed centrifugation, ammonium sulfate precipitation, and affinity chromatography on a lactosyl-Sepharose column. Electrolectin is a dimer composed of two subunits. The molecular weight of the monomer is around 16,500 as determined by sodium dodecyl sulfate-gel electrophoresis and amino acid analysis. The molecular weight of the dimer determined by equilibrium sedimentation is 32,500 +/- 750. The electrolectin monomer is composed of 144 amino acids and 2.2 +/- 0.45 carbohydrate. It contains one tryptophan but cysteine and metals are absent. The exposure of electrolectin to O2 destroys its hemagglutination activity, abolishes its UV fluorescence and shifts its UV absorption maximum from 287 nm to 250 nm. The oxidation of tryptophan to oxindole is prevented by lactose. The strict requirement of reducing agents for the maintenance of electrolectin agglutination activity is explained by the need to prevent the oxidation of a tryptophan residue in the lactose-binding site. The quantum yields of electrolectin and its complex with lactose are pH dependent and reach a maximal value of 0.4 at neutral pH. The binding constant of lactose to electrolectin is also pH dependent. These data and their temperature dependence stress the important contribution of ionizable groups in the binding of lactose. The latter stabilizes the dimeric structure of electrolectin.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D004593:Electrophorus; D005697:Galactosides / Q000302:isolation & purification* / Q000378:metabolism*; D037161:Galectins; D006027:Glycosides / Q000302:isolation & purification* / Q000378:metabolism*; D006386:Hemagglutination Tests; D037102:Lectins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D002241:Carbohydrates; D002352:Carrier Proteins; D005697:Galactosides; D037161:Galectins; D006027:Glycosides; D037102:Lectins; C007816:beta-galactoside