Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass.
Human alpha-L-iduronidase from liver was purified about 20 000-fold with a new rapid three-step, five-column procedure which consisted of a Concanavalin-A-Sepharose/Blue-A-Agarose coupled step, a CM-Sepharose/Bio-Gel HT coupled step followed by a cupric-ion-chelating Sepharose 6B step. The behaviour of alpha-L-iduronidase on gel permeation chromatography was dependent upon both pH and ionic strength of the eluting buffer. The formation of species with enzyme activity which behaved as large-molecular-mass aggregates was favoured under conditions of low ionic strength and neutral pH. The amount of high-Mr species diminished as the pH decreased or the ionic strength increased to favour a single active species of Mr 65 000. A specific monoclonal antibody was generated against liver alpha-L-iduronidase. The antibody specifically immunoprecipitated enzyme activity from both crude and purified sources. The subunit Mr of liver alpha-L-iduronidase was estimated to be 65 000 using SDS-PAGE. Monoclonal antibody immunoprecipitation of radiolabelled enzyme was used to provide definitive confirmation of this subunit size.
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002845:Chromatography / Q000379:methods; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007068:Iduronidase / Q000276:immunology / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology; D008970:Molecular Weight; D009994:Osmolar Concentration; D011489:Protein Denaturation; D012995:Solubility
D000911:Antibodies, Monoclonal; D006026:Glycoside Hydrolases; D007068:Iduronidase