Purification, characterization and subcellular localization of pig liver α‐L‐iduronidase

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Abstract
.alpha.-L-Iduronidase was purified about 100000-fold from pig liver by employing column chromatography on cellulose phosphate (P11), concanavalin A-sepharose 4B, heparin-Sepharose 4B, Toyopearl HW-55, Sephadex G-100 and chelating sepharose 6B charged with cupric ions. The molecular mass of the purified enzyme was estimated to be 70 kDa by Sephadex G-100 column chromatography. The purified enzyme gave a single band on disc polyacrylamide gel electrophoresis without using sodium dodecyl sulfate. However, two separate components of 70 kDa and 62 kDa appeared when it was analyzed by SDS/polyacrylamide gel electrophoresis. These 70-kDa and 62-kDa components were confirmed as .alpha.-L-iduronidase immunochemically. The isoelectric points of these enzymes were both 9.1 measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Km values were 3.0.sbd.3.5 and 65 .mu.M 4-methylumbelliferyl-.alpha.-L-iduronide, respectively. The purified enzyme was stable in the pH range 3.5.sbd.6.0 under conditions with or without 0.5 M NaCl. However, in the presence of 0.5 M NaCl, it was unstable at pH 3.0. Moreover, it was conversely stabilized at pH 7.0 in the presence of 0.5 M NaCl. Immunohistochemically, the enzyme was found in the Kupffer cells and was abundant on their lysosomal membranes. In liver cells, however, the immunohistochemical reaction was weak.