Purification, characterization and subcellular localization of pig liver alpha-L-iduronidase.
alpha-L-Iduronidase was purified about 100,000-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 as measured by isoelectric focusing in a polyacrylamide gel containing ampholine and sucrose. The optimal pH and Km values were 3.0-3.5 and 65 microM 4-methylumbelliferyl-alpha-L-iduronide, respectively. The purified enzyme was stable in the pH range 3.5-6.0 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.
Ohshita
T
T
Department of Pathology, Ryukyu University School of Medicine, Okinawa, Japan.
Sakuda
H
H
Nakasone
S
S
Iwamasa
T
T
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000302:isolation & purification*; D007068:Iduronidase / Q000276:immunology / Q000302:isolation & purification*; D007106:Immune Sera / Q000302:isolation & purification; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D007728:Kupffer Cells / Q000201:enzymology; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008247:Lysosomes / Q000201:enzymology; D008854:Microscopy, Electron; D013552:Swine
D007106:Immune Sera; D006026:Glycoside Hydrolases; D007068:Iduronidase