Human kidney alpha-L-iduronidase: purification and characterization

Rome, L.H.; Garvin, A.J.; Neufeld, E.F.

Archives of Biochemistry and Biophysics 189(2): 344-353

1978


ISSN/ISBN: 0003-9861
PMID: 30407
DOI: 10.1016/0003-9861(78)90221-7
Accession: 068518494

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Abstract
.alpha.-L-Iduronidase (EC 3.2.1.76) was purified 25,000-fold from the soluble proteins of human kidney by chromatography on heparin-Sepharose, hydroxylapatite and Bio-Gel P-100. The .alpha.-L-iduronidase activity is associated with 80% of the protein in the most purified preparation. It has a MW of 60,000 .+-. 6500, determined by sedimentation equilibrium, and can be dissociated by reduction into subunits of MW 31,000 .+-. 6500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate in the presence of dithiothreitol. It contains glucosamine and binds to concanavalin A. the pH optimum, Km and Vmax for 2 substrates, phenyl iduronide and [3H]anhydromannitol iduronide, were found to be 4.0, 1.5 mM, 16 .mu.mol/mg protein per min, and 4.5, 9 mM and 270 .mu.mol/mg protein per min, respectively. The enzyme is of the low uptake, noncorrective form with respect to fibroblasts cultured from the skin of patients with Hurler syndrome. It is inhibited by 10-6 M p-chloromercuribenzoate and 10-3 M Cu2+, but is not significantly affected by other divalent cations, EDTA, or sulfhydryl compounds. Antibodies to .alpha.-L-iduronidase were raised in goats.