Human α-L-iduronidase. I. Purification and properties of the high uptake (higher molecular weight) and the low uptake (processed) forms

The major forms of human α-L-iduronidase have been individually purified over 175,000-fold to apparent homogeneity by sequential anion exchange, lectin affinity, and gel filtration chromatography. The two forms, initially designated as soluble and membrane-associated, were extracted from human lung in approximately equal amounts. Optimal solubilization of the membrane-associated form was facilitated by use of a non-ionic detergent or mannose-6-phosphate and saponin. Following detergent homogenization, the two forms were separated by anion exchange chromatography and then individually purified. The more electronegative form was membrane-associated, had a pI of approximately 5.9, and was selectively taken up (high uptake) by cultured Hurler syndrome fibroblasts; the more electropositive soluble form had a pI of about 6.6 and was incorporated into Hurler fibroblasts at a markedly lower rate (low uptake). After treatment with alkaline phosphatase, the pI values of both enzymes were about 7.8. Using 4-methylumbelliferyl-α-L-iduronide as substrate, the low and high uptake forms were each purified in milligram quantities to specific activities of 284,000 and 202,000 units/mg, respectively, with a combined yield greater than 35%. Each purified enzyme form migrated as a single protein band which also stained for enzymatic activity when electrophoresed in 7% native polyacrylamide disc gels at pH 4.3. By gel filtration, the high uptake form had an M(r) = 85,000 whereas the M(r) for the low uptake form was 68,000. Molecular weight estimates by analytical polyacrylamide gel electrophoresis were 82,000 and 70,000 for the high and low uptake forms, respectively. Rabbit anti-human low uptake α-L-iduronidase antibodies cross-reacted with the high uptake form as demonstrated by both immunotitration and Ouchterlony double immunodiffusion. Amino acid analysis revealed that the high uptake (higher molecular weight) form contained more arginine, glycine, alanine, glutamate or glutamine, leucine, isoleucine, histidine, and proline residues per molecule than the low uptake (lower molecular weight) form. Automated Edman degradation determined that the NH₂-terminal residues of both forms were blocked. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography demonstrated that each purified form was composed of several components; each post-high performance liquid chromatographic component retained catalytic activity and was immunologically cross-reactive with antibodies against the low uptake form. The availability of these forms should permit further biochemical and immunologic investigation of α-L-iduronidase maturation and receptor-mediated enzyme endocytosis as well as studies of the enzymatic defects in the α-L-iduronidase deficiency diseases.