Isolation and characterization of a precursor form of lysosomal α‐glucosidase from human urine

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Abstract
1. A two-step procedure is described for the isolation of lysosomal α-glucosidase from human urine. In the second step, affinity chromatography on Sephadex G-100, two fractions with acid α-glucosidase activity were obtained. 2. Fraction I contained α-glucosidase of Mr 109000, whereas fraction II contained components of Mr 76000 and 70000. 3. α-Glucosidase in fraction I had an Mr similar to that of the precursor of α-glucosidase detected in the medium of fibroblasts after labelling with [14C]leucine. The components in fraction II had Mr identical to those of the mature forms of α-glucosidase found in placenta or cultured human skin fibroblasts. 4. α-Glucosidase in fraction I contained mannose 6-phosphate (3.5 mol/mol polypeptide). No mannose 6-phosphate was present in the components in fraction II. 5. Fraction I, but not fraction II, was avidly endocytosed by α-glucosidase-deficient cultured human skin fibroblasts. Endocytosis of fraction I was inhibited by mannose 6-phosphate. 6. The pH optimum and Km values for p-nitrophenyl α-glucoside, maltose and glycogen of fractions I and II α-glucosidase were almost identical. However, the activity with glycogen relative to that of either p-nitrophenyl α-glucoside or maltose was lower in fraction I than in fraction II. 7. It is concluded that fraction I consists of the precursor form of α-glucosidase and fraction II of the mature forms of the enzyme. 8. The importance of urine as a source of precursors of lysosomal enzymes is discussed.