title: Isolation and characterization of a precursor form of lysosomal alpha-glucosidase from human urine.
abstract: A two-step procedure is described for the isolation of lysosomal alpha-glucosidase from human urine. In the second step, affinity chromatography on Sephadex G-100, two fractions with acid alpha-glucosidase activity were obtained. Fraction I contained alpha-glucosidase of Mr 109000, whereas fraction II contained components of Mr 76000 and 70000. alpha-Glucosidase in fraction I had an Mr similar to that of the precursor of alpha-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 alpha-glucosidase found in placenta or cultured human skin fibroblasts. alpha-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. Fraction I, but not fraction II, was avidly endocytosed by alpha-glucosidase-deficient cultured human skin fibroblasts. Endocytosis of fraction I was inhibited by mannose 6-phosphate. The pH optimum and Km values for p-nitrophenyl alpha-glucoside, maltose and glycogen of fractions I and II alpha-glucosidase were almost identical. However, the activity with glycogen relative to that of either p-nitrophenyl alpha-glucoside or maltose was lower in fraction I than in fraction II. It is concluded that fraction I consists of the precursor form of alpha-glucosidase and fraction II of the mature forms of the enzyme. The importance of urine as a source of precursors of lysosomal enzymes is discussed.
mesh_terms: D000328:Adult; D004792:Enzyme Precursors / Q000652:urine*; D005347:Fibroblasts / Q000201:enzymology; D005959:Glucosidases / Q000652:urine*; D006801:Humans; D007120:Immunochemistry; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism; D012867:Skin / Q000201:enzymology; D000520:alpha-Glucosidases / Q000652:urine*
chemical_list: D004792:Enzyme Precursors; D008360:Mannosephosphates; C027693:mannose-6-phosphate; D005959:Glucosidases; D000520:alpha-Glucosidases