Abstract
Aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which hydrolyzes the N-acetylglucosamine-asparagine linkages in glycoproteins, was purified from human liver to homogeneity. The purification procedure included chromatography on DEAE-cellulose and concanavalin A-Sepharose, gel filtration on Sephadex G-200, and high performance liquid chromatography. The purified enzyme had a final specific activity of 1,200,000 units/mg of protein, a pH optimum of 6.1, a pI of 5.7, a Vmax of 1,240,000 units/mg, and a Km of 1.25 mM toward a natural substrate, aspartylglucosamine. The purified enzyme was remarkably thermostable, retaining 90% of initial activity after 1 h at 60 degrees C. The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and 84,000 by analytical polyacrylamide gel electrophoresis. Under denaturing conditions, the molecular weight was 76,000, indicating that the native enzyme was a monomer. Amino acid composition revealed only 2 methionine residues/enzyme molecule.
Highlights
Aspartylglucosaminidase (EC 3.5.1.26), the lysoso- Finland where itisthe mostcommon lysosomal storage mal enzymewhichhydrolyzes the N-acetylglucosa- disorder [13,14,15]
The enzyme was purified to a specific activity of 1,200,000 units/mg and the finaylield was
Aspartylglucosaminidase (1-aspartamido-P-N-acetylglucos-over the only previous communication (111, which reported amine amidohydrolase, EC 3.5.1.26) is alysosomalenzyme purification of the human hepatiecnzyme to a specific activity that hydrolyzes N-acetylglucosamine-asparagine linkages of 43,000 units/mg of protein with a yield of less than 3%
Summary
From the Divisionof Medical Genetics, Mount SinaiSchool of Medicine, New York, New York 10029 and the Laboratory of Prenatal Genetics, Departmentof Obstetrics and Gynecology, Uniuersity Central Hospital, Helsinki 29, Finland. Aspartylglucosaminidase (EC 3.5.1.26), the lysoso- Finland where itisthe mostcommon lysosomal storage mal enzymewhichhydrolyzes the N-acetylglucosa- disorder [13,14,15]. Patients from other ethnic backmine-asparagine linkages in glycoproteins, was puri- grounds havebeen reported [15,16,17]. Inordertofacilitate fied from human liver to homogeneity. The purified enzyme had a final specific activity of 1,200,000 units/mg of protein, a pH optimum of 6.1, a PI of 5.7, a V,,, of 1,240,000 units/mg, zyme in these patients, we have undertaken the purification and characterization of the normal human enzyme. We describe a method for the purification of human hepatic aspartylglucosaminidase and report the physical and kinetic propertiesof the homogeneous enzyme.
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