Purification and properties of human hepatic aspartylglucosaminidase.

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.