LARGE-SCALE PURIFICATION OF HUMAN ASPARTYLGLUCOSAMINIDASE - UTILIZATION OF EXCEPTIONAL SODIUM DODECYL-SULFATE RESISTANCE

Deficiency of human aspartylglucosaminidase (AGA, glycosylasparaginase, EC 3.5.1.26), a lysosomal amidase, results in the lysosomal storage disease aspartyl-glucosaminuria (AGU). This disorder is most prevalent in the genetically isolated Finnish population. To facilitate the detailed analysis of this important enzyme, which functions in the final degradation step of glycoproteins, we developed a novel purification method which makes possible a simple fivestep 5000-fold purification to apparent homogeneity of human aspartylglucosaminidase from leukocytes. This purification procedure takes advantage of the remarkable SDS resistance of aspartylglucosaminidase as SDS-sensitive proteins aggregate preferentially at low (NH4)(2)SO4 concentrations in the presence of SDS. This new method should beconcentrations in the presence of SDS. This new method should be applicable to the isolation of other SDS-resistant enzymes, e.g., superoxide dismutase. The homogeneous enzyme preparation exhibited a previously unreported fully denaturated 18-kDa form of the cr-subunit of aspartylglucosaminidase on SDS-polyacrylamide gel electrophoresis as a consequence of complete coating by SDS. (C) 1994 Academic Press, Inc.