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 det ailed analysis of this important enzyme, which functions in the final degradation step of glycoproteins, we developed a novel purification m ethod 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 re sistance of aspartylglucosaminidase as SDS-sensitive proteins aggregat e preferentially at low (NH4)(2)SO4 concentrations in the presence of SDS. This new method should beconcentrations in the presence of SDS. T his new method should be applicable to the isolation of other SDS-resi stant enzymes, e.g., superoxide dismutase. The homogeneous enzyme prep aration exhibited a previously unreported fully denaturated 18-kDa for m of the cr-subunit of aspartylglucosaminidase on SDS-polyacrylamide g el electrophoresis as a consequence of complete coating by SDS. (C) 19 94 Academic Press, Inc.