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 aspartylglucosaminuria (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 five-step 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)2SO4 concentrations 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 denatured 19-kDa form of the alpha-subunit of aspartylglucosaminidase on SDS-polyacrylamide gel electrophoresis as a consequence of complete coating by SDS.
Heiskanen
T
T
Department of Human Molecular Genetics, National Public Health Institute, Helsinki, Finland.
Tollersrud
O K
OK
Zhao
M
M
Peltonen
L
L
D000645:Ammonium Sulfate; D001227:Aspartylglucosylaminase / Q000187:drug effects / Q000302:isolation & purification*; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D011489:Protein Denaturation / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D012967:Sodium Dodecyl Sulfate; D001227:Aspartylglucosylaminase; D000645:Ammonium Sulfate