Purification and the effect of peptide N-glycosidase F on lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts.
Glucocerebrosidase was purified from human cultured dermal fibroblasts more than 2200-fold to apparent homogeneity using high performance Alkyl-Superose HR 5/5 hydrophobic interaction and Bio-Sil TSK-250 gel permeation column chromatography. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis and protein staining of the catalytically active and concentrated enzyme fractions from the gel permeation columns revealed the presence of one band of Mr 64,000. The glucocerebrosidase preparation purified to homogeneity was digested with peptide N-glycosidase F that cleaves N-linked oligosaccharide structures from glycoproteins. The molecular weight of glucocerebrosidase after digestion with peptide N-glycosidase F was reduced to Mr 57,000, suggesting that the mature enzyme is a glycoprotein and that N-linked oligosaccharide constitutes a minimum of about 10% of the total molecular weight of the polypeptide. These findings are compatible with the hypothesis that glucocerebrosidase was initially synthesized as a precursor polypeptide which was subsequently glycosylated to become the mature enzyme.
Choy
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Centre for Human Genetics, McGill University-Montreal Children's Hospital Research Institute, Que., Canada.
Woo
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D000581:Amidohydrolases / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000737:chemistry; D005962:Glucosylceramidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
D005962:Glucosylceramidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase