Purification of lysosomal membrane-bound glucocerebrosidase from human cultured fibroblasts using high-performance liquid chromatography.
Glucocerebrosidase from human skin fibroblasts was purified more than 2300-fold to apparent homogeneity with an overall yield of 39% using taurocholate extraction, ammonium sulfate fractionation, and high-performance hydrophobic interaction and gel permeation column chromatography. This relatively high yield is attributed to two modifications from previously published procedures: (i) the elimination of a butanol delipidation step that resulted in substantial loss of enzyme activity; and (ii) the use of 2% (w/v) sodium taurocholate instead of 1-2% sodium cholate that resulted in more than 90% solubilization of total membrane-bound enzyme activity. Confluent monolayers of human cultured skin fibroblasts (approximately 3.6 x 10(8) cells) were harvested from 10 roller bottles. Glucocerebrosidase in the cell pellet was solubilized with 2% (w/v) sodium taurocholate, fractionated in 14% ammonium sulfate, and applied to a high-performance hydrophobic interaction phenyl-5PW column. After an ammonium sulfate descending linear gradient step, glucocerebrosidase was eluted from the column at 4% cholate concentration using a 0-5% linear cholate gradient. There was a 36-fold purification and 80% recovery. In the subsequent step, concentrated glucocerebrosidase fractions from the phenyl column were injected into two Bio-Sil TSK-250 gel permeation columns joined in series. Glucocerebrosidase peak activity was eluted at 263 ml corresponding to Mr 76,000. There was an 18-fold purification and 78% recovery. The enzyme preparation was then recycled through the phenyl-5PW column in order to remove a remaining contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pediatrics, McGill University, Montreal, Quebec, Canada.
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D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D005959:Glucosidases / Q000302:isolation & purification*; D005962:Glucosylceramidase / Q000302:isolation & purification*; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet
D005959:Glucosidases; D005962:Glucosylceramidase