Partial purification and characterization of the glucosidases involved in the processing of asparagine-linked oligosaccharides

J. Mark Michael, Stuart Kornfeld

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Abstract

A glucosidase preparation with activity toward certain glucose-containing oligosaccharides was partially purified from calf liver membranes by Triton X-100 solubilization and DEAE-cellulose and hydroxylapatite chromatography. The enzyme preparation hydrolyzed the glucose residues from (glucose)1,(mannose)9(N-acetylglucosamine)1, and (glucose)2(mannose) 9(N-acetylglucosamine)1 but was totally inactive toward (glucose)3(mannose)9(N-acetylglucosamine) 1. In contrast, crude membrane preparations of the calf liver were active toward all three substrates. The partially purified enzyme had a pH optimum of 6.7 and was very unstable in the absence of added 20% glycerol. The rate of glucose release from the one-and two-glucose-containing oligosaccharides was significantly decreased when four or five of the mannose residues were first removed from the substrate. The release of glucose from (glucose)1(mannose)9(N-acetylglucosamine)1 was inhibited by p-nitrophenyl-α-d-glucoside much more effectively than by p-nitrophenyl-β-d-glucoside, suggesting that this glucose residue may be linked α to the mannose residue. We conclude that during oligosaccharide processing at least two different glucosidases are involved in glucose removal.

Original languageEnglish
Pages (from-to)249-258
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume199
Issue number1
DOIs
StatePublished - Jan 1980

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