Purification to homogeneity and properties of plant glucosidase I.
Glucosidase I was purified about 3600-fold to apparent homogeneity from the microsomal fraction of mung bean seedlings. The purified enzyme removed the terminal alpha1,2-linked glucose from Glc3Man9GlcNAc2-peptide or the endoglucosaminidase H (Endo H)-released oligosaccharide. Glucosidase I activity was inhibited by kojibiose [Glc(alpha1-2)Glc], but not by other glucose disaccharides. Removal of up to four mannose residues from the N-linked oligosaccharide had little effect on its utilization as a substrate for glucosidase I. The enzyme had a subunit molecular weight of 97 kDa on SDS gels and this was shifted to 94 kDa after treatment with Endo H or Endo F, suggesting that glucosidase I is an N-glycoprotein having one oligomannose-type oligosaccharide. Amino acid sequences of this enzyme showed considerable identity to the enzyme cloned from a human hippocampus cDNA library. The enzyme was inhibited by castanospermine, deoxynojirimycin, MDL, and trehazolin, but not by australine or kifunensine. On the other hand, the other processing glucosidase, glucosidase II, is sensitive to inhibition by australine, but not by trehazolin. Thus, these two inhibitors are useful to distinguish glucosidase I from glucosidase II. The mung bean glucosidase I is quite sensitive to the histidine modifying reagent diethyl pyrocarbonate, whereas the pig liver glucosidase I is not. On the other hand, pig liver and pig brain glucosidase I preparations are sensitive to the sulfhydryl reagent NEM (N-ethylmaleimide), whereas the plant enzyme is not. These sensitivities to amino acid modifiers suggest significant differences between the plant and animal glucosidase I, in terms of catalytic site or protein conformation.
Zeng
Y C
YC
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72211, USA.
Elbein
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D007887:Fabaceae / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry; D010946:Plants, Medicinal; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; C042059:glucosidase I; D000520:alpha-Glucosidases