These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Production, purification and characterization of recombinant yeast processing alpha 1,2-mannosidase.
    Author: Lipari F, Herscovics A.
    Journal: Glycobiology; 1994 Oct; 4(5):697-702. PubMed ID: 7881184.
    Abstract:
    The Saccharomyces cerevisiae processing alpha 1,2-mannosidase, which trims Man9GlcNAc to Man8GlcNAc, has a lumenally oriented catalytic domain and an N-terminal transmembrane domain. To obtain sufficient protein to study the structure and mechanism of action of this enzyme, the sequence encoding the catalytic domain was inserted downstream of the alpha-factor promoter and signal peptide in a high-copy vector for expression in S. cerevisiae as a secreted protein. Using oligosaccharide substrate (Glc1Man9GlcNAc or Man9GlcNAc), the medium of cells transformed with this plasmid showed an increase in alpha-mannosidase activity that was directly related to the increase in cell density, whereas no alpha-mannosidase activity was detected in cells transformed with vector alone. SDS-PAGE of the medium showed the presence of a doublet of 63 and 60 kDa that was revealed by Coomassie Blue staining and by Western blotting with antibodies to the endogenous solubilized alpha-mannosidase. The recombinant alpha-mannosidase was present in the medium at a level of approximately 1 mg/l and was purified in a single step by chromatography on S-Sepharose. High-resolution 1H NMR analysis of the Man8GlcNAc formed from Man9GlcNAc in the presence of the recombinant enzyme proved that it retained its specificity and removed only one specific alpha 1,2-mannose residue of the alpha 1,3 branch. Endoglycosidase H treatment decreased the molecular mass of both components of the doublet by approximately 5 kDa, showing that the heterogeneity is not due to differential N-glycosylation. EDTA inhibited the activity of the recombinant enzyme, but the inhibition was reversed by the addition of divalent cations.(ABSTRACT TRUNCATED AT 250 WORDS)
    [Abstract] [Full Text] [Related] [New Search]