Linked Life Data

10929008

Source:http://linkedlifedata.com/resource/pubmed/id/10929008

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
8
pubmed:dateCreated
2000-9-7
pubmed:abstractText
Glucosidase II is an ER heterodimeric enzyme that cleaves sequentially the two innermost alpha-1,3-linked glucose residues from N-linked oligosaccharides on nascent glycoproteins. This processing allows the binding and release of monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins with calnexin and calreticulin, the lectin-like chaperones of the endoplasmic reticulum. We have isolated two cDNA isoforms of the human alpha subunit (alpha1 and alpha2) differing by a 66 bp stretch, and a cDNA for the corresponding beta subunit. The alpha1 and alpha2 forms have distinct mobilities on SDS-PAGE and are expressed in most of the cell lines we have tested, but were absent from the glucosidase II-deficient cell line PHA(R) 2.7. Using COS7 cells, the coexpression of the beta subunit with the catalytic alpha subunit was found to be essential for enzymatic activity, solubilization, and/or stability, and ER retention of the alpha/beta complex. Transfected cell extracts expressing either alpha1 or alpha2 forms with the beta subunit showed similar activities, while mutating( )the nucleophile (D542N) predicted from the glycoside hydrolase Family 31 active site consensus sequence abolished enzymatic activity. In order to compare the kinetic parameters of both alpha1/beta and alpha2/beta forms of human glucosidase II the protein was expressed with the baculovirus expression system. Expression of the human alpha or beta subunit alone led to the formation of active human/insect heteroenzymes, demonstrating functional complementation by the endogenous insect glucosidase II subunits. The activity of both forms of recombinant human glucosidase II was examined with a p-nitrophenyl alpha-D-glucopyranoside substrate, and a two binding site kinetic model for this substrate was shown. The K(M1-2) values and apparent K(i1-2 )for deoxynojirimycin and castanospermine were determined and found to be identical for both isoforms suggesting they have similar catalysis and inhibition characteristics. The substrate specificities of both isoforms using the physiological oligosaccharides were assessed and found to be similar.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0959-6658
pubmed:author
pubmed:issnType
Print
pubmed:volume
10
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
815-27
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2000
pubmed:articleTitle
The heterodimeric structure of glucosidase II is required for its activity, solubility, and localization in vivo.
pubmed:affiliation
Genetics Group and Enzymology Group, Biotechnology Research Institute, National Research Council of Canada, Montreal H4P 2R2, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't