Linked Life Data

1577013

Source:http://linkedlifedata.com/resource/pubmed/id/1577013

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
3
pubmed:dateCreated
1992-6-9
pubmed:abstractText
We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0014-2956
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
205
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
955-60
pubmed:dateRevised
2007-7-23
pubmed:meshHeading
pubmed:year
1992
pubmed:articleTitle
Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.
pubmed:affiliation
Centre de Recherches Roussel-UCLAF, Romainville, France.
pubmed:publicationType
Journal Article