Linked Life Data

8031794

Source:http://linkedlifedata.com/resource/pubmed/id/8031794

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
28
pubmed:dateCreated
1994-8-18
pubmed:abstractText
The cytosolic phospholipase A2 (cPLA2) from the human monocytic cell line U937 contains nine cysteine residues and is subject to oxidation. Iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid) were used to explore the susceptibility of cysteine residues to thiol modification agents as outlined in Schemes 2 and 3. In the absence of thiol reducing agents such as DTT, cPLA2 takes up only 2.8 equiv of [1-14C]iodoacetamide at pH 8.03/37 degrees C. With DTT present, cPLA2 is in its fully reduced form, and 4-5 equiv of acetamide are taken up without altering enzyme activity to give IA-cPLA2. A single equivalent of DTNB suffices to inactivate IA-cPLA2, giving a TNB-labeled enzyme, with the loss of activity correlating with release of an equivalent of 5-thio-2-nitrobenzoate. The TNB-labeled enzyme is quite stable up to 33 degrees C; enzyme activity is recoverable with DTT, even after this disulfide-enzyme adduct is incubated with iodoacetamide at pH 9.5, conditions that inactivate the free enzyme. At pH 9.5/37 degrees C, a single equivalent of 14C-labeled iodoacetamide is incorporated by IA-cPLA2 concomitant with complete loss of enzyme activity. Amino acid analysis of the 14C-labeled enzyme indicates that only cysteine residues are labeled. Lys-C digestion of labeled enzyme with 2 M guanidine at pH 8.0 yields a 40-mer peptide. Amino acid sequencing establishes that the label resides primarily in Cys324, although Cys331 is also labeled. These results identify a region of the enzyme that is susceptible to labeling by group modification reagents and may represent a suitable target for small molecule inhibitors.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
19
pubmed:volume
33
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
8594-603
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1994
pubmed:articleTitle
Inactivation of a cytosolic phospholipase A2 by thiol-modifying reagents: cysteine residues as potential targets of phospholipase A2.
pubmed:affiliation
Syntex Inc., Mississauga, Ontario, Canada.
pubmed:publicationType
Journal Article