Linked Life Data

12384508

Source:http://linkedlifedata.com/resource/pubmed/id/12384508

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
51
pubmed:dateCreated
2002-12-16
pubmed:abstractText
Activated Factor V (FVa) functions as a membrane-bound cofactor to the enzyme Factor Xa (FXa) in the conversion of prothrombin to thrombin, increasing the catalytic efficiency of FXa by several orders of magnitude. To map regions on FVa that are important for binding of FXa, site-directed mutagenesis resulting in novel potential glycosylation sites on FV was used as strategy. The consensus sequence for N-linked glycosylation was introduced at sites, which according to a computer model of the A domains of FVa, were located at the surface of FV. In total, thirteen different regions on the FVa surface were probed, including sites that are homologous to FIXa-binding sites on FVIIIa. The interaction between the FVa variants and FXa and prothrombin were studied in a functional prothrombin activation assay, as well as in a direct binding assay between FVa and FXa. In both assays, the four mutants carrying a carbohydrate side chain at positions 467, 511, 652, or 1683 displayed attenuated FXa binding, whereas the prothrombin affinity was unaffected. The affinity toward FXa could be restored when the mutants were expressed in the presence of tunicamycin to inhibit glycosylation, indicating the lost FXa affinity to be caused by the added carbohydrates. The results suggested regions surrounding residues 467, 511, 652, and 1683 in FVa to be important for FXa binding. This indicates that the enzyme:cofactor assembly of the prothrombinase and the tenase complexes are homologous and provide a useful platform for further investigation of specific structural elements involved in the FVa.FXa complex assembly.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
20
pubmed:volume
277
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
50022-9
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:12384508-Animals, pubmed-meshheading:12384508-Anti-Bacterial Agents, pubmed-meshheading:12384508-Binding Sites, pubmed-meshheading:12384508-COS Cells, pubmed-meshheading:12384508-DNA, Complementary, pubmed-meshheading:12384508-Dose-Response Relationship, Drug, pubmed-meshheading:12384508-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:12384508-Factor Va, pubmed-meshheading:12384508-Factor Xa, pubmed-meshheading:12384508-Glycosylation, pubmed-meshheading:12384508-Humans, pubmed-meshheading:12384508-Kinetics, pubmed-meshheading:12384508-Models, Molecular, pubmed-meshheading:12384508-Mutagenesis, Site-Directed, pubmed-meshheading:12384508-Phospholipids, pubmed-meshheading:12384508-Protein Binding, pubmed-meshheading:12384508-Protein Conformation, pubmed-meshheading:12384508-Protein Structure, Tertiary, pubmed-meshheading:12384508-Recombinant Proteins, pubmed-meshheading:12384508-Thromboplastin, pubmed-meshheading:12384508-Time Factors, pubmed-meshheading:12384508-Tunicamycin
pubmed:year
2002
pubmed:articleTitle
Defining the factor Xa-binding site on factor Va by site-directed glycosylation.
pubmed:affiliation
Department of Clinical Chemistry, Division of Laboratory Medicine, Lund University, The Wallenberg Laboratory, Malmö General Hospital, Malmö SE-205 02, Sweden.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't