Linked Life Data

1498334

Source:http://linkedlifedata.com/resource/pubmed/id/1498334

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
1992-9-14
pubmed:abstractText
To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla-containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla-precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
AIM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0006-4971
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
80
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
942-52
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:1498334-1-Carboxyglutamic Acid, pubmed-meshheading:1498334-Amino Acid Sequence, pubmed-meshheading:1498334-Base Sequence, pubmed-meshheading:1498334-Blood Coagulation, pubmed-meshheading:1498334-Cell Line, pubmed-meshheading:1498334-Chromogenic Compounds, pubmed-meshheading:1498334-DNA, pubmed-meshheading:1498334-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:1498334-Humans, pubmed-meshheading:1498334-Kidney, pubmed-meshheading:1498334-Molecular Sequence Data, pubmed-meshheading:1498334-Mutagenesis, Site-Directed, pubmed-meshheading:1498334-Oligopeptides, pubmed-meshheading:1498334-Partial Thromboplastin Time, pubmed-meshheading:1498334-Protein C, pubmed-meshheading:1498334-Pyrrolidonecarboxylic Acid, pubmed-meshheading:1498334-Structure-Activity Relationship, pubmed-meshheading:1498334-Transfection
pubmed:year
1992
pubmed:articleTitle
Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity.
pubmed:affiliation
Department of Chemistry and Biochemistry, University of Notre Dame, IN 46556.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't