Linked Life Data

1456445

Source:http://linkedlifedata.com/resource/pubmed/id/1456445

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1993-1-7
pubmed:abstractText
Tyrosylprolylarginyl chloromethyl ketone (YPRck) is a radioiodinatable inhibitor that irreversibly binds the active site of tissue plasminogen activator (tPA). A two-step reaction is employed where (1) the YPRck reagent is iodinated and (2) the 125I-YPRck is reacted with the tPA sample; therefore the oxidative effects of conventional protein iodination are avoided. Using fibrin binding as a probe of native tPA binding function, YPRck labeling was shown to be superior to other types of surface iodination. 125I-YPRck was prepared at a high specific radioactivity; i.e., one 125I per 3.5 molecules of peptidyl chloromethyl ketone. Labeled YPRck formed a one to one covalent, sodium dodecyl sulfate stable, complex with tPA resulting in a preparation of 10 mCi per milligram protein, which corresponded to an incorporation ratio of 1:3.5 (125I-YPRck:tPA). Both one-chain and two-chain forms of tPA reacted with YPRck. Radiolabeling tPA with 125I-YPRck occurred in a time-dependent manner with half-maximal incorporation at approximately 30 min under the conditions employed in these studies. The pH optimum for the reaction of tPA with 125I-YPRck was 7.4. Solutions of tPA at less than 1 microgram/ml were efficiently labeled with 125I-YPRck, thus allowing the quantitation of functional protease by incorporation of radiolabel. Significantly, 125I-YPRck specifically labeled tPA in cell culture supernatants after transient transfection of cells with plasmid DNA containing the gene for tPA. Other serine proteases were tested for their relative reactivity with 125I-YPRck. Thrombin and Factor Xa incorporated 125I-YPRck to higher levels than two-chain tPA; whereas plasmin, urokinase, and other plasma proteases were not as efficiently radiolabeled. The use of 125I-YPRck allows rapid and specific radiolabeling of a large number of tPA samples in a nondenaturing environment with a known localization of the radiolabeling reagent.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:volume
206
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
73-83
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1992
pubmed:articleTitle
Radioiodination of the active site of tissue plasminogen activator: a method for radiolabeling serine proteases with tyrosylprolylarginyl chloromethyl ketone.
pubmed:affiliation
Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.
pubmed:publicationType
Journal Article