Document Detail


Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies.
MedLine Citation:
PMID:  3089776     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Two murine monoclonal antibodies (MA-2G6 and MA-1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue-type plasminogen activator (t-PA), inhibited the activity of t-PA on fibrin plates. MA-2G6 inhibited the amidolytic activity of t-PA and did not react with t-PA in which the active-site serine was blocked with diisopropylfluorophosphate nor with t-PA in which the active-site histidine was alkylated by reaction with D-Ile-Pro-Arg-CH2Cl. This indicated that MA-2G6 is directed against an epitope covering the active site of t-PA. MA-1C8 did not inhibit the amidolytic activity of t-PA, but abolished both the binding of t-PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t-PA. Thus MA-1C8 is directed against an epitope which covers the fibrin-binding site of t-PA. The A and B chains of partially reduced two-chain t-PA were separated by immunoadsorption on immobilized MA-1C8 and MA-2G6. The purified B chain reacted with MA-2G6 but not with MA-1C8 and activated plasminogen following Michaelis-Menten kinetics with kinetic constants similar to those of intact t-PA (Km = 100 microM and kcat = 0.02 s-1). However, fibrin or CNBr-digested fibrinogen did not stimulate the activation of plasminogen by the B chain. The purified A chain reacted with MA-1C8 but not with MA-2G6. It bound to fibrin with an affinity similar to that of intact t-PA but did not activate plasminogen. It is concluded that the active center of t-PA is located in the B chain and the fibrin-binding site in the A-chain. Both functional domains are required for the regulation by fibrin of the t-PA-mediated activation of plasminogen.
Authors:
P Holvoet; H R Lijnen; D Collen
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  European journal of biochemistry / FEBS     Volume:  158     ISSN:  0014-2956     ISO Abbreviation:  Eur. J. Biochem.     Publication Date:  1986 Jul 
Date Detail:
Created Date:  1986-09-17     Completed Date:  1986-09-17     Revised Date:  2007-07-23    
Medline Journal Info:
Nlm Unique ID:  0107600     Medline TA:  Eur J Biochem     Country:  GERMANY, WEST    
Other Details:
Languages:  eng     Pagination:  173-7     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Antibodies, Monoclonal / diagnostic use*
Kinetics
Mice
Mice, Inbred BALB C
Plasminogen / metabolism
Tissue Plasminogen Activator / analysis*,  immunology,  physiology
Chemical
Reg. No./Substance:
0/Antibodies, Monoclonal; 9001-91-6/Plasminogen; EC 3.4.21.68/Tissue Plasminogen Activator

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