Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.7 (plasmin)
 9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A urokinase-type plasminogen activator was purified from conditioned media of several human cell cultures, but preferably from the human lung adenocarcinoma line CALU-3 (ATCC, HTB-55), using a combination of chromatography on zinc chelate-Sepharose, SP-Sephadex C-50, and Sephadex G-100. Final yields of 65-100 micrograms/liter of starting material were obtained with a 290-fold purification factor and a recovery of 30%. The purified plasminogen activator consists of a single polypeptide chain with Mr 54,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is very similar or identical to single-chain urokinase-type plasminogen activator on the basis of immunodiffusion, amino acid composition, and the lack of specific binding to fibrin. It has very low amidolytic activity on Pyroglu-Gly-Arg-rho-nitroanilide and is converted to two-chain urokinase by limited exposure to plasmin. It has a specific activity of 60,000 IU/mg on fibrin plates and directly activates plasminogen following Michaelis-Menten kinetics with Km = 1.1 microM and kappa cat = 0.0026 S-1. It is concluded that the plasminogen activator purified from CALU-3-conditioned media is physically and kinetically identical to single-chain urokinase-type plasminogen activator. With the present straightforward purification method and a readily available source, sufficient amounts of single-chain urokinase-type plasminogen activator can be obtained for more detailed investigations of its biochemical, biological, and thrombolytic properties.
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PMID:Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures. 308 Apr 23

A low Mr form (Mr 32,000) of single-chain urokinase-type plasminogen activator (scu-PA) was isolated from conditioned culture medium of a human lung adenocarcinoma cell line, CALU-3 (ATCC, HTB-55). The purified material (scu-PA-32k) consists of a single polypeptide chain and is immunologically similar to Mr 33,000 urokinase. Its NH2-terminal sequence is identical to that beginning at Leu-144 of Mr 54,000 urokinase. Whereas low Mr urokinase is derived from mature Mr 54,000 scu-PA by limited hydrolysis by plasmin first of the Lys-158-Ile-159 peptide bond and then of the Lys-136-Lys-137, scu-PA-32k is generated by specific hydrolysis of the Glu-143-Leu-144 peptide bond by an unidentified protease. scu-PA-32k resembles its Mr 54,000 scu-PA counterpart by its very low activity on chromogenic substrates for urokinase, by plasminogen-dependent fibrinolytic activity on fibrin plates, and by the lack of specific binding to fibrin. It activates plasminogen directly with high affinity, Km = 0.9 microM, but low turnover number, kcat = 0.0028 s-1. It is converted to fully active two-chain urokinase by plasmin with Km = 12 microM and kcat = 0.3 s-1. Like Mr 54,000 scu-PA, it causes significant lysis of a 125I-labeled fibrin clot in human plasma with relatively less fibrinogen breakdown as compared to urokinase. scu-PA-32k, which also has conserved fibrin specificity, represents a molecular variant which may be more suitable for large scale production as a fibrin-specific thrombolytic agent by recombinant DNA technology.
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PMID:Purification and characterization of a novel low molecular weight form of single-chain urokinase-type plasminogen activator. 309 21