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Abstract
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.
Highlights
It is concluded that the plasminogen activator puri- found that this single-chain urokinasepecies obtained either fied from CALU-3-conditioned mediais physically and by recombinant DNA techniques[13]or by purification from kineticallyidenticaltosingle-chainurokinase-type plasminogen activator
First described in 1951 (l), they have been A compelling reason for the continued investigationof the properties of scu-PA is the demonstration bothin vitro
We have devised a method utilizing both readily obtainable materials andmild conditions for the purificationof scu-PA from conditioned medium of a human tumor cell line, CALU-3 (ATCC, HTB-55), which is known to secrete urokinase[20]. We show that this cellular material (CALU-3 UTA) is most likely identical to scu-PA purified from human urine
Summary
Sume that CALU-3 u-PA is identical to scu-PA. The purpose of this study was 2-fold. We sought t o it is concluded that natural scu-PA,purified from identify sufficiently enriched and widely available sources of both human urine and conditionemdedia from CALU-3cells, scu-PA and todevelop a straightforward purification method possesses potent plasminogen-activating properties distinct avoiding extremes of pH or denaturing conditions.To achieve from thoseof its immunological relative,two-chain urokinase. Thisend, we screened several human cell lines for UK-Ag Whether other purified forms of single-chain plasminogen secretion and identified one,CALU-3, a human lung adeno- activators [8,9,10,11,12], some of which are characterized ashaving carcinoma cell line which secretes relatively large amounts of high fibrin affinity [7,11,12], are identical to scu-PA remains UK-Ag into serum-freemedia containing aprotinin. Sephadex C-50 in the step provided quantitative separation of UK-Ag from 95% of the total protein, which after
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