Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteolytic enzymes released from granulocytes upon stimulation with the chemotactic N-formyl peptide FNLPNTL (in the presence of cytochalasin B) prevented activation of tumor cell single-chain urokinase-type plasminogen activator (pro-uPA) by plasmin. Elastase was identified by the use of eglin C (elastase inhibitor) and a monoclonal antibody to elastase as the functional proteolytic enzyme in granulocyte supernatants. Action of purified granulocyte elastase on pro-uPA generated enzymatically inactive two-chain uPA linked by disulfide bridges which was indistinguishable by SDS-PAGE from plasmin-generated HMW-uPA. The major elastase cleavage site in pro-uPA was located between Ile159 and Ile160. a minor one between Thr165 and Thr166. Elastase cannot substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. However, when pro-uPA was first activated by plasmin to form enzymatically active HMW-uPA, this enzymatic activity was not impaired by subsequent elastase treatment.
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PMID:Elastase released from human granulocytes stimulated with N-formyl-chemotactic peptide prevents activation of tumor cell prourokinase (pro-uPA). 252 37

Equimolar mixtures of recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody (MA-15C5) directed against fragment-D dimer of human cross-linked fibrin were conjugated, using the cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (PySSProSu). The conjugate (rscu-PA/MA-15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment-D dimer with a yield of 42 +/- 15% (mean +/- SD, n = 3), contained an average of 1.2 +/- 0.3 IgG molecules/rscu-PA molecule. On non-reduced SDS/PAGE it migrated as a main band with apparent Mr of 200,000. Specific amidolytic activities expressed/mass of u-PA were less than 250 IU/mg for rscu-PA/MA-15C5 and rscu-PA, 140,000 +/- 13,000 IU/mg and 100,000 +/- 17,000 IU/mg for their plasmin-generated two chain derivatives rtcu-PA/MA-15C5 and rtcu-PA respectively. Specific activities on fibrin plates were 100,000 +/- 24,000 IU/mg and 130,000 +/- 49,000 IU/mg for rscu-PA/MA-15C5 and rtcu-PA/MA-15C5 respectively, as compared to 180,000 +/- 15,000 IU/mg for both rscu-PA and rtcu-PA. Activation of plasminogen with rscu-PA/MA-15C5 (Km = 0.37 +/- 0.16 microM, k2 = 0.0063 +/- 0.0030 s-1 or rtcu-PA/MA-15C5 (Km = 19 +/- 3.0 microM, k2 = 2.0 +/- 0.10 s-1) in purified systems followed Michaelis-Menten kinetics with Km and k2 values comparable to those of rscu-PA and rtcu-PA. In an in vitro system composed of a 125I-fibrin-labeled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained; 50% lysis in 2 h required 1.4 microgram/ml of rscu-PA or 0.33 microgram/ml of rtcu-PA, but only 0.22 microgram u-PA/ml of rscu-PA/MA-15C5 or 0.15 microgram u-PA/ml of rtcu-PA/MA-15C5. Addition of purified fragment-D dimer reversed the increased fibrinolytic potency of rscu-PA/MA-15C5 in a concentration-dependent way (50% inhibition at 7.2 micrograms fragment-D dimer/ml). Thus, conjugation of u-PA moieties with the fibrin-specific antibody MA-15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4-fold for rscu-PA and 2.2-fold for rtcu-PA.
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PMID:Biochemical properties of conjugates of urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin. 253 85

Metastatic spread of malignant tumor appears to correlate with activation of the fibrolytic system. The role of fibrinolysis in growth and metastasis was examined in Lewis lung carcinoma of mice. The inhibition of fibrinolysis or proteases decreased the primary tumor growth and pulmonary metastasis, whereas the activation of fibrinolysis or proteases increased the number of metastatic foci in the lung. Electronmicroscopically, thrombus formation in the primary site prevented tumor invasion and metastasis formation. Plasminogen activator (PA) content of excised tumors was determined by SDS-PAGE, and major PA was found to be urokinase (UK) type. Immunohistochemical study with specific antisera was done. When tumor cells possessed a high level of UK, laminin and type IV collagen, components of the basement membrane, disappeared from tumor tissues. These findings suggest that PA through protease cascade plays a role in tumor invasion and metastasis. Clinically, patients with advanced cancer are usually in a hypercoagulable state with elevated fibrinogen, and fibrin deposition around tumor mass is a serious problem in cancer chemotherapy. UK infusion prior to 5-fluorouracil increased tissue concentration of antitumor agent. However, development of consumption coagulopathy characterized by progression from hypercoagulable state to disseminated intravascular coagulation has also been found in several cases.
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PMID:[Tumor metastasis and the fibrinolytic system]. 273 23

Ascitic fluid from tumour patients (hepatoma, gastric cancer, gallbladder cancer, colorectal cancer, ovarian cancer) and from non-malignant diseases (liver cirrhosis, congestive heart failure) were compared with respect to their content of determinants of the fibrinolytic system, tissue-type plasminogen activator antigen (t-PAag) and activity (t-PAact), urokinase-type plasminogen activator antigen (u-PA) and plasminogen activator inhibitor activity (PAI). Furthermore, SDS-polyacrylamide slab-gel electrophoresis (SDS-PAGE) was performed to evaluate molecular weight distribution of the detectable fibrinolytic parameters. In malignant ascites, PAI activity was three to four times higher, and increased complex formation of PAI with t-PA could be demonstrated, compared with non-malignant ascitic fluid. Tissue-type plasminogen activator antigen and activity showed a similar concentration in ascites of both study groups. Urokinase-type plasminogen activator antigen was detectable neither in ascites of malignant nor in ascites of non-malignant origin. It is concluded that t-PA is the physiological plasminogen activator in ascites and that increased PAI levels followed by increased complex formation between t-PA and PAI might reflect a reaction of the peritoneum.
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PMID:Plasminogen activators and plasminogen activator inhibitor in malignant and non-malignant ascitic fluid. 285 12

Neutrophils (PMN) are important in the cellular phase of blood fibrino lytic activity (FA). The contribution of monocytes (MC), which have FA, is unclear. To determine the relative roles of these cells to activity in normal blood, we examined, by solid phase radiofibrin assay, FA of normal blood and plasma, and of purified PMN and MC, with and without plasminogen (PLG), mini-plasminogen (mPLG), the other major elastase fragment of PLG, or autologous plasma. PMN alone (0.5 x 10(6)/ml) had striking activity (292 +/- 25 SEM ng fibrin lysed/h; n = 10 normal subjects) while MC alone (0.5 x 10(6)/ml) had mean FA of 32 +/- 4 ng/h, which could be accounted for by contaminating PMN (36 +/- 8 ng/h). Thus, in a 1 h assay (when cellular FA accounts for 70-80% of FA in whole blood), normal numbers of MC (0.5 x 10(6)/ml) had no detectable FA when assayed with PLG or normal plasma. With longer assay times (2-6 h), PLG-dependent (plasminogen activator, PA) activity was demonstrated with mixtures of MC and PLG or plasma. This PA activity was released into the medium and required prior contact of MC and an intact, soluble PLG molecule for PA activity to be detected in medium (suggesting a PLG-MC triggering mechanism), since activity was reduced or absent when MC were exposed to mPLG, the other major elastase fragment of PLG, or solid phase PLG. Exposure of MC to solid phase fibrin did not result in PA release. MC PA activity was little affected by cycloheximide pretreatment, indicating preformed rather than newly synthesized PA. By SDS-PAGE and fibrin zymography, MC extracts revealed a single PA band with features of pro-urokinase (single chain urinary-type PA): Mr 55,000, inhibition by antiurokinase antibody (but not by anti-tPA), and resistance to inhibition by DFP. By ELISA assay, approximate normal monocyte content of this PA (as Mr 55,000 urokinase) was 0.03 fg (3.3 x 10(8) molecules) per cell.
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PMID:Fibrinolytic activity of normal human blood monocytes. 292 5

Expression of plasminogen activator (PA) activity may be an important factor in the ability of tumour cells to metastasize; however, not all metastatic cells produce detectable PA activity. Conditioned culture media from revertant metastatic clones of cells derived by fusion of metastatic and non-metastatic rat mammary adenocarcinoma cells were found to contain a potent inhibitor of PA. This inhibited thrombin, human urokinase (UK) and tumour-derived PA, but not plasmin or trypsin. Inhibition was still obtained after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) of mixtures of PA and inhibitor, followed by development of PA activity on fibrin overlays. The PA inhibitor eluted from Sephadex G-200 over a broad M.wt. range (35,000-80,000) and was inactivated by heating to 70 degrees for 30 min. The appearance of inhibitory activity in the culture media was time-dependent and could be reduced by incubation of cells with cycloheximide. Because of these findings, the possible presence of inhibitors should be considered in investigations into the role of PA in the metastatic process.
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PMID:An inhibitor of plasminogen activator produced by tumour cell fusion hybrids. 293 23

A proteinase inhibitor has been isolated from human colorectal adenocarcinomas by extraction with a low-ionic-strength buffer and a combination of Con A-Sepharose, Sephadex G-200, DEAE-cellulose and chromatofocusing steps. The preparation appeared to be homogeneous upon gel exclusion chromatography and SDS-polyacrylamide gel electrophoresis and had an estimated molecular weight of 66,000. The inhibitor was able to bind and inhibit urokinase, plasmin, trypsin, tissue plasminogen activator and thrombin. The binding appeared to be stoichiometric and relatively fast. The isoelectric point of the protein was 4.6-4.7. The inhibitor did not crossreact with antisera elicited against alpha 2-macroglobulin, alpha 2-antiplasmin, antithrombin III or C1-inhibitor, but it did crossreact with an antiserum against alpha 1-antitrypsin in double immunodiffusion. The antiserum only partially attenuated the activity of the inhibitor. Whereas alpha 1-antitrypsin completely inhibited the amidolytic activity of elastase, the tumor inhibitor had no effect on elastase under the same conditions.
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PMID:Isolation and partial characterization of a proteinase inhibitor from human colorectal adenocarcinoma. 293 82

A hybrid human cDNA was constructed by splicing of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5'-untranslated, the pre-pro region and amino acids Ser1-Thr263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu144-Leu411. The cDNA fragments were obtained from full length t-PA cDNA, cloned from Bowes melanoma poly(A)+ mRNA, and from full length u-PA cDNA, cloned from CALU-3 lung adenocarcinoma poly(A)+ mRNA. The hybrid (t-PA/u-PA) cDNA was expressed in Chinese hamster ovary cells and the translation product purified from the conditioned cell culture media. On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000. On immunoblotting, it reacted both with rabbit antisera raised against human t-PA and against human u-PA. The urokinase-like amidolytic activity of the protein was only 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single-chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA). The specific activity of the protein on fibrin plates was 57,000 IU/mg by comparison with the International Reference Preparation for Urokinase. Both the single-chain hybrid (t-PA/scu-PA) and the two-chain plasmin derivative (t-PA/tcu-PA) bound specifically to fibrin, albeit more weakly than t-PA. The t-PA/tcu-PA hybrid had a higher selectivity for fibrin than tcu-PA, measured in a system composed of a whole human 125I-fibrin-labeled plasma clot immersed in human plasma. Both hybrid proteins activated plasminogen directly with Km = 1.5 microM and k2 = 0.0058 s-1 for t-PA/scu-PA and with Km = 80 microM and k2 = 5.6 s-1 for t-PA/tcu-PA. CNBr-digested fibrinogen stimulated the activation of plasminogen with t-PA/tcu-PA (Km = 0.20 microM and k2 = 1.2 s-1). It is concluded that these t-PA/u-PA hybrid proteins combine, at least to some extent, the fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), which in some assays result in improved fibrin-mediated plasminogen activation.
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PMID:Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator. 295 60

The human hepatoma line Hep G2 produces an acid- and SDS-sensitive plasminogen activator inhibitor (PAI). This protein has been previously purified and used to raise polyclonal antibodies. This antiserum has been used to isolate cDNA clones from a human placental lambda gt11 cDNA library. The immunologically positive clones were screened for expression of recombinant proteins which inhibit urokinase activity and form an inhibitor-enzyme complex with 125I-urokinase. Two positives (lambda PAI 11.1 and lambda PAI 14.1) have been obtained. The cDNA insert of the longer isolate (lambda PAI 14.1) consists of 1962 base pairs encoding the entire mature Hep G2 PAI and a 3'-noncoding region of 801 base pairs. The clone apparently lacks portions of 5'- and 3'-untranslated sequences. The translated amino acid sequence matches the sequence obtained for the mature Hep G2 PAI and consists of 379 amino acids with a molecular mass of 42 770 Da. Interestingly, this PAI clone is quite different from the placental-type PAI-2 sequence as expected, but matches the sequence of the endothelial-type PAI (PAI-1) reported to be acid-insensitive and SDS-enhancible.
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PMID:cDNA cloning and expression in E. coli of a plasminogen activator inhibitor (PAI) related to a PAI produced by Hep G2 hepatoma cell. 302 37

In the present studies we have compared the structural and biochemical properties of human protease nexin-I (PN-I) and a protease inhibitor present in the serum-free culture fluid of normal rat brain astrocytes. The inhibitor binds to and forms covalent complexes with human urokinase and thrombin. The inhibitor has an approximate Mr = 43,000 based on the size of the complexes (deduced from SDS-PAGE) and mediates the cellular binding and uptake of the proteases to which it links. Binding is heparin sensitive and occurs on a cell surface receptor that also binds complexes formed between proteases and a well-characterized cell-secreted protease inhibitor, human PN-I. In addition, the inhibitor co-migrates with PN-I on SDS-PAGE and cross-reacts with anti-PN-I antibody on immunoblots. A similar molecule, designated NPF, is produced by C6 glioma cells in culture and has neurite promoting activity on a neuroblastoma cell line.
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PMID:Identification of a protease inhibitor produced by astrocytes that is structurally and functionally homologous to human protease nexin-I. 304 Jan 75


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