Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Chick embryo fibroblast cultures develop fibrinolytic activity after transformation by Rous sarcoma virus (RSV). This fibrinolytic activity is not present in normal cultures, and it does not appear after infection with either nontransforming strains of avian leukosis viruses or cytocidal RNA and DNA viruses. In cultures infected with a temperature sensitive mutant of RSV the onset of fibrinolysis appears after exposure to permissive temperatures and precedes by a short interval the appearance of morphological evidence of transformation. See PDF for Structure The rate of fibrinolysis in transformed cultures depends on the nature of the serum that is present in the growth medium: some sera (e.g., monkey or chicken serum) promote high enzymatic activity, while others (calf, fetal bovine) do not. Some sera contain inhibitors of the fibrinolysin. Based on the effect of a small number of known inhibitors, at least one step of the fibrinolytic process shows specificity resembling that of trypsin. The sera of sarcoma-bearing chickens contain an inhibitor of the fibrinolysin, whereas normal chicken sera do not. For general discussion, conclusions, and summary see the accompanying paper, part II, (J. Exp. Med. 137:112).
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PMID:An enzymatic function associated with transformation of fibroblasts by oncogenic viruses. I. Chick embryo fibroblast cultures transformed by avian RNA tumor viruses. 434 90

Many tumors contain elevated levels of plasminogen activator and thus produce elevated levels of the protease plasmin in the milieu of the tumor. We have hypothesized, therefore, that it should be possible to prepare peptidyl prodrug derivatives of anticancer drugs that would be locally activated by tumor-associated plasmin. As an initial test of this hypothesis, we synthesized the peptidyl prodrugs of the anticancer drugs (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin, AT-125) and N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard) by mixed anhydride coupling of the parent drug with the protected tripeptide, Boc-D-Val-Leu-Lys(Boc)-OH, followed by deprotection with trifluoroacetic acid. The prodrugs showed an increased selective in vitro cytotoxicity for Rous sarcoma virus transformed chicken embryo fibroblasts (which produce elevated levels of plasminogen activator) compared to nontransformed fibroblasts (which produce low levels of plasminogen activator). In the presence of the plasmin inhibitor, p-nitrophenyl p'-guanidinobenzoate at 2 micrograms/mL, the selectivity of the phenylenediamine mustard prodrug was reduced, but there was no effect on the cytotoxicity of the free drug. Furthermore, the prodrug analogue D-valylleucyl-D-lysylphenylenediamine mustard (in which L-Lys has been replaced by D-Lys) was inactive. Finally, the prodrug derivative of acivicin did not display selective toxicity for transformed cells when the cells were cultured in plasminogen-free medium. These results suggest that plasmin hydrolysis is necessary for the activation of the prodrugs. The prodrugs were tested in vivo for antitumor activity. The prodrug of acivicin, like acivicin itself, was inactive against the B16 melanoma, a murine tumor that produces high levels of plasminogen activator. This prodrug was active against the M5076 carcinoma, a tumor that displays only moderate levels of plasminogen activator; however, despite the fact that the prodrug was 2- to 3-fold less toxic on a molar basis than acivicin, there was no evidence of an increased therapeutic index. The prodrug of phenylenediamine mustard was also slightly less toxic than the parent drug, but again there was no evidence for an improved therapeutic index against the B16 tumor.
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PMID:Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard. 622 Oct 99

We have synthesized peptidyl prodrugs of doxorubicin (Dox) designed to be selective substrates of plasmin. Such prodrugs might be locally activated by the elevated levels of plasmin produced near many solid tumors under the action of tumor-associated plasminogen activators. One such prodrug, 3'-(D-Val-Leu-Lys)-Dox, was obtained via a mixed-anhydride coupling with isobutyl chloroformate between the protected peptide Fmoc-D-Val-Leu-N epsilon-Fmoc-Lys-OH and doxorubicin, followed by removal of the Fmoc groups with anhydrous ammonia. Compared to doxorubicin, the prodrug showed about a 7-fold improved selective cytotoxicity against chicken embryo fibroblasts transformed with the Rous sarcoma virus (which produce high levels of plasminogen activator) compared to normal cells (which produce low levels of plasminogen activator). However, the prodrug was a very poor plasmin substrate, and although in vivo tests against the murine B16 melanoma showed that the prodrug was active, the maximum T/C obtained was less than that achieved by doxorubicin even at 25 times the molar concentration of prodrug. Qualitatively similar results were obtained for a far more hydrophobic prodrug, 3'-(Boc-Val-Leu-Lys)-Dox. These results demonstrate that peptidyl prodrugs of doxorubicin designed as plasmin substrates are more selective anticancer agents in vitro than doxorubicin itself but that the bulky anthracycline moiety probably prevents efficient plasmin-catalyzed conversion to the active parent drug, so that, in their present form, these drugs are not potent enough to allow a determination as to whether or not they are more selective in vivo.
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PMID:Plasmin-activated prodrugs for cancer chemotherapy. 2. Synthesis and biological activity of peptidyl derivatives of doxorubicin. 630 53

Studies have established that cryoprecipitates of the plasma of tumor patients contain a biological activity enhancing morphological cell transformation (transformation-enhancing factor; TEF) in cultures of chicken embryo fibroblasts infected with temperature-sensitive mutants of Rous sarcoma virus. We report here that similar TEF activity is effected by defined fragments of human plasma fibronectin obtained by limited digestion with major humoral or tissue proteinases. TEF activity was obtained from plasminolytic fragments of fibronectin and from cathepsin G-treated fibronectin. No activity was recorded from intact dimeric fibronectin or its reduced and alkylated subunits, from fibrinogen or its plasminolytic fragments, or from plasmin (EC 3.4.21.7) or cathepsin G (EC 3.4.21.20) treated or untreated with proteinase inhibitors. All of the TEF activity of the proteolytic fragments of fibronectin was located on the gelatin-binding peptides. The minimum effective doses in the TEF assay were 750 ng/ml of plasmin-treated fibronectin, 100 ng/ml of gelatin-binding plasminolytic fibronectin (enriched in Mr 180,000--190,000 polypeptides), and 100 ng/ml of gelatin-binding fragments of cathepsin G-treated fibronectin (enriched in a Mr 30,000 fragment). TEF activity of proteinase-treated fibronectin was inhibited by gelatin and by intact dimeric fibronectin. The potent TEF activity of proteolytic fragments of fibronectin raises the possibility that they may have a role in malignant transformation.
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PMID:Transformation-enhancing activity of gelatin-binding fragments of fibronectin. 645 38

We report the isolation of a specific protease zymogen from chicken plasma. The purification procedure involves barium citrate precipitation, ammonium sulfate fractionation, removal of plasminogen and plasmin on lysine-Sepharose, followed by anion and cation exchange, and gel permeation chromatography. Based on quantitative radioimmunoassay the zymogen is present in plasma at a concentration of 160 mg/liter, and it is obtained by our procedure in highly purified form with a yield of 1.4%. The single polypeptide chain contains an NH2-terminal alanine residue. The native molecule migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 84,000 under reducing conditions. It can be identified as an inactive proenzyme because it has very low amidolytic activity, does not react with the fluorescent active site titrant 4-methyl-lumbelliferyl p-guanidinobenzoate, and does not incorporate radioactive [3H]diisopropylfluorophosphate. It is very susceptible to limited proteolysis which converts it to an active enzyme with trypsin-like specificity. The active enzyme, likewise a single polypeptide chain, migrates as a doublet with apparent molecular weights of 39,000 and 40,000. Its amidolytic activity with synthetic peptide substrates is at least 40-fold higher than that of the proenzyme, it reacts efficiently with 4-methylumbelliferyl p-guanidinobenzoate, and incorporates [3H]diisopropylfluorophosphate while undergoing irreversible inactivation. The enzyme appears to be a reasonably efficient plasminogen activator in zymographic gels, but not in solution. With human high molecular weight kininogen as substrate the enzyme was about 25% as efficient as human plasma kallikrein. It lacks any plasminogen-independent proteolytic activity with other protein substrates, and it hydrolyzes small peptide substrates designed for both human kallikrein and urinary urokinase, respectively. Inhibition studies with peptide chloromethyl ketones indicate enzymatic properties closer to human plasma kallikrein than to the human plasminogen activator urokinase (EC 3.4.21.31). The chicken plasma enzyme and the plasminogen activator from the conditioned media of Rous sarcoma virus-transformed chick embryo fibroblasts treated with tumor promoter are different by criteria of tryptic peptide maps, and amino acid composition and enzymatic specificity. The designations chicken plasma prekallikrein plasminogen proactivator and chicken plasma kallikrein plasminogen activator are proposed for the zymogen and enzyme forms, respectively. Using rabbit antibodies against the proenzyme we developed a solid phase immunoadsorption procedure that allowed us to isolate the protein with an overall yield of 11.4%.
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PMID:A proenzyme from chicken plasma similar to human plasma prekallikrein. 655 13

The antifibrinolytic activity of cytosols obtained from cultured rabbit endothelial cells was studied to determine whether it resulted from the presence of an antiplasmin or an antiactivator. These cytosol preparations inhibited the fibrinolytic activity initiated by some plasminogen activators (e.g. urokinase, rabbit endothelial cell activator), but not others (e.g. activators associated with bovine endothelial cells and Rous sarcoma virus-infected chick embryo fibroblasts), suggesting that inhibition occurred at the level of plasmin formation, not plasmin activity. The fibrinolytic activity of plasmin itself was unaffected by concentrations of cytosol that completely blocked urokinase-mediated fibrinolysis consistent with this conclusion. In addition, the ability of urokinase to cleave 125I-plasminogen into its characteristic activation fragments was inhibited by cytosol in a dose-dependent manner. When urokinase was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two peaks of activity were detected, corresponding to Mr = 55,000 and 32,000. Urokinase preincubated with cytosol and analyzed in a similar manner demonstrated no activity in any portion of the gel, suggesting that its ability to function as a plasminogen activator was irreversibly lost following its interaction with cytosol. These results indicate that the antifibrinolytic activity of rabbit endothelial cells results from the presence of a molecule or molecules with antiactivator activity. The cellular location and unusual degree of specificity distinguish the endothelial cell inhibitor(s) from antifibrinolytic agents observed in other cells and in plasma and platelets.
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PMID:An inhibitor of plasminogen activator in rabbit endothelial cells. 678 54

The chicken gene, 9E3/CEF4, is a small inducible cytokine highly homologous to human IL-8 and gro alpha. It is overexpressed during wound healing and in the tissues around tumours induced by Rous sarcoma virus. More is known about the expression of 9E3 in vivo than any other of the small cytokines, yet little is known about its biochemical characteristics and functions. Here we report on some of the biochemical properties of the 9E3 gene product, the kinetics of protein secretion, the post-secretory processing of the protein, and on its association with ECM molecules. The protein: (1) is synthesized and secreted in < 10 min; (2) is not glycosylated and does not bind heparin with high affinity; (3) is secreted as a 9 kDa form and is processed to a 6-7 kDa form by plasmin, an enzyme released at wound sites and produced in association with tumours; (4) the small form binds to interstitial collagen, laminin and to a lesser extent to proteoglycan, and does not bind to collagen IV or fibronectin. This is the most rapidly secreted protein yet described in eukaryotic cells and is the first of the small inducible cytokines to be found to associate with ECM molecules other than glycosaminoglycans. Our results suggest that, given the appropriate stimulus, the level of the 9E3 cytokine could be elevated very rapidly, resulting in similarly rapid biological responses. The different modes of availability of the two forms of the molecule suggest that the two isoforms may play different roles in vivo.
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PMID:The 9E3/CEF4 cytokine: kinetics of secretion, processing by plasmin, and interaction with extracellular matrix. 881 41

Plasminogen activator (PLAU) is a serine protease that converts plasminogen to plasmin, a general protease, which promotes fibrinolysis and degradation of extracellular matrix. PLAU was reported in 1970s as one of the robustly induced enzymatic activities in Rous sarcoma virus (RSV)-transformed chicken cells. More than three decades later, with the completion of the sequencing of the chicken genome and the subsequent availability of Affymetrix GeneChip genome arrays, several laboratories have surveyed the transcriptional program affected by the RSV transformation. Interestingly, the PLAU gene was shown to be the most highly upregulated transcript. The induction of PLAU was a transformation-dependent process because viruses with deleted Src gene did not induce the transcription of the PLAU gene. Both Src and PLAU genes are associated with and contribute to the complex phenotype of human cancer. Although the activity and structures of these two enzymes are well characterized, the precise molecular function of these gene products in signaling networks is still not fully understood. Yet, the knowledge of their association with cancer is already translated into the clinical setting. Src kinase inhibitors are being tested in clinical trials of cancer therapy, and PLAU gene and its inhibitor have been included as biomarkers with strong prognostic and therapeutic predictive values. This vignette reviews the history of PLAU and Src discovery, and illuminates the complexity of their relationship, but also points to their emerging impact on public health.
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PMID:From Rous sarcoma virus to plasminogen activator, src oncogene and cancer management. 2138 93


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