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)

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.
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PMID:Vitronectin modulates glycosaminoglycan dependent reactions of protein C inhibitor. 128 93

Two types of plasminogen activator (PAs) are present in human endometrium, and their contents vary with the different phases of menstrual cycle, i.e. high in the proliferative phase and low in the secretory phase. In the present study by immunohistochemical technique, both uPA and tPA antigens were demonstrated in the stromal and glandular cells of the endometrium. In cell culture, tPA was released only from stromal cells and uPA only from glandular cells as determined by SDS-PAGE followed by fibrin overlay technique, but PA inhibitor type-1 (PAI-1) was secreted by both stromal and glandular cells. Furthermore, secretion of PAs from endometrial cells was enhanced by adding estradiol and markedly inhibited by progesterone in a dose dependent manner, while the PAI reacted just in the opposite way. The effect of the peptide hormones, hCG, GnRH, PRL, as well as cAMP in cell culture on the secretion of PAs and PAI was similar to that of estradiol, while forskolin demonstrated definitely more stimulative effect on tPA than uPA. Taking into account of the finding of the present study, it appears that, under hormonal control, a balance between PAs and PAI in the endometrium exists. The physiological roles of the PAs and PAI in the endometrium were discussed.
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PMID:[Plasminogen activators and plasminogen activator inhibitor type-1 in human endometrium]. 129 66

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase-type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single-chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro-uPA. As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage of the Lys158-Ile159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro-uPA of 1:2,000). Nevertheless, even at pH 7.0, pro-uPA was activated by cathepsin L, although a 10-fold higher concentration of cathepsin L was required. As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways of activation of pro-uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro-uPA; (ii) activation by serine proteases (plasmin, kallikrein, Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).
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PMID:Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L. 155 16

When human granulocytes were stimulated with the chemotactic peptide FNLPNTL (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosinyl- lysin; in the presence of cytochalasin B), proteolytic enzymes were released which prevented activation of tumor-cell derived pro-uPA by plasmin. Elastase was identified by use of eglin C (elastase inhibitor) and an inhibitory monoclonal antibody to elastase as the functional proteolytic enzyme in these granulocyte supernatants. Purified human granulocyte elastase cleaves pro-uPA at amino acid position lle159-lle160 thus generating an enzymatically inactive two-chain form of uPA, as judged by N-terminal amino acid sequence analysis. An additional minor elastase-mediated cleavage site was detected at position Thr165-Thr166. This form of uPA was indistinguishable by SDS-PAGE from plasmin-generated enzymatically active HMW-uPA. Action of plasmin on the proenzyme form of uPA (pro-uPA) generates an enzymatically active uPA-molecule (high molecular weight form; HMW-uPA) which is cleaved at amino acid position Lys158-lle159 (Mr = 33,000 (B-chain) and 22,000 (A-chain). Thus elastase cannot substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. Enzymatically active HMW-uPA, however, was not affected by elastase. Elastase-containing granulocytes were identified by immunohistochemical staining of elastase in breast cancer tissue. Granulocytes were located close to the tumor cells and also in the tumor stroma surrounding the tumor nests. These tumor cells contain pro-uPA. Evidently, the conversion of tumor cell pro-uPA into enzymatically active HMW-uPA is controlled by elastase released from granulocytes into the tumor tissue.
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PMID:Human tumor cell urokinase-type plasminogen activator (uPA): degradation of the proenzyme form (pro-uPA) by granulocyte elastase prevents subsequent activation by plasmin. 183 19

Expression of plasminogen activator inhibitor 2 (PAI-2) under the control of the protease B gene promoter in a mutant strain of Saccharomyces cerevisiae, DS569, resulted in its accumulation intracellularly at up to 20% of the soluble cell protein. Provision of an N-terminal signal sequence resulted in the secretion of a hyperglycosylated molecule. The intracellularly produced PAI-2 was purified by copper-chelate and anion-exchange chromatography to greater than 95% pure and was fully active. The recombinant PAI-2 formed SDS-stable complexes with urokinase and tissue-type plasminogen activator and inhibited the proteases with similar reaction kinetics to placental PAI-2 (second-order rate constant for uPA, 2.4 x 10(6) M-1 s-1, and for two-chain tPA, 0.7 x 10(5) M-1 s-1). As is the case for placental PAI-2, the N-terminus of the yeast-derived recombinant PAI-2 was blocked. The high productivity and consequent ease of purification mean that S. cerevisiae provides an excellent source of recombinant PAI-2 for investigation of its therapeutic potential in the treatment of neoplastic and inflammatory diseases.
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PMID:Purification and characterisation of plasminogen activator inhibitor 2 produced in Saccharomyces cerevisiae. 190 Oct 39

Increasing evidence suggests the involvement of leukocytes in the fibrinolytic system. Monocytes secrete pro-urokinase (Grau, Thromb Res 1989; 53: 145) and it has been shown that these cells have specific receptors for urokinase and plasminogen (Miles, Thromb Haemostas 1987; 58: 936). The aim of this study was to analyse the presence of plasminogen activator inhibitor(s) in platelet-free suspensions of human peripheral blood monocytes and polymorphonuclear leukocytes (PMN). SDS-PAGE and reverse fibrin autography showed an inhibitory band of 50 kDa in the monocyte extracts (Triton X-100) but not in the PMN extracts. Urokinase (u-PA) was mixed with increasing amounts of monocyte extract for 10 min and the mixtures were added to 125I-fibrin coated wells containing plasminogen. A dose-dependent decrease in the u-PA fibrinolytic activity was observed. The amount of inhibition increased when the monocyte releasates were preincubated with u-PA (40% inhibition after 5 min preincubation and 80% after 15 min), indicating a direct interaction between this activator and an inhibitor(s). After SDS-PAGE of monocyte extracts, immunoblotting and peroxidase staining identified both PAI1 and PAI2, with an apparent molecular weight of 47-50 kDa. Monocyte-associated PAI1 formed complexes with single chain t-PA with a molecular mass 50 kDa higher than the molecular mass of the free PAI1. However, a significant amount of PAI1 remained unbound to t-PA. This inactive PAI1 could have come from a rapid inactivation of the primary active PAI1. These PAI1 and PAI2 detected in human monocytes may be transcendent in the regulation of the fibrinolytic system.
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PMID:Detection of both type 1 and type 2 plasminogen activator inhibitors in human monocytes. 233 62

Immature mice were injected subcutaneously with 5 IU PMSG for 2 days to stimulate follicle development, which was followed by administration of 5 IU hCG to induce ovulation. The ovaries were removed at various periovulatory stages for preparing ovarian homogenates, granulosa cells and cumulus-oocyte complexes. The activity of plasminogen activator in the samples, separated by SDS-PAGE, were determined by fibrin-overlay technique. The results show that 15% of the gonadotropin-treated animals were ovulated 8h after hCG administration, about 6-8h earlier than that occurred in rat. Moreover, both tPA, and uPA activity were stimulated following PMSG treatment in ovarian homogenates and granulosa cells. Subsequent hCG injection further increased the two types of PA activity in a time-dependent manner, reaching maximum 4-8h after hCG treatment, and declined following ovulation. Greater uPA activity (70%) in the cultured mouse granulosa cells was found. It is, therefore, suggested that both tPA and uPA may be involved in the regulation of ovulation in mouse. The cumulus-oocyte complexes contained mainly tPA, which activity showed a time-dependent increase and reached a maximum between 12-24h after hCG treatment. Since cumulus-oocyte complexes collected from oviducts post ovulation still retain a considerable amount of tPA, the enzyme in the complexes may also play a role in the process of cumulus dispersion, oocyte transportation and implantation.
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PMID:[Plasminogen activator activity in mouse ovaries during periovulatory period]. 250 47

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

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

A single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified approximately 18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 micrograms of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pI 9.05 and pI 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I. U./mg.
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PMID:Purification and characterization of single-chain urokinase-type plasminogen activator (pro-urokinase) from human A431 cells. 310 Dec 22


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