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Query: EC:3.4.21.68 (tissue plasminogen activator)
 11,311 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have expressed human tissue plasminogen activator (t-PA) gene at high levels in a mouse cell line. The t-PA cDNA with deletion of the long 3' untranslated region was inserted into a bovine papilloma virus (BPV) derived vector under the control of a mouse metallothionein promoter. The mouse metallothionein (mMT) gene also provided signals for splicing and polyadenylation. Mouse C127 cells transfected with this construct secreted t-PA at high levels into the cell culture medium. When an SV40 polyadenylation signal was inserted between the t-PA cDNA and the mMT splicing signals, the expression level increased by several fold. The expression levels did not increase further upon either introduction of Rous sarcoma virus LTR into the plasmid or mutation of the translation initiation context sequence to conform with the consensus one. Most of the plasmid appears to be integrated into the host chromosome. Cells producing high levels of t-PA tend to detach from the dish in a few days after passage. When grown on porous microcarriers, however, such cells can be maintained in culture for months and t-PA can be harvested continuously.
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PMID:High level expression of human tissue-type plasminogen activator gene in mouse C127 cell. 136 38

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.
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PMID:Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli. 138 96

The role of W74 in stabilization of the binding of omega-amino acids to the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) has been assessed by examination of the binding (dissociation) constants (Kd) of epsilon-aminocaproic acid (EACA) and one of its structural analogues, 7-aminoheptanoic acid (7-AHpA), to variants of r-[K2tPA] generated by site-directed mutagenesis of the wild-type kringle domain. Two nonconservative mutations at W74 of r-[K2tPA] have been constructed, expressed, and purified, resulting in one variant molecule containing a W74L mutation (r-[K2tPA/W74L]) and another containing a W74S mutation (r-[K2tPA/W74S]). In both cases, binding of EACA and 7-AHpA was virtually eliminated in the mutated kringles. Two additional conservative mutations at W74 of r-[K2tPA] have been similarly generated, resulting in r-[K2tPA/W74F] and r-[K2tPA/W74Y]. For these mutants, binding of the same ligands to the variant recombinant kringle domain is retained, although it is significantly weaker in nature. The 1H-NMR spectra of each of the variant kringles demonstrates that all retain the general gross conformations of their wild-type counterpart but that some environmental changes of proton resonances occur at particular aromatic amino acid residues that may be involved in omega-amino acid binding. Differential scanning calorimetric analyses of each of the variant kringles suggest that none of the mutations led to substantial destabilization of their structures, again suggestive of gross conformational similarities in all r-[K2tPA] molecules constructed. We conclude that the aromatic character present at position 74 of wild-type r-[K2tPA] is of great importance to its ability to interact with omega-amino acid ligands, with tryptophan being the most effective amino acid at that position.
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PMID:Role of tryptophan-74 of the recombinant kringle 2 domain of tissue-type plasminogen activator in its omega-amino acid binding properties. 155 17

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.
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PMID:Localization in the fibrinogen gamma-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen. 156 67

Seven healthy male volunteers were subjected to exercise of short (STR; 1.7 km), middle (MTR; 4.8 km) and long (LTR; 10.5 km) term runs at a speed close to maximal capacity. Blood samples were drawn before, immediately after exercise and at intervals over the next 10 h. FVIIIR:Ag (von Willebrand factor) rose 2.2-3.2 fold and persisted at higher levels than baseline during the observation time. A spontaneous drop in FVII (p less than 0.03) was found immediately after STR (13.5 +/- 2.5%) and LTR (18.3 +/- 2.4%), whereas only a minor decrease (7.5 +/- 6.5%) occurred in MTR. The procoagulant activity of monocytes isolated from whole blood exposed to LPS showed a striking enhancement in STR and MTR. An immediate enhancement in fibrinolytic activity was found in all groups (p less than 0.03) assessed by increased plasma levels of t-PA and shortened whole blood clot lysis time (WBCLT). The transient shortening of WBCLT was succeeded by a tendency to prolongation of the lysis time. A 45-year old male differed markedly from the others by demonstrating an extreme and consistent prolongation of WBCLT. Thus, it has been speculated that strenuous exercise possibly makes a subject more susceptible to a thrombotic event.
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PMID:Formation and persistence of procoagulant and fibrinolytic activities in circulation after strenuous physical exercise. 209 90

In our very recent ESR study we reported that upon rt-PA binding to platelets the H+1/h0 ratios of 16-doxylstearate and 5-doxylstearate spin labels incorporated into the lipid bilayer of platelet membranes were significantly decreased. It corresponded to the increased rigidity of platelet lipid bilayer. In order to further explore this phenomenon we employed a fluorescence-quenching technique which enabled us to estimate the energy transfer efficiency and the apparent interchromophore distance between membrane protein tryptophan and 1-anilino-8-naphthalenesulphonate (ANS) molecules embedded in the membrane lipid bilayer. As t-PA interacts with the platelet membrane this distance decreases, resulting in the relevant increase of energy transfer efficiency. Thus, the data indicate that upon t-PA binding the membrane tryptophan residues are more exposed to the external environment and the quenchable fraction of membrane tryptophan becomes greater. Furthermore, the spectrum of ANS is slightly shifted towards longer wavelengths, which can be accounted for by an increase in the polarity of the environment. It suggests a diminished contact of membrane tryptophan with phospholipid fatty acids. Based on these observations we concluded that the interaction of rt-PA with platelet membranes might induce conformational changes in the membrane proteins, and consequently result in rearrangements of lipid matrix and the alterations in lipid-protein interactions in platelet membranes.
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PMID:Tissue-type plasminogen activator induces alterations in structure and conformation of membrane proteins upon its interaction with human platelets. 826 41

The involvement of the strictly conserved tryptophan-25 (W25) residue in the structural stability and omega-amino acid ligand binding properties of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been investigated. Two conservative mutants were constructed and expressed that contained W25-->F and W25-->Y substitutions. The binding (dissociation) constants (Kd) for three ligands, viz., 6-aminohexanoic acid (EACA), 7-aminoheptanoic acid (7-AHpA), and L-lysine (Lys), to these polypeptides were determined by intrinsic fluorescence titrations. In the case of r-[K2tPA/W25F], the Kd values for these ligands were found to be 37, 16, and 89 microM for EACA, 7-AHpA, and Lys, respectively. For r-[K2tPA/W25Y], the Kd values for these same ligands were 64, 9, and 115 microM, respectively. The wild-type (wt) kringle domain possessed Kd values of 43, 6, and 85 microM for EACA, 7-AHpA, and Lys, respectively. The effect of these mutations on the stability of the r-[K2tPA] domain has been examined by differential scanning colorimetry. The temperature of maximum heat capacity (Tm) of wt-r-[K2tPA] (75.6 degrees C) was dramatically reduced to 50.8 and 58.0 degrees C for r-[K2tPA/W25F] and r-[K2tPA/W25Y], respectively. In the presence of EACA, the Tm values were increased to 86.1, 61.7, and 68.7 degrees C, respectively, indicating that EACA does interact with the r-[K2tPA] mutants and stabilizes their native conformations, similar to the case with wt-r-[K2tPA].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Role of the strictly conserved tryptophan-25 residue in the stabilization of the structure and in the ligand binding properties of the kringle 2 domain of tissue-type plasminogen activator. 831 51

Murine embryonal carcinoma cells do not express detectable cell-surface epidermal growth factor receptors (EGF-R) but after 2 days of differentiation induced by retinoic acid (RA) increasingly express mRNA and protein encoded by the EGF receptor gene (Joh et al., Cell Growth and Differentiation 3, 315, 1992). The effect on morphology, growth, and differentiation of the introduction of expression vectors that produce either a truncated, kinase-negative mouse EGF receptor or an antisense mRNA was studied in P19 embryonal carcinoma (EC) cells before and after differentiation. The presence of either construct should lead to the reduction of EGF-R expression by either the dominant negative effects of a truncated protein or the inhibition of endogenous EGF-R mRNA production/translation by complementary RNA, respectively. Cells were cotransfected with the bacterial neomycin resistance gene and constitutively expressing clones were selected with G418. The cytomegalovirus LTR promoter/enhancer was found to be very inefficiently activated in P19 EC cells. After RA addition, changes in gene expression included induction of both the exogenous truncated constructs and endogenous EGF-R. Differentiation was gauged by the expression of tissue-type plasminogen activator and intermediate filament protein markers of neural tissues, as well as EGF-R. The expression of 120-kDa truncated EGF-Rs was high in four clones, but all 10 clones examined had diminished abilities to differentiate after RA induction compared to four control cell lines. Similarly, the majority of antisense transfected clones was unable to differentiate normally. The results indicate that the reduced expression of EGF-Rs in differentiating EC cells inhibits the rate, frequency, and extent of differentiation after RA induction. We conclude that the expression of EGF-Rs plays a role in the stimulation of differentiation and we speculate that the mechanism involves the tyrosine kinase activity of the receptor.
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PMID:Inhibition of differentiation in P19 embryonal carcinoma cells by the expression of vectors encoding truncated or antisense EGF receptor. 836 61

Maspin is a tumor suppressor protein expressed by normal human mammary epithelium but not by many breast tumor cell lines. Recombinant human maspin (rMaspin) inhibits tumor cell motility, invasion, and metastasis and thus has potential value as an anti-cancer therapeutic. Maspin is a member of the serpin family and, although the molecular mechanism by which maspin acts is unknown, recent work suggests that tissue plasminogen activator is a potential target. A puzzling observation in previous cell culture studies was loss of rMaspin activity at higher protein concentrations. One hypothesis to explain these results is self-association of rMaspin at the higher concentrations, which would be consistent with the tendency of serpins to form noncovalent polymers. This hypothesis is addressed by examining the relationship between rMaspin stability and self-association. Urea denaturation of rMaspin at pH 7 and 25 degrees C and at protein concentrations ranging from 0.01 to 0.2 mg/ml has been monitored by circular dichroism and intrinsic tryptophan fluorescence. Denaturation profiles show a protein concentration dependence and indicate the presence of at least one unfolding intermediate. The results suggest that destabilization of native monomeric rMaspin leads to partial unfolding and formation of an intermediate which can self-associate.
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PMID:Three-state unfolding and self-association of maspin, a tumor-suppressing serpin. 1051 31

The effect of methylglyoxal on the plasminogen-plasmin system is studied. Treatment of plasminogen with methylglyoxal at a 20-fold molar excess results in covalent modification of the molecule as evidenced by the decreased number of NH(2) side chains, arginine side chain residues and the new band in the non-tryptophan dependent fluorescent spectrum. This structural modification is associated with profound functional alterations: the rate of activation by streptokinase, tissue-type plasminogen activator, urokinase-type plasminogen activator and trypsin decreases and the amidolytic activity of the generated plasmin is impaired. Plasmin treatment with methylglyoxal on the other hand does not alter its steady-state kinetic parameters on a peptidyl-anilide synthetic substrate, indicating that modification susceptible side chains are sensitive to methylglyoxal only in the zymogen. Our data suggest that in vivo fibrinolysis could be impaired under pathological conditions, e.g. increased methylglyoxal formation in diabetes mellitus.
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PMID:Modulation of plasminogen activation and plasmin activity by methylglyoxal modification of the zymogen. 1089 32


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