Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tissue-type plasminogen activator (t-PA), purified from the culture fluid of a stable human melanoma cell line, is a serine protease, different from urokinase, with a molecular weight of about 70,000. It is composed of one polypeptide chain, which is converted to a two-chain molecule by limited plasmic action. Activation of plasminogen to plasmin occurs by cleavage of the Arg 560-Val 561 peptide bond. Kinetic analysis has shown that the activation obeys Michaelis-Menten kinetics and that the presence of fibrin strikingly enhances the activation rate by increasing the affinity of plasminogen for fibrin-bound t-PA. The directed action of plasmin toward fibrin in vivo, might be explained by the low Michaelis constant in the presence of fibrin (0.16 microM), which allows efficient plasminogen activation on a fibrin clot, while its high value in the absence of fibrin (65 microM) prevents efficient activation in plasma. Plasmin formed on the fibrin surface would then be protected from rapid inactivation by alpha 2-antiplasmin. An important consequence of this molecular model for physiological fibrinolysis is that specific thrombolysis is only expected with the use of a specific plasminogen activator, which confines activation to the fibrin surface. Studies on the thrombolytic properties of purified t-PA in various animal species and in humans have revealed a higher specific thrombolytic activity than urokinase. Thrombolysis could be achieved without causing significant plasminogen activation, alpha 2-antiplasmin consumption, or fibrinogen breakdown. Alternatively, pro-urokinase, the zymogen precursor of urokinase, also displays a certain degree of fibrin specificity. Its mechanism of action and potential therapeutic value remain to be established.
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PMID:New approaches to thrombolytic therapy. 643 77

Preparations of outer membrane of two strains of Escherichia coli contain a protease that can activate the serum zymogen plasminogen to the active protease plasmin. The amount of plasmin formed is proportional to the membrane concentration. The kinetics of plasminogen activation are linear and obey the Michaelis--Menten rate equation. The Km(app) for the activation of dog plasminogen by E. coli outer membrane preparations is similar to the Km(app) for the activation of dog plasminogen by human urokinase. The E. coli enzyme is active in a membrane-associated form, as opposed to a secreted or soluble form, and is most likely a serine protease because it is inhibited by diisopropyl fluorophosphate. It is also inhibited from activating plasminogen by p-nitrophenyl-p-guanidinobenzoate and aprotinin. Analysis of the activation of plasminogen by the E. coli enzyme by NaDodSO4/polyacrylamide gel electrophoresis showed that the cleavage of plasminogen to plasmin was as specific as that exhibited in the activation of plasminogen to plasmin by urokinase. Possible in vivo roles for this plasminogen activator in E. coli outer membranes are discussed.
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PMID:Activation of plasminogen to plasmin by a protease associated with the outer membrane of Escherichia coli. 645 46

Four criteria were used to examine serum-free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS-stable approximately 77 K Da complexes between a medium component and [125I]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 micrograms/10(6) cells), rat embryo heart muscle cells (0.13 micrograms/10(6) cells), mouse myotubes (0.1 micrograms/10(6) cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV-40)-transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus-transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells. Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin-binding factors. Lysates from all of these cell types contained a factor that formed approximately 60-65 K Da sodium dodecyl sulfate (SDS)-stable complexes with [125I] thrombin. This MW is significantly lower than that of [125I] thrombin-PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI-26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease-binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.
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PMID:Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor. 657 53

Conditioned medium from cultures of human umbilical vein endothelial cells was analyzed for the presence of tissue plasminogen activator (tPA) and urokinase. Immunoprecipitation studies using metabolically labeled conditioned medium and anti-tPA IgG revealed a single band on autoradiographs corresponding to a Mr of 100,000. No bands were observed after immunoprecipitation with anti-urokinase IgG. The Mr 100,000 tPA was found to be inactive and did not bind to fibrin clots. However, exposure of this tPA to 1% NaDodSO4 resulted in the appearance of plasminogen activator activity with no apparent change in its Mr. Treatment with 10 mM diisopropylfluorophosphate prior to NaDodSO4 activation did not inhibit the NaDodSO4-induced appearance of plasminogen activator activity, indicating that the active site was not available for diisopropylfluorophosphate binding. The possibility that the properties of this Mr 100,000 tPA reflected a tPA-inhibitor complex was examined. Attempts to dissociate such a complex by denaturation, reduction, or extremes of temperature were not successful. However, after treatment of conditioned medium with 1 M hydroxylamine in the presence of 0.1% NaDodSO4, the Mr of the anti-tPA immunoprecipitable material declined by 40,000 to Mr 60,000, a Mr consistent with that of other human tPAs. Hydroxylamine has been shown previously to dissociate covalently coupled serine protease-inhibitor complexes. Furthermore, incubation of purified human melanoma cell tPA with conditioned medium resulted in an increase in its Mr by 40,000 with a concomitant decline in tPA activity. The data suggest that the latent tPA present in the conditioned medium of endothelial cells is composed of a Mr 60,000 tPA associated with an inhibitor.
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PMID:Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex. 658 Jun 16

The activity of the serine protease plasminogen activator (PA) was measured in cell lysates from primary mouse keratinocyte cultures as well as from a number of established mouse keratinocyte lines. Enzyme activity was generally higher in the transformed lines than in the primary cultures; however, among the lines tested, those that expressed the highest degree of morphologic differentiation had the highest levels of cell-associated PA. In both the normal (primary) and transformed (established) keratinocyte cultures, PA activity increased when cultures reached confluence and morphologic evidence of differentiation was noted. The highest specific activity of the enzyme was found in cells shed from differentiating cultures, which consisted predominantly of detergent-resistant cornified envelopes. As the cultures differentiated and these cells were shed from the culture surface, the total cell-associated PA activity of the culture decreased accordingly. In both the normal and transformed keratinocyte cultures, peak PA activity occurred at a time when DNA synthesis was declining. These findings indicate that as keratinocytes differentiate, their intracellular levels of PA increase. The modulation of this endogenous keratinocyte enzyme may play an important, although as yet undefined, role in the normal maturation and terminal differentiation of these cells.
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PMID:Plasminogen activator in differentiating mouse keratinocytes. 668 29

By incubation of human citrated plasma with acetone 25% v/v kallikrein inhibitors were destroyed and prekallikrein activated to kallikrein. When the incubation was carried out in the presence of benzamidine 7 mM, the cofactor capacity of high molecular weight kininogen (HMrK) was protected against destruction by a serine protease which was not plasma kallikrein. By analogy with studies in rat plasma this protease might be a plasminogen activator (Berstad & Briseid 1982; Johansen & Briseid 1983). Factor XII in the plasma preparation was activated to unfragmented factor XIIa by adsorption to kaolin, and assayed as prekallikrein activator (PKA). The extent of activation of factor XII was only insignificantly influenced by the 1 + 1 (v/v) dilution of the plasma preparation with a suspension of kaolin. When, however, the preparation was diluted greater than 1 + 5 (v/v) before incubation with the suspension, a stoichiometric HMrK concentration-effect curve could be established, allowing the assay of cofactor-active HMrK. Assays of HMrK in plasma preparations from healthy men and women demonstrated an average lower level of cofactor-active HMrK in the preparations from women. It is suggested that benzamidine is not capable of providing a complete protection of HMrK during the procedure in all plasma samples.
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PMID:Activation of factor XII in human plasma: protection by benzamidine of the cofactor function of high molecular weight kininogen. 668 67

The intracellular localization of the serine protease plasminogen activator was analyzed in homogenates of bovine brain cortex using differential fractionation procedures. The distribution of the enzyme was clearly different from that of cytosol and mitochondrial markers, and was similar to that of plasma membrane proteins and of the muscarinic acetylcholine receptor, which is a specific marker for the synaptic membrane. The specific activity of plasminogen activator was increased in fractions enriched in intact synaptosomes. Most of the enzyme in intact synaptosomes was found to be firmly associated with the synaptosomal membrane, and could be solubilized by high concentrations of salt or by non-ionic detergent. Purified synaptic vesicles, however, did not contain large amounts of plasminogen activator. Bovine brain synaptosomes were shown to contain two species of the enzyme, having apparent molecular weights of 80,000 and 55,000. The presence of plasminogen activator in the synaptosomal membrane may indicate its possible involvement in the functioning of nerve terminals.
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PMID:Plasminogen activator is enriched in the synaptosomal plasma membranes. 675 70

Plasma membranes isolated from normal thymocytes of hamster and rats were found to exhibit neutral protease activity toward 125I-labeled casein. The plasma membrane-associated proteases were completely inhibited by the serine protease inhibitors, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride and p-nitrophenyl-p-guanidinobenzoate, partially inhibited by soybean trypsin inhibitor and antipain, but were only weakly inhibited by L-1-tosylamino-2-phenylethyl chloromethyl ketone. The plasma membrane-associated proteases were also completely inhibited by ZnCl2 (75--100 mu M), but they were not affected by several other divalent cations. The plasma membrane fraction contained a plasminogen activator activity which was specifically localized in this fraction. The plasma membrane-associated plasminogen activator activity was inhibited by all of the inhibitors which inhibited plasma membrane-associated proteases except L-1-tosylamido-2-phenylethyl chloromethyl ketone. Labeling of plasma membrane-associated serine esterases with [3H] diisopropyl fluorophosphate followed by separation of the proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that this fraction contained a single major 3H-labeled protein of Mr 105 000. Both the plasminogen activator and the Mr 105 000 esterase were shown to be glycoproteins by affinity chromatography on lentil lectin-Sepharose. These results indicate that the plasminogen activator of thymocytes is a glycosylated serine protease with an active site-containing subunit of Mr 105 000 which is specifically localized in the plasma membrane.
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PMID:Characterization of a plasma membrane-associated plasminogen activator on thymocytes. 679 69

Incorporation of the serine protease active site reagent diisopropyl fluorophosphate (DFP) into a plasminogen activator with an Mr of approximately 52000 released from cultured human glioblastoma cells was strongly enhanced by incubation with plasmin. This observation led to the isolation of an inactive form of the enzyme from serum-free conditioned culture fluid by affinity chromatography on a column of a Sepharose-bound monoclonal antibody raised against urokinase. An 831-fold purification was obtained with a yield of 41%. The purified molecule was homogeneous as evaluated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (NaDodSO4), having one stainable band under nonreducing as well as reducing conditions with an Mr of approximately 52000. It was unable to activate plasminogen, but catalytic amounts of plasmin converted it into active enzyme. After NaDodSO4-polyacrylamide gel electrophoresis, the active enzyme showed one band under nonreducing conditions, but after reduction, two bands with Mr values of approximately 20000 and 32000 were observed. The active enzyme incorporated [3H]DFP into the approximately Mr 32000 band, while no incorporation was observed into the inactive form. These findings show that the Mr 52000 human plasminogen activator exists in a proenzyme form consisting of a single polypeptide chain that by proteolysis between half-cystine residues is converted into the active enzyme consisting of two chains with molecular weights of approximately 20000 and 32000, the active site being on the latter chain. The results are consistent with the active form of the enzyme being identical with the higher molecular weight form of urokinase, and together with recent observations that a murine plasminogen activator is released from sarcoma virus transformed cells as an inactive proenzyme, they suggest that zymogens to plasminogen activators are of more general occurrence.
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PMID:Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody. 689 Dec 64

The tissue activator was extracted with 2 M ammonium thiocyanate and purified by L-arginine methyl ester, concanavalin A and ion-exchange chromatographies, and Sephacryl S-200 gel filtration in buffers containing Triton X-100 and/or ammonium thiocyanate. The final preparations had specific activities of 25 000-40 000 IU/mg protein and were shown to be a single band with an apparent molecular weight of 54 00 by SDS-polyacrylamide gel electrophoresis with or without reducing agent. When subjected to isoelectric focusing, its major component had an isoelectric point of approx. 8.2 with minor components. (7.8-8.6). The purified tissue activator was a serine protease, dissimilar to urokinase in some respects including antigenicity, strong affinity to insoluble fibrin monomer and hydrolytic activities for synthetic substrates. The crude extract contained another plasminogen activator with antigen identity to urokinase, which constituted approx. 15% of the total activity in crude extract. These findings indicated that human kidney would produce at least two plasminogen activators, namely, the tissue activator as a major plasminogen activator and urokinase.
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PMID:Purification and characterization of human kidney plasminogen activator dissimilar to urokinase. 705 41


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