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

The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.
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PMID:Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody. 653 82

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 plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. The cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three-step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine-Sepharose and Sephadex G-150. All solvents contained Tween-80 (0.01%) and, except for the last step, aprotinin. The final product had a specific activity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single-chain molecules. Evidence was obtained that four different types of activator variants occur. First and known previously, the one-chain form can be proteolytically cleaved into a two-chain form. Secondly, both the one-chain and two-chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one-chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N-terminal amino acids. Finally, a possible positional microheterogeneity was detected. Digestion with plasmin yields the two-chain form with disulfide-bonded polypeptide chains, 'A' and 'B' (from the N-terminal and C-terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains the active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one-chain form. A speculative explanation for this activity may possibly be the presence of an epsilon-amino group of a lysine residue at a position close to the bond cleaved in the two-chain form.
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PMID:Purification and characterization of a melanoma cell plasminogen activator. 668 60

A culture of the human epidermoid carcinoma HEp 3 produces a plasminogen activator of Mr = 53,000 which we have purified to apparent homogeneity from serum-free conditioned medium by the combination of immunoaffinity chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The highly purified protein has the following properties: 1) It is indistinguishable from urinary urokinase in electrophoretic mobility, in immunodiffusion, and in autoradiographically visualized tryptic peptide maps obtained from the 125I-labeled proteins. 2) The HEp 3 protein differs from urinary urokinase in the following respects: (a) although the apparent molecular weights of the two are identical (Mr = 53,000), the urinary enzyme consists of two polypeptide chains, whereas the HEp 3 protein is a single chain form. (b) Urinary urokinase can be labeled easily by incubation with radioactive diisopropylfluorophosphate but the HEp 3 protein cannot. (c) When assayed by the hydrolysis of a synthetic chromogenic peptide substrate, the HEp 3 enzyme has less than 1% of the catalytic activity of urinary urokinase. 3) On controlled exposure to plasmin, the HEp 3 protein is converted to an active enzyme that is identical with urinary urokinase in molecular weight, polypeptide chain composition, diisopropylfluorophosphate labeling, and specific catalytic activity. We conclude that the HEp 3 protein is a proenzyme that can be converted to active two-chain urokinase by plasmin, probably by a single proteolytic nick in the polypeptide chain.
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PMID:A proenzyme form of human urokinase. 680 70

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

Two spontaneously arising variant clones were selected from the N18 neuroblastoma cell line solely on the basis of their flattened morphology and tight adherence to the culture flask. Two other clones having the round loosely adherent morphology typical of the parent line were also selected, and flat variants were shown to arise in them upon prolonged cultivation. The flat variant clones have slower growth rates in culture, lower cloning efficiencies in suspension, and reduced acetylcholinesterase inducibility when compared with either the parent N18 line or the round cell clones. Cells of both morphologic types have high levels of plasminogen activator and are tumorigenic, although the variants have a slower growth rate in vivo, consistent with their slower growth rate in culture. SDS-polyacrylamide gel electrophoresis of total protein from the two cell types shows that the flat variants have increased amounts of a 200,000 molecular weight polypeptide that has tentatively been identified as the heavy chain of myosin. Round morphological revertants from one of the flat variant clones exhibited growth characteristics typical of the parent N18 line, but their content of myosin heavy chain, although reduced, was not so low as that in the round cell clones originally isolated. The possibility of a causal relationship between flat morphology, reduced suspension cloning efficiency, and increased content of myosin heavy chain is discussed.
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PMID:Clonal variation in cultured neuroblastoma cells. I. Isolation and characterization of variants. 719 8

Urokinase (u-PA) is synthesized and secreted as a single-chain polypeptide (single-chain u-PA, scu-PA), which has such little enzymatic activity in solution that it has been considered essentially enzymatically inert. We found that plasminogen activator inhibitor type 1 (PAI-1), the major PAI in plasma, demonstrated concentration-dependent inhibition of this solution-phase scu-PA enzymatic activity. 125I-scu-PA formed complexes with PAI-1 in a concentration- and time-dependent manner, as detected by SDS-polyacrylamide gel electrophoresis under reducing conditions. Among a given population of scu-PA molecules, all measurable enzymatic activity was inhibited by a 10-fold molar excess of PAI-1. However, at this stoichiometry, only a minority of 125I-scu-PA molecules formed SDS-stable complexes with PAI-1 (i.e. complexes that formed a covalent bond upon denaturation), even though the uncomplexed PAI-1 molecules remained competent to inhibit u-PA enzymatic activity. Neither the extent nor the time course of complex formation was altered by using PAI-1 that had been pre-incubated with native human vitronectin, compared with native PAI-1 alone. 125I-scu-PA.PAI-1 complexes that would form a covalent bond if denatured were reversible and existed in equilibrium with either non-complexed or loosely complexed reactants. These data suggest that scu-PA has more enzyme-like properties than previously appreciated and raises the possibility that it resembles single-chain tissue type-plasminogen activator in lacking a complete zymogen conformation.
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PMID:Interaction of single-chain urokinase and plasminogen activator inhibitor type 1. 754 49

The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.
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PMID:Binding of tissue plasminogen activator to endothelial cells. The effect on functional properties. Localization of a ligand in the B-chain of tPA. 774 May 7

The addition of N-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum. However, the presence of the sequon does not automatically ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent. To investigate whether intracellular protein folding can influence sequon utilization, we have expressed tissue-type plasminogen activator (t-PA) in cell culture in the presence of mild concentrations of the reducing agent dithiothreitol to prevent co-translational disulfide bond formation in the endoplasmic reticulum. We show that conditions that prevent disulfide bond formation lead to complete glycosylation of a sequon that otherwise undergoes variable glycosylation in untreated cells. This demonstrated that folding and disulfide bond formation of t-PA determines its extent of core N-linked glycosylation. When dithiothreitol was removed from the cells, the reduced and overglycosylated t-PA formed disulfide bonds, folded, and was secreted. We also show t-PA present within cells is more susceptible to reduction with low concentrations of dithiothreitol than secreted t-PA.
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PMID:Intracellular folding of tissue-type plasminogen activator. Effects of disulfide bond formation on N-linked glycosylation and secretion. 787 53


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