UNIPROT:P00750 - FACTA Search

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)

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

1. Human uterine cervical stroma was found to contain a Ca(2+)-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein-Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4-8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4x10(5) by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10mum) or leupeptin (10mum) was also found to be inhibitory, but chymostatin (40mug/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.
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PMID:Partial purification and characterization of a novel neutral proteinase from human uterine cervix. 699 9

A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor. We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (Kd approximately 8-10 nM). These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-methylamine (alpha 2M*) binding whereas GST/115-319 only potently inhibits t-PA binding. Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding. In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and alpha 2M* binding to LRP. The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined. Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and alpha 2M* binding. These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells. Within domain 18-24, arginine 21 is required for inhibition of t-PA and alpha 2M* binding as well as for the direct binding of amino-terminal constructs to LRP. Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding. Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of alpha 2M* binding. Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.
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PMID:Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein. 753 37

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the P1 and P1' positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at P1 including Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P1'. Kinetic analyses of selected variants identified P1Tyr and P1His as the most efficient tPA-specific inhibitors, with second-order rate constants (ki) of 4.0 x 10(5) M-1s-1 and 3.6 x 10(5) M-1s-1, respectively. Additional PA-specific PAI-1 variants containing substitutions at P3 through P1' were constructed. P3Tyr-P2Ser-P1Lys-P1'Trp and P3Tyr-P2Ser-P1Tyr-P1'Met had ki values of 1.7 x 10(6) M-1s-1 and 2.5 x 10(6) M-1s-1 against tPA, respectively, but both were inactive against uPA. In contrast, P2Arg-P1Lys-P1'Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several P1Arg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.
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PMID:Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity. 772 51

A recombinant DNA Chinese hamster ovary (CHO) cell line which produces tissue-type plasminogen activator (t-PA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day-1. The cultivation consisted of four phases with four different ammonium chloride concentrations (0, 2.5, 5, and 7.5 mM) in the feed medium, causing a reactor ammonium concentration of up to 8 mM. Cell growth was not inhibited by these high ammonium concentrations, as cell densities of around 2.3 x 10(6) cells mL-1 were established. In contrast, the production of t-PA was reduced under high ammonium concentration. The decrease in specific t-PA production could be due to either a negative ammonium influence on productivity or a limitation of medium components, e.g., amino acids. Cell metabolism was changed under high ammonium concentrations, seen most clearly by a decrease in specific ammonium production by a factor of 8 and an increase in specific alanine production of 30%.
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PMID:Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture. 776 23

A recombinant DNA Chinese hamster ovary (CHO) cell line that produces tissue-type plasminogen activator (tPA) was cultivated continuously in suspension with a constant dilution rate of 0.5 day with three different asparagine concentrations in the feed (0.05, 2.55 and 7.55 mM). The up-shift in asparagine concentration caused an up-shift in asparagine consumption [15.7 and 31.4 nmol (10(6) cells)-1 h-1] and intracellular concentration (2.19 and 18.7 mM). The up-shift was accompanied by an increased production of ammonium, glycine and alanine, and a metabolic shift whereby the cells began to produce aspartate and glutamate, which were consumed before the shift. The tPA production was reduced in the up-shift culture. This might be explained by ammonium inhibition, but alternatively by a surprising down-shift in the intracellular concentration of many amino acids, a down-shift that was not observed in the extracellular concentrations or consumption rates. For efficient physiological engineering of mammalian cells it is necessary to include both extracellular and intracellular measurements and to consider the transport into and out of the cells.
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PMID:Extra- and intracellular amino acid concentrations in continuous Chinese hamster ovary cell culture. 776 83

rt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagenized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent. The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 +/- 100% versus 140 +/- 40% within the 2 h observation period per mg of compound administered per kg body weight (mean +/- SEM, p = 0.004) and a significantly lower dose of 0.08 +/- 0.01 versus 0.21 +/- 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparative thrombolytic properties of tissue-type plasminogen activator and of a plasminogen activator inhibitor-1-resistant glycosylation variant, in a combined arterial and venous thrombosis model in the dog. 797 84

Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA), may occur in three interconvertible conformations: active, latent, and substrate. To delineate specific domains in the PAI-1 molecule responsible for its conformational flexibility and associated functional diversity, four mutants of PAI-1 (with the amino acids at positions P12, P10, P8, and P6, respectively, substituted with proline) were expressed in Escherichia coli, purified, and characterized. Wild-type PAI-1 (wtPAI-1) had a specific activity of 21 +/- 10% (mean +/- S.D., n = 3) of the theoretical maximum value. PAI-1-P12 (Ala-->Pro at P12), PAI-1-P10 (Ser-->Pro at P10), and PAI-1-P8 (Thr-->Pro at P8) had specific activities of 0.06 +/- 0.03% (n = 3), 2.6 +/- 1.0% (n = 4), and 2.7 +/- 1.1% (n = 3), respectively (p < 0.03 versus wtPAI-1). PAI-1-P6 (Val-->Pro at P6) has a specific activity of 12 +/- 3.3% (n = 3) of the theoretical maximum value (p = not significant versus wtPAI-1). SDS-polyacrylamide gel electrophoresis of mixtures of wtPAI-1 or PAI-1-P6 with a 2-fold molar excess of t-PA yielded a mixture of a covalent 110-kDa t-PA.PAI-1 complex (15-25%), nonreactive 45-kDa material (44-67%), and a 41-kDa band (18-31%) representing cleaved PAI-1. PAI-1-P12, PAI-1-P10, and PAI-1-P8 behaved as substrates, yielding predominantly the 41-kDa cleavage product (85-91%) and a small amount (9-15%) of non-reactive material. NH2-terminal amino acid sequencing revealed that cleavage occurred at the P1-P1' bond (Arg346-Met347). Incubation of PAI-1-P12, PAI-1-P10, or PAI-1-P8 with a 2-fold molar excess of urokinase-type plasminogen activator, plasmin, or thrombin also primarily generated a 41-kDa cleavage product (62-89%). Incubation of wtPAI-1 and PAI-1-P6 at 37 degrees C resulted in a loss of inhibitory activity, whereas the substrate behavior of PAI-1-P12, PAI-1-P10, and PAI-1-P8 remained unaltered. Treatment of the three substrate-like mutants with guanidinium Cl did not induce inhibitory activity. In conclusion, point mutations at positions P12, P10, and P8 yield PAI-1 variants with stable substrate properties, which may facilitate more detailed structure/function studies.
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PMID:Conversion of plasminogen activator inhibitor-1 from inhibitor to substrate by point mutations in the reactive-site loop. 803 24

We have used the molecular dynamics (MD) simulation package AMBER4 to search the conformation of a peptide predicted as a leucine zipper motif for the human immunodeficiency virus type 1 integrase protein (HIV IN-LZM). The peptide is composed of 22 amino acid residues and its location is from Val 151 to Leu 172. The searching procedure also includes two known alpha-helices that served as positive controls--namely, a 22-residue GCN4-p1 (LZM) and a 20-residue poly (L-alanine) (PLA). A 21-residue peptide extracted from a cytochrome C crystal (CCC-t) with determined conformation as a beta-turn is also included as a negative control. At the beginning of the search, two starting conformations--namely, the standard right-handed alpha-helix and the fully stretched conformations--are generated for each peptide. Structures generated as standard alpha-helix are equilibrated at room temperature for 90 ps while structures generated as a fully stretched one are equilibrated at 600 K for 120 ps. The CCC-t and PLA helices are nearly destroyed from the beginning of equilibration. However, for both the HIV IN-LZM and the GCN4-p1 LZM structures, there is substantial helicity being retained throughout the entire course of equilibration. Although helix propagation profiles calculated indicate that both peptides possess about the same propensity to form an alpha-helix, the HIV IN-LZM helix appears to be more stable than the GCN4-p1 one as judged by a variety of analyses on both structures generated during the equilibration course. The fact that predicted HIV IN-LZM can exist as an alpha-helix is also supported by the results of high temperature equilibration run on the fully stretched structures generated. In this run, the RMS deviations between the backbone atoms of the structures with the lowest potential energy (PE) identified within every 2 ps and the structure with the lowest PE searched in the same course of simulation are calculated. For both the HIV IN-LZM and the GCN4-p1 LZM, these rms values decrease with the decrease of PE, which indicates that both structures are closer in conformations as their PEs are moved deeper into the PE well.
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PMID:Molecular dynamics simulation of a leucine zipper motif predicted for the integrase of human immunodeficiency virus type 1. 807 85

The developing neural retina expresses a set of extracellular proteases including plasminogen activator and gelatinases. Since neurites of retina cells cultured on fluorescent gelatin digest the substrate in their paths, we have suggested that the proteases are used by the tips of growing fibers to allow them to migrate within the mass of the tissue in vivo. In order to obtain further information about relationships between extracellular proteases and fiber growth, we have examined the effects of the specific inhibitors HS-LFA (HS-Leu-Phenylala-Ala, enantiomeric forms 1 and 2), bathophenanthroline sulfonate (BPS), phenylmethyl sulfonyl fluoride (PMSF), and relevant controls on the activity of retinal growth cones in vitro, monitored by time lapse video microscopy. Of the inhibitors tested, only the two enantiomeric forms of HS-LFA caused a reproducible cessation of both spike extension and filopodial processes at the growth cone ruffling, while control media had no effect. In some cases, the growth cone swelled and exhibited small protrusions. The behavior of growth cones was in sharp distinction to that of the cytoplasm of neural cells, and membrane ruffling of flat cells, which continued in activity throughout. Growth cone activity returned after several hours in the presence of the agent. BPS was toxic at concentrations above 2.5 mM. Below that, it had no effect. L-cysteine, PMSF, and control media had no effect. The relevance of these results to the possible role of proteases in fiber outgrowth from retinal cells is discussed.
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PMID:Inhibition of retinal growth cone activity by specific metalloproteinase inhibitors in vitro. 808 Oct 16

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