Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 have used differential scanning calorimetry to measure the effect of replacements of valine 65 on thermal stability of the isolated kringle-2 domain of tissue plasminogen activator (t-PA). The role of this site in stability was examined because a human t-PA variant having this valine (residue 245 in t-PA numbering) replaced with a methionine has been described [Johnston, M.D., & Berger, H. (1987) U.K. Patent Application GB 2176702A]. Mutants of kringle-2 having valine 65 replaced with Met, Leu, Ile, Thr, Ala, or Ser were constructed by using site-directed mutagenesis in conjunction with a restricted site selection strategy. Isolated kringle-2 domains were expressed in Escherichia coli and purified as previously described for the wild-type domain [Cleary, S., Mulkerrin, M.G., & Kelley, R.F. (1989) Biochemistry 28, 1884-1891]. None of these substitutions results in a significant perturbation of the native conformation of kringle-2 as judged by far-UV circular dichroism and equilibrium dialysis measurements of L-lysine affinity. A two-state analysis of the heat capacity profile observed for heating a solution of wild-type (w-t) kringle-2 containing 100 mM citrate, pH 4.5, provides values of 64.3 +/- 0.8 degrees C for Tg (melting temperature), 81 +/- 5 kcal/mol for delta H g, and 1.2 +/- 0.9 kcal/(mol-deg) for delta C p. Thermal denaturation of w-t kringle-2 is reversible in the pH range 3-6 as indicated by the observation of similar heat capacity profiles for consecutive heating cycles and also recovery of spectroscopic and lysine binding properties upon cooling the heat-denatured protein.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of residue 65 substitutions on thermal stability of tissue plasminogen activator kringle-2 domain. 250 77

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another.
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PMID:Mutant and chimeric recombinant plasminogen activators. Production in eukaryotic cells and preliminary characterization. 311 52

The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.
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PMID:Specific binding of urinary-type plasminogen activator (u-PA) to vitronectin and its role in mediating u-PA-dependent adhesion of U937 cells. 753 60

The relative contribution of the finger/growth factor domains of tissue-type plasminogen activator (t-PA) and of the other t-PA domains to the clearance of t-PA by hepatocytes was investigated. A recombinant finger/growth factor construct inhibited t-PA and t-PA/plasminogen activator inhibitor type-1 degradation with an IC50 of 1800 nM, whereas a t-PA mutant lacking the finger and growth factor domains inhibited degradation with an estimated IC50 of 1200 nM. In comparison the IC50 of t-PA was found to be approximately 10 nM. Clearance of t-PA by human or rat hepatoma cells was not inhibited by high concentrations of fucose (50 mM), which suggests that the fucose on Thr-61 is not involved in clearance by these cells. These results suggest that the binding of t-PA involves several low affinity binding sites located on distinct domains of the t-PA molecule.
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PMID:The role of the finger and growth factor domains in the clearance of tissue-type plasminogen activator by hepatocytes. 759 2

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

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

One feature that distinguishes all of the inhibitory members of the serpin gene family is the presence of a small uncharged residue at the P14 position of the reactive center loop. In this report we examine the effects of mutations at this position, in the serpin, plasminogen activator inhibitor type 1 (PAI-1). Replacement of the native P14 Thr-333 residue by an Arg (Thr-333-->Arg) resulted in complete loss of inhibitory activity toward tissue-type plasminogen activator and urokinase-type plasminogen activator. Comparison of the binding of the mutant inhibitor and wild type PAI-1 (WTPAI-1) to anhydrotrypsin indicated that the initial interaction of the two inhibitors with proteases was identical. However, whereas WTPAI-1 forms SDS-stable complexes with both plasminogen activators, the mutant PAI-1 was efficiently cleaved as a substrate. Amino-terminal sequence analysis indicated that cleavage of the mutant PAI-1 occurred at its reactive center P1-P1' Arg-Met bond. Thermal denaturation studies of native and cleaved PAIs indicated that native Thr-333-->Arg mutant had a thermal stability identical to active WTPAI-1 and that both proteins became significantly more stable following cleavage by elastase (cleaved at the P4-P3 bond). Finally, the function of recombinant PAI-1 variants containing 15 of the possible 19 amino acid substitutions at P14 were analyzed. While residue size appeared to have little effect on inhibitory activity, the presence of either a positive or a negative charge at P14, converted PAI-1 to a substrate. Taken together, these results suggest that while insertion of the reactive center loop is not essential for protease binding, it is a necessary second step required for inhibitor function. The presence of a charged residue at P14 can retard this insertion, resulting in conversion of the serpin to a substrate.
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PMID:Serpin reactive center loop mobility is required for inhibitor function but not for enzyme recognition. 796 84

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

The glycoprotein tissue-type plasminogen activator (t-PA) is subject to hepatic clearance in humans. Here, the interaction of t-PA with a well-differentiated hepatoma cell line (HepG2) was examined. Suspended HepG2 cells bound 125I-t-PA in a specific, saturable, and reversible fashion through a Ca(2+)-dependent, active site-independent mechanism. Binding isotherms indicated a high affinity system with a single class of saturable binding sites (Kd 39 nM; maximum binding capacity 493,000 sites per cell). Bound t-PA was rapidly degraded at 37 degrees C in a manner inhibited by lysosomotropic agents or metabolic inhibitors. Pretreatment of t-PA with monoclonal antibodies against the EGF/fibronectin finger domain, but not kringle 2 or kringle 1, reduced total binding by 86%. Binding of 125I-t-PA to HepG2 cells was inhibited by monosaccharides fucose and galactose and by the neoglycoprotein fucosyl-albumin. Enzymatic removal of alpha-fucose residues, but not alpha-galactose, high mannose, or complex oligosaccharide from 125I-t-PA, reduced specific binding by 60 +/- 5%. Binding was also inhibited by high, but not low, molecular weight urokinase, which contains an EGF-based threonine-linked alpha-fucose homologous to that of t-PA. These data suggest that EGF-associated O-linked alpha-fucose may mediate t-PA binding and degradation by HepG2 cells. This mechanism may be relevant to other proteins with analogous structures.
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PMID:alpha-Fucose-mediated binding and degradation of tissue-type plasminogen activator by HepG2 cells. 811 82


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