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)

Recombinant BM 06.022 (M(r) 39,589) is a domain-deletion mutant of the human tissue-type plasminogen activator (tPA) structured by the kringle 2 and protease modules. Unfolding under various conditions was investigated via 1H-NMR spectroscopy by monitoring the well-resolved high-field methyl resonances at approximately -0.97 ppm (kringle 2) and approximately -0.29 and -0.54 ppm (protease). Reversible acid/base unfolding is manifest under low pH (< 4.8) conditions. It is observed that, relative to the protease, the kringle exhibits higher overall stability at low pH. At pH 4.6, BM 06.022 undergoes two distinct thermal melting transitions, at approximately 334 and approximately 352 K, assigned to an irreversible denaturation of the protease and a reversible unfolding of the kringle 2, respectively. Under the same conditions, the protease reacted with the active site inhibitor 1,5 dansyl-L-glutamylglycyl-L-arginine chloromethyl ketone (EGRck) exhibits a higher (approximately 10 K) thermal stability than the inhibitor-free protease. Upon acidification, the EGRck-modified protease unfolds irreversibly around pH 3.4. As exemplified by BM 06.022, a single-chain protein, as defined by continuity of the polypeptide backbone, can exhibit simultaneous folding reversibility and irreversibility for autonomous segments of the sequence. Conversion of the isolated (single-chain) protease or intact BM 06.022 to their catalytically active two-chain forms via plasminolytic cleavage of the Arg275-Ile276 peptide bond leaves the kringle 2 spectrum unaffected while perturbing the resolved high-field methyl resonances stemming from the protease. The latter also shift when the protease is reacted with EGRck, indicating that these signals are sensitive to events at the binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tissue-type plasminogen activator domain-deletion mutant BM 06.022: modular stability, inhibitor binding, and activation cleavage. 791 92

By means of gel filtration, ionic exchange chromatography and DEAE-column HPLC, an acidic phospholipase A2 (PLA2) was purified from beaded lizard (Heloderma horridum) venom. The purified PLA is a single-chain polypeptide, consisting of about 163 amino acid residues with a molecular mass of 19,000 Da as calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analysis. HHV-PLA showed a rather specific inhibitory effect on platelet aggregation induced by U46619 and epinephrine in human platelet-rich plasma in a dose- and time-dependent manner, whereas it had little effect on collagen- and ADP-induced aggregation. ATP-release reaction induced by various agonists were dose- and time-dependently inhibited by HHV-PLA, even though platelet aggregation was apparently not affected in human washed platelets. When HHV-PLA was chemically modified with p-bromophenacyl bromide, both of its enzymatic activity and antiplatelet activity were lost. Furthermore, exogenous lysophosphatidylcholine and HHV-PLA treated phosphatidylcholine inhibited platelet aggregation induced by U46619 in human washed platelets. In conclusion, PLA enzyme from H. horridum venom inhibits exclusively U46619- or thromboxane-induced platelet aggregation of human platelet-rich plasma probably by virtue of their PLA enzymatic activity on plasma phospholipids, converting phospholipids (e.g., phosphatidylcholine) into lysophospholipids, which in turn interfere with the coupling of TXA2 receptor and its signalling transduction system.
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PMID:Effect on human platelet aggregation of phospholipase A2 purified from Heloderma horridum (beaded lizard) venom. 812 83

A series of strategically designed recombinant (r) mutants of the kringle 1 region of human plasminogen ([K1HPg]) have been constructed and the resulting gene products employed to reveal the identities of the residues that contribute to stabilization of the binding of omega-amino acid ligands to this domain. On the basis of determinations of the binding constants of the ligands, 6-aminohexanoic acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic acid, to a variety of these mutants, we find that the anionic site of the polypeptide responsible for stabilization of the amino group of the ligands consists of both D54 and D56 and the cationic site of the polypeptide that interacts with the carboxylate group of the ligand is composed solely of R70. The main hydrophobic interactions that stabilize binding of these ligands, likely by interactions with the ligand hydrophobic regions, are principally due to W61, Y63, and Y71. The results obtained are consistent with conclusions that could be made from analysis of the X-ray crystal structure of r-[K1HPg] and from previous studies from this laboratory regarding the binding of ligands of this type to the kringle 2 region of tissue-type plasminogen activator ([K2tPA]). It thus appears as though a common ligand binding site has evolved in different kringles with ligand specificity differences between r-[K2tPA] and r-[K1HPg] perhaps explainable by the different nature of the cationic sites on these polypeptides that are involved in coordination to the ligand carboxylate groups.
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PMID:Amino acids of the recombinant kringle 1 domain of human plasminogen that stabilize its interaction with omega-amino acids. 821 59

A new type of gamma Gly-268 (GGA) to Glu (GAA) substitution has been identified in a homozygous dysfibrinogen by analyses of the affected polypeptide and its encoding gene derived from a 58 year-old man manifesting no major bleeding or thrombosis. The functional abnormality was characterized by impaired fibrin assembly most likely due to failure to construct properly aligned double-stranded fibrin protofibrils. This presumption was deduced from the following findings: (1) Factor XIIIa-catalyzed cross-linking of the fibrin gamma-chains progressed in a normal fashion, indicating that the contact between the central E domain of one fibrin monomer and the D domain of another took place normally; (2) Nevertheless, factor XIIIa-catalyzed cross-linking of the fibrinogen gamma-chains was obviously delayed, suggesting that longitudinal association of D domains of different fibrin monomers, ie, D:D association was perturbed; (3) Plasminogen activation catalyzed by tissue-type plasminogen activator was not as efficiently facilitated by polymerizing fibrin monomer derived from the patient as by the normal counterpart. Therefore, gamma Gly-268 would not be involved in the 'a' site residing in the D domain, which functions as a complementary binding site with the thrombin-activated 'A' site in the central E domain, but would be rather involved in the D:D self association sites recently proposed for human fibrinogen. Thus, the gamma Glu-268 substitution newly identified in this homozygous dysfibrinogen seems to impair proper alignment of adjacent D domains of neighboring fibrin molecules in the double-stranded fibrin protofibril, resulting in delayed fibrin gel formation.
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PMID:A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I. 863 38

Platelet-derived growth factor (PDGF) is a chemotactic and mitogenic agent for fibroblasts and smooth muscle cells and plays a key role in the development of atherosclerotic lesions. PDGF is produced by a number of normal and transformed cell types and occurs as homo- or heterodimers of A and B polypeptide chains. Using Chinese hamster ovary (CHO) cells transfected with various forms of PDGF, we have previously shown that PDGF A(s) (short splice version) is secreted, PDGF A(l) (long splice version) predominantly extracellular matrix-associated, and PDGF B divided between medium, cells, and matrix. In the present study we have demonstrated the mitogenic activity of matrix-localized PDGF in artificial and more physiologically relevant models by culturing Balb/c-3T3 cells (3T3), human foreskin fibroblasts (HFF), and rabbit aortic smooth muscle cells (SMC) on extracellular matrix (ECM) laid down by PDGF-expressing CHO cells and human umbilical vein endothelial cells (HUVEC). These cells responded to the local growth stimulus of PDGF-containing CHO ECM and HUVEC ECM. We showed that 3T3 cells required proteolytic activity to utilize matrix-localized PDGF, as aprotinin and epsilon-ACA inhibited growth and 3T3 cells were shown to possess plasminogen activator activity. HFF and SMC did not appear to require proteolytic activity (including metalloproteinase and serine protease activity) as a prerequisite for mitogenesis but were able to access immobilized PDGF by contact with the matrix. An understanding of the mechanisms whereby the utilization of stored PDGF is controlled in situations of excessive cellular proliferation will aid in the development of therapy for these conditions.
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PMID:Extracellular matrix is a source of mitogenically active platelet-derived growth factor. 870 68

The primary structure of a 2671 bp DNA fragment between the pla gene (encoding plasminogen activator) and the origin of replication of the wild-type Yersinia pestis plasmid pYP358 was determined. Two ORFs of 1074 and 426 bp with opposite transcription polarities were identified on both strands. They encode a 357 aa pesticin activity protein (Pst) and a 141 aa pesticin immunity polypeptide (Pim). A GC-rich palindromic structure located between pst and pim can form a hairpin loop and serve as rho-independent transcription terminator sequences for both genes. The site for the interaction with the LexA repressor of the SOS system was found in another palindromic structure preceding the pst structural gene. A deduced 39.9 kDa Pst polypeptide is devoid of a signal peptide, indicating a Sec-independent mode of export. Pst carries a pentapeptide typical of TonB-dependent colicins (TonB box) that is necessary for the interaction with the yersiniabactin/pesticin receptor and for active energy-dependent transport through the outer membrane. The substitution of the last five C-terminal amino acids did not significantly influence the bactericidal activity of the truncated pesticin. The pesticin lost its ability to kill sensitive bacteria and to bind to a pesticin receptor after deletion of the last 57 C-terminal amino acids. A deduced 16 kDa Pim protein has an N-terminal hydrophobic amino acid stretch with features typical of prokaryotic signal peptides. Pim is a slightly hydrophilic protein with a basic pl. The immunity protein was localized in the periplasmic space and in the outer-membrane fraction after its overexpression under the polymerase T7 promoter. Several other ORFs were identified on the sequenced 2671 bp fragment, but none of them seemed to encode a typical lysis peptide, which is necessary for the release of the pesticin. In the promoter region and in the regions preceding and following the pst operon, the DNA sequence has high (> 70%) identity with other colicin genes. The DNA sequence located 284 bp upstream of the pim gene showed more than 90% similarity to antisense RNA I of the ColE1 replicon. This defined the location of the pYP358 origin of ColE1-type replication.
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PMID:Structural and functional organization of the Yersinia pestis bacteriocin pesticin gene cluster. 900 4

The kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been expressed in Pichia pastoris cell lines GSI 15 and KM71. This construct contained a hexahistidine sequence at the C-terminus of the kringle to aid in purification by immobilized metalion-affinity chromatography. The exact amino acid sequence of the isolated kringle was EAEAYV-[K2tPA]SR(H)6, where [K2tPA] represents amino acid sequence residues C1-C82 of the kringle domain (residues 180-261 of tPA). The clones of the yeast transformants provided large amounts of the recombinant (r)-[K2tPA]-containing polypeptide at levels that allowed ready purification of several hundred mg from shake flasks and near-gram levels from a high-biomass fermenter. Purification of the kringle domain directly from cell-conditioned media was accomplished in a single step by either immobilized Ni(+)-affinity chromatography or lysine-Sepharose affinity chromatography. N-linked glycans were present on approx. 30% of this yeast-expressed material, at N5 of the kringle (corresponds to N11 of the particular construct, N184 of full-length tPA). The expressed recombinant kringle recognized a conformation-specific monoclonal antibody generated against tPA that is directed to the K2 domain of the protein, interacted properly with various omega-amino acid ligands, and showed signature conformational properties when studied by differential scanning calorimetry and high-resolution 1H-NMR. The results demonstrate that the P. pastoris system can be employed to obtain large amounts of secreted and properly folded kringle domains.
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PMID:High-level secretion in Pichia pastoris and biochemical characterization of the recombinant kringle 2 domain of tissue-type plasminogen activator. 903 37

The recombinant plasminogen activator (rDSPA alpha 1) from the vampire bat Desmodus rotundus is a promising new thrombolytic agent that exhibits a superior pharmacological profile if compared to tissue-type plasminogen activator (t-PA) or streptokinase. In the present study the structures of the carbohydrate moieties at the two N-glycosylation sites (Asn-117, Asn-362) of rDSPA alpha 1 expressed in Chinese hamster ovary cells were determined. N-Linked glycans were enzymatically released from isolated tryptic glycopeptides by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F digestion and separated by two-dimensional HPLC. Oligosaccharide structures were characterized by analysis of carbohydrate composition and linkage, by mass spectrometry, and by sequence analysis in which the fluorescently labeled glycans were cleaved with an array of specific exoglycosidases. More than 30 different oligosaccharides were identified. The results revealed that Asn-117 carried a mixture of one high-mannose structure (17% of site-specific glycosylation), three hybrid glycans (26%) and predominantly biantennary complex N-glycans (54%). Glycosylation site Asn-362 was found to comprise complex glycans with biantennary (50%), 2,4- and 2,6-branched triantennary (21%, 11%), and tetraantennary structures (10%), which were fucosylated at the innermost residue of N-acetylglucosamine. Mainly neutral and monosialylated glycans, and smaller quantities of disialylated glycans, were detected at both glycosylation sites. Sialic acid was alpha 2-3 linked to galactose exclusively. As shown in this study the N-glycans attached to Asn-117 of rDSPA alpha 1 are more processed during biosynthesis than the high-mannose structures linked to Asn-117 of t-PA, to which the polypeptide backbone of rDSPA alpha 1 is structurally closely related.
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PMID:Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA alpha 1 expressed in Chinese hamster ovary cells. 906 66

The oligosaccharide structures present on Asn5 of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator [(r)-[K2tPA]] have been determined by a combination of techniques, including HPLC, FPLC, gel filtration, endoglycosidase digestions and mass spectrometry. The major oligosaccharides identified after their liberation by either hydrazinolysis or by the enzyme peptide:N4-(N-acetyl-beta-glucosaminyl)asparaginyl amidase, were in the oligomer range of (mannose)8(N-acetylglucosamine)2 (Man8GN2) to Man18GN2. The preponderance of these glycans spanned Man9GN2 to Man12GN2, and the major overall product was Man10GN2. An additional (less than 5%) amount of the polypeptide was hyperglycosylated. In contrast with glycoproteins produced in Saccharomyces cerevisiae, our results with specific mannosidase digestions were consistent with previous studies showing that (alpha 1,3)-linked mannose residues were not present in extensions of the core Man8GN2 unit. The results show that the N-linked glycosylation pathways in P. pastoris are substantially different from those found in S. cerevisiae, with shorter Man(alpha 1,6) extensions to the core Man8GN2 and the apparent lack of significant Man(alpha 1,3) additions representing the major processing modality of N-linked glycans in P. pastoris.
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PMID:Glycosylation properties of the Pichia pastoris-expressed recombinant kringle 2 domain of tissue-type plasminogen activator. 912 88

Erythrina variegata trypsin inhibitor ETIa belongs to the Kunitz inhibitor family, but is unique in its ability to bind and inhibit tissue-type plasminogen activator (tPA). A cDNA clone encoding ETIa was isolated from the lambda gt11 cDNA library using specific antiserum as a probe and characterized by nucleotide sequencing. The cloned ETIa cDNA consists of 762 nucleotides and includes an open reading frame encoding a polypeptide of 198 amino acids. Comparison of the deduced protein sequence and the determined protein sequence indicated the presence of two signal peptides composed of 24 and 2 amino acids at the N- and C-termini, respectively. The cDNA encoding mature ETIa was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3). The recombinant ETIa (rETIa) was expressed in E. coli as inclusion bodies; it was purified to homogeneity by gel filtration on Sephadex G-75. The rETIa exhibited almost the same inhibitory activity toward trypsin and tPA as ETIa. Six mutants, in which the amino acids Arg61, Leu62, Arg63, and Ala65 were replaced by Pro, Phe, Leu/Asp, and Tyr, respectively, were constructed by site-specific mutagenesis and expressed in E. coli. The site-specific mutation of Arg63 to Leu (aR63L) or Asp (aR63D) in ETIa resulted in abolition of the inhibitory activities toward both trypsin and tPA. The mutants aR61P and aL62F showed significantly reduced tPA-inhibitory activity, and furthermore the double mutant aR61P/L62F lacked tPA-inhibitory activity, despite retaining the trypsin-inhibitory activity. In contrast, the mutant aA65Y exhibited tPA-inhibitory activity to the same extent as rETIa. This result suggests that Arg61 and Leu62 in ETIa, in addition to Arg63, may play an important role in the interaction with tPA.
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PMID:The tissue-type plasminogen activator inhibitor ETIa from Erythrina variegata: structural basis for the inhibitory activity by cloning, expression, and mutagenesis of the cDNA encoding ETIa. 913 14

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