UNIPROT:P00750 - FACTA Search

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Query: UNIPROT:P00750 (PLA)
16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We analyzed the tissue-type plasminogen activator (TPA)-binding proteoglycans (PGs) on human umbilical vein endothelial cells (HUVECs), which were metabolically labeled with [35S]NA2SO4. Cell extracts were then prepared and subjected to affinity chromatography on diisopropyl fluorophosphate (DFP)-inactivated TPA-Sepharose 4B. Approximately 6% of the incorporated 35S radioactivity bound to DFP-treated TPA-Sepharose 4B and was eluted with 2 mol/L NaCl. In addition to NaCl, heparin, arginine, and lysine but not glycine, epsilon-amino-n-caproic acid or aspartic acid inhibited this binding and eluted the bound 35S radioactivity. Urea-containing polyacrylamide gel electrophoresis of the eluted material consistently revealed two main signals of 35S radioactivity (one with an M(r) between 600,000 and 750,000 [PGA] and the other with an M(r) between 120,000 and 180,000 [PGC]). Occasionally a less intense signal with an M(r) between 340,000 and 440,000 (PGB) was seen. Heparitinase treatment markedly decreased the intensities of both 35S signals (PGA and PGB), and chondroitinases AC and ABC abolished the 35S signal of PGC, indicating that most of the HUVEC-incorporated radioactivity with an affinity for TPA could be attributed to heparan sulfate- and chondroitin sulfate-like structures. Reductive elimination, which was performed to separate the possible glycosaminoglycan moieties from the core proteins, confirmed the PG-like nature of this material and again revealed heparan sulfate and chondroitin sulfate as the major glycosaminoglycan components. We therefore conclude that HUVECs synthesize TPA-binding, heparan sulfate- and chondroitin sulfate-containing PGs. In vivo, similar PGs may play a role in TPA binding to endothelial cells and thereby possibly influence TPA activity and/or provide an intravascular storage pool of TPA.
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PMID:Isolation and characterization of tissue-type plasminogen activator- binding proteoglycans from human umbilical vein endothelial cells. 896 24

Plasminogen activator inhibitor type 1 (PAI-1), the primary physiologic inhibitor of plasminogen activation, is associated with the adhesive glycoprotein vitronectin (Vn) in plasma and the extracellular matrix. In this study we examined the binding of different conformational forms of PAI-1 to both native and urea-purified vitronectin using a solid-phase binding assay. These results demonstrate that active PAI-1 binds to urea-purified Vn with approximately 6-fold higher affinity than to native Vn. In contrast, inactive forms of PAI-1 (latent, elastase-cleaved, synthetic reactive center loop peptide-annealed, or complexed to plasminogen activators) display greatly reduced affinities for both forms of adsorbed Vn, with relative affinities reduced by more than 2 orders of magnitude. Structurally, these inactive conformations all differ from active PAI-1 by insertion of an additional strand into beta-sheet A, suggesting that it is the rearrangement of sheet A that results in reduced Vn affinity. This is supported by the observation that PAI-1 associated with beta-anhydrotrypsin, which does not undergo rearrangement of beta-sheet A, shows no such decrease in affinity, whereas PAI-1 complexed to beta-trypsin, which does undergo sheet A rearrangement, displays reduced affinity for Vn similar to PAI-1.plasminogen activator complexes. Together these data demonstrate that the interaction between PAI-1 and Vn depends on the conformational state of both proteins and suggest that the Vn binding site on PAI-1 is sensitive to structural changes associated with loss of inhibitory activity.
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PMID:Characterization of the binding of different conformational forms of plasminogen activator inhibitor-1 to vitronectin. Implications for the regulation of pericellular proteolysis. 906 24

Myoepithelial cells in situ and in vitro exert important paracrine effects on carcinoma cells which are mediated by high expression of extracellular matrix molecules, proteinase inhibitors and angiogenic inhibitors. Myoepithelial xenografts (human matrix secreting (HMS)-X, HMS-3X and HMS-4X) established from benign human salivary gland and breast myoepithelial tumors accumulate an abundant extracellular matrix which can be extracted with 6 M urea and 2 M guanidinium hydrochloride to form a gel at 25-37 degrees C. This gel, termed Humatrix, exhibits different biochemical and biological properties than the conventional non-human matrical gels in existence, i.e. Matrigel and Vitrogen 100. Whereas Matrigel consists mainly of basement membrane molecules, e.g. laminin, type IV collagen and heparan sulfate proteoglycan, and Vitrogen 100 consists mainly of non-basement membrane molecules, e.g. type I and type III collagen, Humatrix contains significant amounts of both basement membrane and non-basement membrane molecules, including large amounts of chondroitin sulfate proteoglycan. Like Matrigel, Humatrix contains bound growth factors, including epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I); unlike Matrigel, which contains predominantly significant quantities of bound proteinases, including tissue-type plasminogen activator (tPA), matrix metalloproteinase (MMP)-2 and MMP-9, and angiogenic factors, including basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta, Humatrix contains predominantly bound proteinase inhibitors such as protease nexin II (PN-II) and alpha1-antitrypsin and angiogenic inhibitors such as thrombospondin-1. Humatrix selectively stimulates the growth and tumorigenicity of human myoepithelial cell lines but inhibits invasion, angiogenesis and metastasis of other non-myoepithelial malignant cell lines. Because of its myoepithelial origin Humatrix represents a more natural source of extracellular matrix molecules and bound factors that carcinoma cells encounter in vivo.
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PMID:Humatrix, a novel myoepithelial matrical gel with unique biochemical and biological properties. 948 91

Functional assembly of the plasminogen-dependent proteolytic system on the cell surface requires multiple interactions involving urokinase (uPA), urokinase receptor (uPAR), plasminogen activator inhibitors, and other molecules that mediate cell migration and adhesion. We analyzed the in vitro interaction of uPAR-containing particulate cell fractions with the amino-terminal fragment (ATF) of human urokinase and the matrix-like form of vitronectin. Binding and cross-linking of 125I-labeled ATF to crude membrane extracts from LB6-19 mouse cells overexpressing human uPARs in the presence of 25 nM urea-denatured vitronectin led to the formation of Mr 137,000, 92, 000, and 82,000 covalent complexes. Immunoprecipitation of the preformed cross-linked 125I-labeled complexes with anti-vitronectin, anti-uPA, or anti-uPAR antibodies revealed that the Mr 82,000 and 92, 000 species do contain ATF and vitronectin and identified the Mr 137, 000 species as a ternary complex formed by ATF, uPAR, and vitronectin. A similar electrophoretic pattern was displayed by acid-pretreated membranes extracted from MCF-7 breast carcinoma or HT1080 fibrosarcoma cell lines, as well as a ductal breast carcinoma specimen; the latter exhibited complex formation at concentrations of vitronectin lower than 10 nM. Finally, uPAR-vitronectin interaction was further documented by the decreased reactivity of an anti-uPAR polyclonal antibody to acid-pretreated sections of 10 breast carcinomas that had been preincubated with vitronectin. Our findings highlight the ability of uPAR to interact simultaneously with vitronectin and uPA in breast cancer, supporting a dynamic coupling of the molecular mechanisms underlying plasminogen-dependent matrix degradation and cell adhesion.
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PMID:Vitronectin binding to urokinase receptor in human breast cancer. 981 12

The crystal structure of annexin A3 (human annexin III) solved recently revealed a well-ordered folding of its N-terminus with the side chain of tryptophan 5 interacting with residues at the extremity of the central pore. Since the pore of annexins has been suggested as the ion pathway involved in membrane permeabilization by these proteins, we investigated the effect of the N-terminal tryptophan on the channel activity of annexin A3 by a comparative study of the wild-type and the W5A mutant in structural and functional aspects. Calcium influx and patch-clamp recordings revealed that the mutant exhibited an enhanced membrane permeabilization activity as compared to the wild-type protein. Analysis of the phospholipid binding behavior of wild-type and mutant protein was carried out by cosedimentation with lipids and inhibition of PLA(2) activity. Both methods reveal a much stronger binding of the mutant to phospholipids. The structure is very similar for the wild-type and the mutant protein. The exchange of the tryptophan for an alanine results in a disordered N-terminal segment. Urea-induced denaturation of the wild-type and mutant monitored by intrinsic fluorescence indicates a separate unfolding of the N-terminal region which occurs at lower urea concentrations than unfolding of the protein core. We therefore conclude that the N-terminal domain of annexin A3, and especially tryptophan 5, is involved in the modulation of membrane binding and permeabilization by annexin A3.
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PMID:The annexin A3-membrane interaction is modulated by an N-terminal tryptophan. 1086 76

The hyaluronic acid binding serine protease (PHBSP), an enzyme with the ability to activate the coagulation factor FVII and the plasminogen activator precursors and to inactivate factor VIII and factor V, could be isolated from human plasma in the presence of 6M urea as a single-chain zymogen, whereas under native conditions only its activated two-chain form was obtained. The total yield of proenzyme (proPHBSP) was 5-6 mg/l, corresponding to a concentration of at least 80-100nM in plasma. Upon removal of urea, even in the absence of charged surfaces a rapid development of amidolytic activity was observed that correlated with the appearance of the two-chain enzyme. The highest activation rate was observed at pH 6. ProPHBSP processing was concentration-dependent following a second order kinetic and was accelerated by catalytic amounts of active PHBSP, indicating an intermolecular autocatalytic activation. Charged macromolecules like poly-L-lysine, heparin, and dextran sulfate strongly accelerated the autoactivation, suggesting that in vivo proPHBSP activation might be a surface-bound process. The intrinsic activity of the proenzyme was determined to be 0.25-0.3%, most likely due to traces of PHBSP. The presence of physiological concentrations of known plasma inhibitors of PHBSP, like alpha2 antiplasmin and C1 esterase inhibitor, but not antithrombin III/heparin, slowed down zymogen processing. Our in vitro data suggest that the autoactivation of proPHBSP during plasma fractionation is induced by the removal of inhibitors of PHBSP and is accelerated by charged surfaces of the chromatographic resins.
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PMID:Activation of proPHBSP, the zymogen of a plasma hyaluronan binding serine protease, by an intermolecular autocatalytic mechanism. 1120 57

Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris--HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.
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PMID:Bacterial expression and characterization of starfish phospholipase A(2). 1125 May 52

The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.
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PMID:Solution and interface aggregation states of Crotalus atrox venom phospholipase A2 by two-photon excitation fluorescence correlation spectroscopy. 1138 5

Patatin is the major protein constituent of potato tubers and displays broad esterase activity. The native enzyme actually belongs to a highly homologous multigene family of vacuolar glycoproteins. From these, the patB2 patatin gene was selected and cloned into pUC19 without its signal sequence but with an N-terminal histidine-tag. This patatin was overexpressed under the control of the lac promotor in Escherichia coli strain DH5alpha. The protein was recovered as inclusion bodies, folded into its native state by solubilization in urea and purified to homogeneity. Starting with one gram of inclusion bodies, 19 mg of pure and active recombinant patatin was isolated, with even higher specific activity than the glycosylated wild-type patatin purified from potato tubers. The purified enzyme showed esterolytic activity with p-nitrophenylesters dissolved in Triton X-100 micelles. The activity of patatin on p-nitrophenylesters with different carbon chain lengths showed an optimum for p-nitrophenylesters with 10 carbon atoms. Besides general esterolytic activity, the pure enzyme was found to display high phospholipase A activity in particular with the substrates 1,2-dioctanoyl-sn-glycero-3-phosphocholine (diC(8)PCho) (127 U.mg(-1)) and 1,2-dinonanoyl-sn-glycero-3-phosphocholine (diC(9)PCho) (109 U.mg(-1)). Recently, the structure of human cytosolic PLA(2) (cPLA(2)) was solved, showing a novel Ser-Asp active site dyad [1]. Based on a partial sequence alignment of patatin with human cPLA(2), we propose that patatin contains a similar active site dyad. To verify this assumption, conserved Ser, Asp and His residues in the family of patatins have been modified in patatin B2. Identification of active site residues was based on the observation of correctly folded but inactive variants. This led to the assignment of Ser54 and Asp192 as the active site serine and aspartate residues in patatin B2, respectively.
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PMID:Cloning, expression, purification and characterization of patatin, a novel phospholipase A. 1158 94

We investigated the effects of bovine somatotropin (bST) on mammary gland function and composition in the declining phase of lactation in goats. Sixteen Saanen goats, 180 +/- 11 days in milk (DIM), were divided equally into control and treated groups. The treated group received 120 mg/2 wk of slow-release bST for three cycles. Milk yield, milk composition, milk clotting measures, and plasmin-plasminogen activator activities were recorded weekly. Milk Na and K were determined in individual milk samples collected weekly during the third cycle. Blood samples were collected weekly during the second cycle and the plasma analyzed for nonesterified fatty acids (NEFA), glucose, and urea. At the end of the 6 wk, three goats from each group were slaughtered, and the udders were removed. Mammary gland weight, composition, and total DNA content were determined. The histological effects of bST on mammary tissue were investigated. The analyzed parameters included numbers of alveoli, corpora amylacea, apoptotic cells, and laminin fibronectin distribution and localization. An extensive morphological analysis on the epithelial and stromal components was performed. Milk yield was significantly higher in the treated group, fat content was not affected, but protein and nonprotein nitrogen were lower in treated goats milk. Treatment with bST did not influence milk pH but reduced coagulation time. Plasmin and plasminogen activator activities were not affected. Milk K levels were higher and the Na/K ratio was lower in treated animals. Plasma glucose, NEFA, and urea were unaffected. Mammary gland weight and total DNA were higher in treated than control animals, suggesting that with advancing lactation bST treatment maintains cells. Fat, protein, and collagen content of the mammary tissue did not differ between the groups. Treatment with bST significantly increased the number of lactating alveoli (LA) and significantly reduced the number of regressing alveoli (RA) and corpora amylacea, both within and outside the alveolar lumen. Laminin and fibronectin localization were not affected, and very few apoptotic cells were found in both treated and control samples. Our findings suggest that bST administration to dairy goats in late lactation can modulate mammary gland activity and improve lactation persistency; this is associated with maintained total mammary parenchyma weight and lactating alveoli.
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PMID:Bovine somatotropin administration to dairy goats in late lactation: effects on mammary gland function, composition and morphology. 1208 43

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