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 a novel phospholipase A(2) (PLA(2)), group XII (GXII) PLA(2), distinct from other cysteine-rich groups with a catalytic histidine motif, by its 20-kDa size and distribution of the 14 cysteine residues within the protein. Alternative spliced forms with distinct subcellular localization, designated GXII-1 and GXII-2, were identified by reverse transcription-polymerase chain reaction. Importantly, GXII PLA(2)s, in particular GXII-2 PLA(2), and group V PLA(2), but not group X PLA(2), were selectively expressed in murine type 2 helper T (Th2) clones and in vitro differentiated mouse CD4 Th2 cells as compared with type 1 helper T clones and cells. Stimulation with anti-CD3 appreciably up-regulated expression of GXII PLA(2)s and group V PLA(2) by steady state analysis of the Th2 cells as compared with type 1 helper T cells. These results suggest that group XII and group V PLA(2)s might participate in helper T cell immune response through release of immediate second signals and generation of downstream eicosanoids.
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PMID:A novel group of phospholipase A2s preferentially expressed in type 2 helper T cells. 1127 38

A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.
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PMID:Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding. 1129 44

Crotoxin B, the basic Asp49-PLA(2) subunit from crotoxin, the main component of Crotalus durissus terrificus venom, displays myotoxic, edema-inducing, bactericidal (upon Escherichia coli), liposomal-disrupting and anticoagulant activities. Chemical modifications of His (with 4-bromophenacyl bromide, BPB), Tyr (with 2-nitrobenzenesulphonyl fluoride, NBSF), Trp (with o-nitrophenylsulphenyl chloride, NPSC) and Lys (with acetic anhydride) residues of this protein, in addition to cleavage with cyanogen bromide (CNBr) and inhibition with ethylenediaminetetraacetic acid (EDTA), were carried out in order to study their effects on enzymatic and pharmacological activities. Lethality was reduced after modification of His or Lys residues, as well as after cleavage with CNBr, while enzymatic activity was completely abolished after modification of His or incubation with EDTA. Modification of Lys or Tyr, or cleavage with CNBr, partially reduced enzymatic activity. Anticoagulant activity was modified similarly to enzymatic activity, evidencing the dependency of this pharmacological effect on catalytic activity. Myotoxicity was reduced after modification of His or Lys, as well as after cleavage with CNBr, whereas EDTA reduced this effect to a lesser extent. Bactericidal effect was significantly reduced only after modification of Lys and after cleavage with CNBr. Edema-inducing activity was partially inhibited after treatment with EDTA and strongly reduced after acetylation of Lys residues and cleavage with CNBr, being only partially reduced after His alkylation. On the other hand, liposome disrupting activity was only partially reduced after modification of His and Tyr or after cleavage with CNBr. Modification of Trp residue partially reduced lethality and myotoxicity but did not affect enzymatic or anticoagulant activities. These data indicate that enzymatic activity is relevant for some pharmacological effects induced by crotoxin B (mainly lethal, myotoxic and anticoagulant activities), and also evidence that this subunit of crotoxin displays regions different from the active catalytic site which are involved in some of the toxic and pharmacological effects induced by this phospholipase A(2).
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PMID:Effects of chemical modifications of crotoxin B, the phospholipase A(2) subunit of crotoxin from Crotalus durissus terrificus snake venom, on its enzymatic and pharmacological activities. 1146 30

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

Nanoparticles were formed by the complexation of poly(L-arginine) (PLA), poly(L-histidine) (PLH) and poly(L-lysine) (PLL) with dodecanoic acid (C12). Dynamic light scattering, zeta potential measurements, atomic force microscopy, fluorescence, and circular dichroism spectroscopy were used for their characterization. It was found that the diameters of the poly(L-arginine) dodecanoate (PLA-C12), poly(L-histidine) dodecanoate (PLH-C12), and poly(L-lysine) dodecanoate (PLL-C12) complex nanoparticles were in the range 120-200 nm. Furthermore, the pH-sensitive dissolution and the surface charges can be adjusted by choosing PLA, PLH and PLL. The particle stability against basic pH values increases with increasing pK(a) value of the poly(amino acid) in the series PLH-C12, PLL-C12 and PLA-C12. The particles as such show a core-shell morphology. Their cores are formed by stoichiometric poly(amino acid) dodecanoate complexes while the shells stabilizing the particles are formed by cationic poly(amino acid) chains in an uncomplexed state. The particles were tested as containers for hydrophobic molecules such as pyrene, which served as a fluorescence probe for measuring the polarity within the particles, and Q(10) which functioned as a model drug. The maximum uptake of Q(10) into the nanoparticles is about 13% (w/w), thereby making the complexes attractive as simple drug carriers for controlled release purposes. Circular dichroism measurements revealed that the poly(amino acid) chains of PLA-C12 and PLL-C12 adopt predominantly an alpha-helix and that of PLH-C12 a beta-sheet.
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PMID:pH-sensitive nanoparticles of poly(amino acid) dodecanoate complexes. 1167 52

A formaldehyde-mediated aggregation pathway (FMAP) has been shown to be primarily responsible for the solid-state aggregation of lyophilized formalinized protein antigens [e.g., tetanus toxoid (TT) and formalinized bovine serum albumin (f-BSA)] in the presence of moisture and physiological temperature. Coincorporation of the formaldehyde-interacting amino acid, histidine, strongly inhibits the FMAP. The purpose of this study was to test whether previous solid-state data are applicable toward the stabilization of formalinized antigens encapsulated in poly(lactide-co-glycolide) (PLGA)-based microspheres. Formaldehyde-treated bovine serum albumin (f-BSA) and BSA were selected as a model formalinized protein antigen and a nonformalinized control, respectively. As in the solid state, we found that the FMAP was dominant in the aggregation of f-BSA encapsulated in PLGA 50/50 microspheres, whereas the aggregation mechanism of encapsulated BSA was mostly converted from thiol-disulfide interchange to an acid-catalyzed noncovalent pathway. The lack of noncovalent aggregation in encapsulated f-BSA could be explained by its higher thermodynamic stability after formalinization, which inhibits protein unfolding. Targeting the FMAP, coencapsulation of histidine and trehalose successfully inhibited the aggregation of f-BSA in microspheres. By combining the use of an optimized oil-in-oil (o/o) encapsulation method, coencapsulation of histidine and trehalose, and use of low-acid-content poly(D,L-lactide) (PLA) and poly(ethylene glycol) (PEG) blends, a 2-month continuous release of f-BSA was achieved with the absence of aggregation.
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PMID:Stabilization of a model formalinized protein antigen encapsulated in poly(lactide-co-glycolide)-based microspheres. 1174 14

Bothropstoxin-I (BthTx-I) is a myotoxic phospholipase A(2) variant present in the venom of Bothrops jararacussu, in which the Asp(49) residue is replaced with a lysine, which damages artificial membranes by a Ca(2+)-independent mechanism. Wild-type BthTx-I and the mutants Lys(49)-->Asp, His(48)-->Gln and Lys(122)-->Ala were expressed in Escherichia coli BL21(DE3) cells, and the hydrolytic, myotoxic and membrane-damaging activities of the recombinant proteins were compared with native BthTx-I purified from whole venom. The Ca(2+)-independent membrane-damaging and myotoxic activities of the native and wild-type recombinant BthTx-I, His(48)Gln and Lys(49)Asp mutants were similar; however, the Lys(122)Ala mutant demonstrated reduced levels of both activities. Although a low hydrolytic activity against a mixed phospholipid substrate was observed with native BthTx-I, no substrate hydrolysis was detected with the wild-type recombinant enzyme or any of the mutants. In the case of the Lys(49)Asp mutant, this demonstrates that the absence of catalytic activity in Lys(49)-PLA(2) is not a consequence of the single Asp(49)-->Lys replacement. Furthermore, these results provide unambiguous evidence that the Ca(2+)-independent membrane-damaging and myotoxic activities are maintained in the absence of hydrolysis. The evidence favours a model for a hydrolysis-independent, membrane-damaging mechanism involving an interaction of the C-terminal region of BthTx-I with the target membrane.
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PMID:Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis. 1182 43

Apolipoprotein(a) [apo(a)] is the distinctive glycoprotein of lipoprotein Lp(a), which is disulfide linked to the apo B100 of a low density lipoprotein particle. Apo(a) possesses a high degree of sequence homology with plasminogen, the precursor of plasmin, a fibrinolytic and pericellular proteolytic enzyme. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen-like kringle 4 and single copies of kringle 5, and the protease region including the backbone positions for the catalytic triad (Ser, His, Asp). A lysine-binding site that is similar to that of plasminogen kringle 4 is present in apo(a) kringle IV type 10. These kringle motifs share some amino acid residues (Asp55, Asp57, Phe64, Tyr62, Trp72, Arg71) that are key components of their lysine-binding site. The spatial conformation and the function of this site in plasminogen kringle 4 and in apo(a) kringle IV-10 seem to be identical as indicated by (i) the ability of apo(a) to compete with plasminogen for binding to fibrin, and (ii) the neutralisation of the lysine-binding function of these kringles by a monoclonal antibody that recognises key components of the lysine-binding site. In contrast, the lysine-binding site of plasminogen kringle 1 contains a Tyr residue at positions 64 and 72 and is not recognised by this antibody. Plasminogen bound to fibrin is specifically recognised and cleaved by the tissue-type plasminogen activator at Arg561-Val562, and is thereby transformed into plasmin. A Ser-Ile substitution at the activation cleavage site is present in apo(a). Reinstallation of the Arg-Val peptide bond does not ensure cleavage of apo(a) by plasminogen activators. These data suggest that the stringent specificity of tissue-type plasminogen activator for plasminogen requires molecular interactions with structures located remotely from the activation disulfide loop. These structures ensure second site interactions that are most probably absent in apo(a).
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PMID:Apolipoprotein(a): structure-function relationship at the lysine-binding site and plasminogen activator cleavage site. 1192 26

Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300-->His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by approximately 600 microg/kg M5 versus approximately 1200 microg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.
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PMID:Prourokinase mutant that induces highly effective clot lysis without interfering with hemostasis. 1196 67

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.
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PMID:Enzymatic properties of human cytosolic phospholipase A(2)gamma. 1203 69


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