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)

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.
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PMID:Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution. 1554 28

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.
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PMID:Solid phase peptide synthesis on epoxy-bearing methacrylate monoliths. 1563 24

The patient was a 34-year-old man with life-long bleeding episodes, whose hemorrhage problem was characterized predominantly by prolonged bleeding at surgical or traumatic sites. All routine coagulation parameters were within normal ranges. The patient's bleeding tendency was not caused by factor XIII deficiency, alpha2-antiplasmin deficiency, or tissue type-plasminogen activator increase. His characteristic abnormalities of fibrinolysis included shortened euglobulin clot lysis time, low plasminogen activator inhibitor-1 (PAI-1) activity and antigen in plasma, which were remarkably reduced to only about 10% of control. An operation was performed in order to clear two hematomas in the patient's left leg and hip, and subsequent bleeding episodes were well controlled with adjuvant administration of intravenous aminomethylbenoic acid after surgery. PAI-1 gene analysis by polymerase chain reaction product sequencing revealed that the patient had a heterozygous missense mutation G to A transition at nucleotide position 4497 in exon 2, causing replacement of alanine 15 (GCC) to threonine (ACC) at signal peptide. The restriction endonuclease analysis showed that this gene mutation also existed in the patient's father, but not in his mother and 60 normal subjects. The wild-type and mutant plasmids were constructed and transiently transfected into Chinese hamster ovary cell lines; the levels of PAI-1 activity and antigen in the media of the mutant were approximately 70% of the wild type, and the levels of PAI-1 protein in cell lysates were almost equal in wild-type and mutant plasmids. These results indicate that the mutation in signal peptide may partly impair the secretion of PAI-1.
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PMID:A case of deficiency of plasma plasminogen activator inhibitor-1 related to Ala15Thr mutation in its signal peptide. 1565 May 51

Yersinia pestis plasminogen activator (Pla), a surface virulence factor contributes to the highly invasive nature of the pathogen by binding various tissue matrix components. In this study we characterised the laminin-binding site(s) of Pla via phage display and alanine-scanning mutagenesis. Previously we isolated 18 different heptamer peptide sequences from a phage display library with biopanning on laminin, and have shown that two of them with sequences of WSLLTPA or YPYIPTL completely inhibited laminin binding of a Pla-expressing recombinant Escherichia coli strain. These phages themselves strongly bound laminin in an ELISA assay utilising horseradish peroxidase-labelled anti-M 13 antibody. In the present study, with the application of synthetic peptides, a 55% and a 33% inhibition was demonstrated using WSLLTPA and YPYIPTL, respectively. Peptide pattern alignment showed two homologous regions for WSLLTPA and one for YPYIPTL inside the Pla sequence. Amino acids were changed for alanine in one of the WSLLTPA regions and in the YPYIPTL region in order to prove the role of the LTP/PTL motifs in laminin binding. Of the four constructed mutants, the triple mutant L65A-T66A-L67A in the WSLLTPA region and the double mutant G178A-L179A in the YPYIPTL region decreased the laminin binding capacity of the Pla-expressing recombinant E. coli by about 50%.
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PMID:Identification of laminin-binding motifs of Yersinia pestis plasminogen activator by phage display. 1596 69

Previous studies have suggested that thrombin interacts with integrins in endothelial cells through its RGD (Arg-187, Gly-188, Asp-189) sequence. All existing crystal structures of thrombin show that most of this sequence is buried under the 220-loop and therefore interaction via RGD implies either partial unfolding of the enzyme or its proteolytic digestion. Here, we demonstrate that surface-absorbed thrombin promotes attachment and migration of endothelial cells through interaction with alpha(v)beta(3) and alpha(5)beta(1) integrins. Using site-directed mutants of thrombin we prove that this effect is mediated by the RGD sequence and does not require catalytic activity. The effect is abrogated when residues of the RGD sequence are mutated to Ala and is not observed with proteases like trypsin and tissue-type plasminogen activator, unless the RGD sequence is introduced at position 187-189. The potent inhibitor hirudin does not abrogate the effect, suggesting that thrombin functions through its RGD sequence in a non-canonical conformation. A 1.9-Angstroms resolution crystal structure of free thrombin grown in the presence of high salt (400 mm KCl) shows two molecules in the asymmetric unit, one of which assumes an unprecedented conformation with the autolysis loop shifted 20 Angstroms away from its canonical position, the 220-loop entirely disordered, and the RGD sequence exposed to the solvent.
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PMID:Thrombin functions through its RGD sequence in a non-canonical conformation. 1599 37

The charge of Lys300(c143) located within a flexible loop(297-313) of sc-uPA has been identified as an important determinant for its high intrinsic activity. Mutations affecting the flexibility of the loop also modulate the intrinsic activity. Glu-plasminogen activation by sc-uPA is strongly promoted by fibrin fragment E but not fibrin fragment D-dimer, whereas plasminogen activation by t-PA is strongly promoted by fragment D-dimer but not fragment E. To further investigate the effect of conformation changes in the flexible loop on catalytic properties of sc-uPA, cassette mutations at Pro309(c152) were made and characterized. It was found that the activation of Pro309(c152) mutants by Lys-plasmin was only moderately affected. In contrast, the intrinsic and two-chain activities of Pro309(c152) mutants against S2444 were both significantly decreased. The two-chain activities of these mutants against Glu-plasminogen were also reduced in a range of 1.1- to 127-fold. The mutations of Pro309(c152) to Trp/Phe and Arg/Asp more significantly affected both intrinsic and two-chain activities, while only a moderate decrease in activity was found with mutations to Ala/Ser/Thr. In contrast to wild-type sc-uPA, plasminogen activation by Pro309(c152) mutants was found to be promoted by both fibrin fragment E and D-dimer. In the presence of 2.0 microM D-dimer, plasminogen activation by mutant Pro309(c152) --> His was promoted by 22-fold, while only 2.0-fold promotion was found with mutant Pro309(c152) --> Gly. In conclusion, these findings demonstrated that conformation changes in the flexible loop of sc-uPA not only affect its intrinsic and two-chain activity, but also extend its promotion of plasminogen activation by fragment E to D-dimer.
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PMID:Mutagenesis at Pro309 of single-chain urokinase-type plasminogen activator alters its catalytic properties. 1623 30

Phospholipases A(2)s (PLA(2)s) are widely distributed in mammals and snake venoms. They catalyze the production of arachidonic acid from membrane phospholipids leading to the bioynthesis of pro-inflammatory eicosanoids. A peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) was designed and synthesized as a specific inhibitor of PLA(2). It was shown earlier that the peptide bound to group II PLA(2) specifically and had a dissociation constant (K(d)) of 8.8 x 10(-9) M. In the present studies for the binding of LAIYS with a group I PLA(2) from Naja naja sagittifera using surface plasmon resonance the dissociation constant was found to be 4.5 x 10(-5) M which is considerably lower than the value found for the group II PLA(2). In order to determine the details of binding at the molecular level, a group I PLA(2) from the venom of Naja naja sagittifera was crystallized with peptide LAIYS. The crystal structure showed the presence of LAIYS at the substrate-binding site but has fewer interactions than those observed with group II PLA(2) from Daboia russelli pulchella. The observed difference in the binding affinity is caused primarily due to poor fitting of the peptide LAIYS in the binding site of group I PLA(2). Apparently, the location of Trp 19 in group I PLA(2) is not favourable for the binding of LAIYS. The two complexes also differ drastically in the formation of intermolecular interactions. In the present structure, the side chain of Ser (P) interacts with His 48 and Asp 49 while in the complex with group II PLA(2) it was Tyr (P) OH that formed the corresponding interactions. Tyr (P) in group I PLA(2) is the main contributor of the hydrophobic interactions whereas in the complex of LAIYS with group II PLA(2) it was the peptide segment Leu-Ala-Ile that produced the bulk of hydrophobic forces. The structures further showed that the peptide LAIYS was fully inside the substrate-binding region of the group II PLA(2) while a significant portion of the peptide LAIYS was hanging outside the surface of the group I PLA(2). The buried area in the complex with group II PLA(2) was 811 A(2) whereas, the corresponding area in group I PLA(2) was 449 A(2). This shows that the peptide LAIYS is very compatible with the substrate-binding site of group II PLA(2) and rather poorly fits into the substrate-binding site of group I PLA(2). This indicates that a highly specific ligand for one form of PLA(2) may be a poor partner for another form of enzyme.
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PMID:Crystal structure of the complex of group I PLA2 with a group II-specific peptide Leu-Ala-Ile-Tyr-Ser (LAIYS) at 2.6 A resolution. 1627 56

The purpose of this study was to examine the effects of 28 days of beta-alanine (b-Ala) and creatine monohydrate (CrM) supplementation on the onset of neuromuscular fatigue by using the physical working capacity at neuromuscular fatigue threshold (PWC(FT)) test in untrained men. Fifty-one men (mean age +/- SD = 24.5 +/- 5.3 years) volunteered to participate in this 28-day, double-blind, placebo-controlled study and were randomly assigned to 1 of 4 groups: placebo (PLA; 34 g dextrose; n = 13), CrM (5.25 g CrM plus 34 g dextrose; n = 12), b-Ala (1.6 g b-Ala plus 34 g of dextrose; n = 12), or b-Ala plus CrM (CrBA; 5.25 g CrM plus 1.6 g b-Ala plus 34 g dextrose; n = 14). The supplement was ingested 4 times per day for 6 consecutive days, then twice per day for 22 days before posttesting. Before and after the supplementation, subjects performed a continuous incremental cycle ergometry test while a surface electromyographic signal was recorded from the vastus lateralis muscle to determine PWC(FT). The adjusted mean posttest PWC(FT) values (covaried for pretest PWC(FT) values) for the b-Ala and CrBA groups were greater than those for the PLA group (p < or = 0.05). However, there were no differences between the CrM vs. PLA, CrBA vs. b-Ala, CrM vs. b-Ala, or CrM vs. CrBA groups (p > 0.05). These findings suggested that b-Ala supplementation may delay the onset of neuromuscular fatigue. Furthermore, there appeared to be no additive or unique effects of CrM vs. b-Ala alone on PWC(FT).
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PMID:Effects of twenty-eight days of beta-alanine and creatine monohydrate supplementation on the physical working capacity at neuromuscular fatigue threshold. 1719 55

Staphylokinase (SAK) expresses plasminogen activator (PA) activity by forming a complex with plasmin. The interaction between the plasmin-SAK complex and plasminogen was investigated using synthesized peptides, which were constructed according to the amino acid sequence of the SAK molecule. A synthetic nonadecapeptide (SAK22-40) corresponding to Glu22-Leu40 by the SAK molecule enhanced the activation of Glu-plasminogen by the plasmin-SAK complex. Analysis of IAsys resonant mirror biosensor showed that SAK22-40 bound to Glu-plasminogen. This binding was completely inhibited by IgG against the B-chain in the plasminogen molecule. But, this binding was not inhibited by IgG against lysine-binding sites (LBS) of the A-chain in the plasminogen molecule. The substitution of Lys35 with Ala in SAK22-40 did not enhance the activation of Glu-plasminogen by the plasmin-SAK complex. When SAK22-40 was administrated in a mouse thrombosis model, earlier recanalization was observed than in mice with vehicle administration. Thus, a newly synthesized peptide, SAK22-40 enhanced Glu-plasminogen activation and induced effective thrombolysis.
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PMID:Effect of staphylokinase-derived nonadecapeptide on the activation of plasminogen. 1747 90

A novel serine protease with fibrinolytic activity named CSP was purified from the culture supernatant of the fungus Cordyceps sinensis, a kind of Chinese herbal medicine. Analysis of the purified enzyme by SDS-PAGE indicated that CSP was a single polypeptide chain with an apparent molecular weight of 31 kDa, and N-terminal sequencing revealed that the first ten amino acid residues of the enzyme were Ala-Leu-Ala-Thr-Gln-His-Gly-Ala-Pro-Trp-. When casein was used as a substrate, the proteolytic activity of CSP reached its maximum at pH 7.0 and 40 degrees C. The effect of chemical agents on the enzyme activity indicated that CSP is a serine protease with a free cysteine residue near the active site. It hydrolysed fibrinogen, fibrin and casein with a high efficiency, while hydrolysing bovine serum albumin (BSA) and human serum albumin (HSA) to a lesser extent. CSP was found to be a plasmin-like protease, but not a plasminogen activator, and it preferentially cleaved the A alpha chain of fibrinogen and the alpha-chain of fibrin. Therefore, the extracellular protein CSP may represent a potential new therapeutic agent for the treatment of thrombosis.
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PMID:A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization. 1766 28


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