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)

Bovine pancreatic PLA(2) (phospholipase A(2)) is a 14 kDa protein whose structure is highly cross-linked by seven disulphide bonds. We investigated the structural stability of this enzyme by the method of 'disulphide-scrambling' with denaturants such as urea, GdmCl (guanidine hydrochloride), GdmSCN (guanidine thiocyanate) and at high temperatures in the presence of 2-mercaptoethanol (0.2 mM) as thiol initiator. Reverse-phase HPLC was used to follow denaturation. To denature 50% of the native protein, 1.25 M GdmSCN, approx. 3 M GdmCl and higher than 8 M urea were required. Only 20% of the protein was denatured after 2 h at 60 degrees C, whereas complete denaturation was seen after 2 h at 70 degrees C and within 30 min at 80 degrees C. A distinct enhancement of stability was observed when denaturation was conducted in the presence of 10 mM calcium chloride, which has not been reported previously. CD studies of GdmCl denaturation of bovine PLA(2) showed that 2.5 M GdmCl was required to denature 50% of the protein in the presence of 0.2 mM 2-mercaptoethanol (in agreement with the HPLC analysis), whereas 6.4 M GdmCl was necessary to denature 50% of the protein in the absence of a thiol initiator. Conformational stability (Delta G (water)) was estimated to be 8.7 kcal/mol (1 cal=4.184 J) by 'disulphide-intact' denaturation (where 'native' disulphide framework was unaffected) and 2.5 kcal/mol by 'disulphide-scrambling' denaturation (involved breaking of native disulphides and formation of 'non-native' ones). The difference, Delta(Delta G (water)), of 6.2 kcal/mol was the conformational stability contributed by the 'native-framework' of seven disulphides. Using bovine PLA(2) as an example, we have demonstrated a novel comparative technique, where the conformational stability study of a disulphide-containing protein, with a common denaturant, in both the presence and absence of catalytic amounts of a thiol initiator can be used as a convenient method to estimate selectively and quantitatively the actual contribution of the 'native disulphide bond network' towards the global conformational stability of the protein.
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PMID:Investigating conformational stability of bovine pancreatic phospholipase A2: a novel concept in evaluating the contribution of the 'native-framework' of disulphides to the global conformational stability of proteins. 1453 80

The purpose of this study was to design and characterize a zero-order bioresorbable reservoir delivery system (BRDS) for diffusional or osmotically controlled delivery of model drugs including macromolecules. The BRDS was manufactured by casting hollow cylindrical poly (lactic acid) (PLA): polyethylene glycol (PEG) membranes (10 x 1.6 mm) on a stainless steel mold. Physical properties of the PLA:PEG membranes were characterized by solid-state thermal analysis. After filling with drug (5 fluorouracil [5FU] or fluorescein isothiocyanate [FITC]-dextran:mannitol, 5:95 wt/wt mixture) and sealing with viscous PLA solution, cumulative in vitro dissolution studies were performed and drug release monitored by ultraviolet (UV) or florescence spectroscopy. Statistical analysis was performed using Minitab (Version 12). Differential scanning calorimetry thermograms of PLA:PEG membranes dried at 25 degrees C lacked the crystallization exotherms, dual endothermal melting peaks, and endothermal glass transition observed in PLA membranes dried at -25 degrees C. In vitro release studies demonstrated zero-order release of 5FU for up to 6 weeks from BRDS manufactured with 50% wt/wt PEG (drying temperature, 25 degrees C). The release of FITC dextrans of molecular weights 4400, 42 000, 148 000, and 464 000 followed zero-order kinetics that were independent of the dextran molecular weight. When monitored under different concentrations of urea in the dissolution medium, the release rate of FITC dextran 42 000 showed a linear correlation with the calculated osmotic gradient(DeltaPi). This study concludes that PEG inclusion at 25 degrees C enables manufacture of uniform, cylindrical PLA membranes of controlled permeability. The absence of molecular weight effects and a linear dependence of FITC-dextran release rate on DeltaPi confirm that the BRDS can be modified to release model macromolecules by an osmotically controlled mechanism.
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PMID:A bioresorbable, polylactide reservoir for diffusional and osmotically controlled drug delivery. 1472 94

A post-synaptic neurotoxic phospholipase A(2) (PLA(2)) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD(50) 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA(2) by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A(2) activity of NN-XIa-PLA(2,) isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA(2): WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA(2) isoforms from the venom to varying extent. The interaction of the WSG with the PLA(2) is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA(2) using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA(2) has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant.
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PMID:Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera. 1649 89

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 M: guanidine hydrochloride (Gdm.HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm.HCl with a G25 column and simultaneously dissolved in 8 M: urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding.
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PMID:A comparative investigation on different refolding strategies of recombinant human tissue-type plasminogen activator derivative. 1661 26

A secreted phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber A-2688, previously identified by us, is the first PLA(2) identified in prokaryotes. Genome sequence data of Streptomyces coelicolor A3(2) indicates that the bacterium carries two genes encoding hypothetical PLA(2)s, which exhibit 100 and 78% identity, respectively, to the S. violaceoruber PLA(2). In this study, we named the former and latter proteins as the first and second PLA(2)s, respectively. When the second PLA(2) was expressed in Escherichia coli cells, it formed an inclusion body. The present study demonstrates a method to purify it to homogeneity without the disappearance of the enzymatic activity: the inclusion body was washed with sodium deoxycholate and dissolved in the presence of 2 M urea at pH 12, then refolded by the dilution method. The refolding of enzyme was confirmed by the circular dichroism spectrum. The second PLA(2) purified to homogeneity had the same specific activity as that of the S. violaceoruber PLA(2) and the yield was approximately 6.8 mg/L culture. The second PLA(2) exhibits similar enzymatic properties to the S. violaceoruber PLA(2), except that the former enzyme does not utilize phophatidic acid as a substrate. The surface electrostatic potential of the S. coelicolor PLA(2) model, which is created by the computer-homology modeling, suggests that the positively charged surface of the enzyme does not affect the substrate specificity.
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PMID:Purification and characterization of the second Streptomyces phospholipase A2 refolded from an inclusion body. 1680 51

The fusion protein of enterokinase light chain, DsbA-rEKL, was expressed mainly in inclusion body in E. coli. The recombinant bacteria was fermented to high density, with high expression of the fusion protein. After being washed with 0.5% Triton X-100 and 4mol/L urea, the inclusion body was dissolved in 6mol/L guanidine and 100mmol/L DTP, derivatized by cystine and refolded by pulse refolding. The strategy of pulse refolding involved the addition of 0.03mg/mL of fusion protein until its final concentration reached 0.3mg/mL. The refolded protein was autocleaved and the active EKL molecule was released after adding 2mmol/L CaCl2. Using the two-step purification processes of IDA-Sepharose chromatography and Q-Sepharose chromatography, the purity of rEKL was found to be above 95%, with a high activity to cleave the recombinant reteplase fusion protein Trx-rPA. The yield of purified rEKL was more than 60mg/L of cultures. As a result, the therapeutic proteins like rPA could be produced on a large-scale in a way such as expressed in the form of fusion proteins.
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PMID:[Refolding of the fusion protein of recombinant enterokinase light chain rEKL]. 1703 7

Streptococcus agalactiae is an etiological agent of several infective diseases in humans. We previously demonstrated that FbsA, a fibrinogen-binding protein expressed by this bacterium, elicits a fibrinogen-dependent aggregation of platelets. In the present communication, we show that the binding of FbsA to fibrinogen is specific and saturable, and that the FbsA-binding site resides in the D region of fibrinogen. In accordance with the repetitive nature of the protein, we found that FbsA contains multiple binding sites for fibrinogen. By using several biophysical methods, we provide evidence that the addition of FbsA induces extensive fibrinogen aggregation and has noticeable effects on thrombin-catalyzed fibrin clot formation. Fibrinogen aggregation was also found to depend on FbsA concentration and on the number of FbsA repeat units. Scanning electron microscopy evidentiated that, while fibrin clot is made of a fine fibrillar network, FbsA-induced Fbg aggregates consist of thicker fibers organized in a cage-like structure. The structural difference of the two structures was further indicated by the diverse immunological reactivity and capability to bind tissue-type plasminogen activator or plasminogen. The mechanisms of FbsA-induced fibrinogen aggregation and fibrin polymerization followed distinct pathways since Fbg assembly was not inhibited by GPRP, a specific inhibitor of fibrin polymerization. This finding was supported by the different sensitivity of the aggregates to the disruptive effects of urea and guanidine hydrochloride. We suggest that FbsA and fibrinogen play complementary roles in contributing to thrombogenesis associated with S. agalactiae infection.
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PMID:Multiple interactions of FbsA, a surface protein from Streptococcus agalactiae, with fibrinogen: affinity, stoichiometry, and structural characterization. 1704 2

In snake venoms, non-covalent protein-protein interaction leads to protein complexes with synergistic and, at times, distinct pharmacological activities. Here we describe a new protein complex containing phospholipaseA(2) (PLA(2)), protease, and a trypsin inhibitor. It is isolated from the venom of Daboia russelii by gel permeation chromatography, on a Sephadex G-75 column. This 44.6 kDa complex exhibits only phospholipase A(2) activity. In the presence of 8M urea it is well resolved into protease (29.1 kDa), PLA(2) (13 kDa), and trypsin inhibitor (6.5 kDa) peaks. The complex showed an LD(50) of 5.06 mg/kg body weight in mice. It inhibited the frequency of spontaneous release of neurotransmitter in hippocampal neurons. It also caused peritoneal bleeding, and edema in the mouse foot pads. Interestingly, the complex caused degeneration of both the germ cells and the mouse Leydig cells of mouse testis. A significant reduction in both the diameter of the seminiferous tubules and height of the seminiferous epithelia were observed following intraperitoneal injection of the sub-lethal dose (3 mg/kg body weight). This effect of the toxin is supported by the increase in the activities of acid and alkaline phosphatases and the nitric oxide content in the testes, and a decrease in the ATPase activity. Because of its potent organ atrophic effects on the reproductive organs, the toxin is named "Reprotoxin". This is the first report demonstrating toxicity to the reproductive system by a toxin isolated from snake venom.
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PMID:Isolation and characterization of "Reprotoxin", a novel protein complex from Daboia russelii snake venom. 1857 7

This study is to investigate the long-term effects of PEG-PLA nano artificial cells containing hemoglobin (NanoRBC) on renal function and renal histology after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: NanoRBC in Ringer lactate, Ringer lactate, stroma-free hemoglobin (SFHB), polyhemoglobin (PolyHb), autologous rat whole blood (rat RBC). Blood samples were taken before infusions and on days 1, 7 and 21 after infusions for biochemistry analysis. Rats were sacrificed on day 21 after infusions and kidneys were excised for histology examination. Infusion of SFHB induced significant decrease in renal function damage evidenced by elevated serum urea, creatinine and uric acid throughout the 21 days. Kidney histology in SFHb infusion group revealed focal tubular necrosis and intraluminal cellular debris in the proximal tubules, whereas the glomeruli were not observed damaged. In all the other groups, NanoRBC, PolyHb, Ringer lactate and rat RBC, there were no abnormalities in renal biochemistry or histology. In conclusion, injection of NanoRBC did not have adverse effects on renal function nor renal histology.
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PMID:Effects of PEG-PLA-nano artificial cells containing hemoglobin on kidney function and renal histology in rats. 1897 92

The aim of this study is to investigate the effect of poly(D, L-lactic acid) (PLA) nanoparticles as triptolide carrier on abating renal toxicity for Sprague Dawley rats after oral administration. Triptolide has severe toxicities on digestive, urogenital and blood circulatory system. High-resolution 600-MHz 1H-nuclear magnetic resonance (1H-NMR)-based metabolic analysis was performed on urine samples obtained from five groups of Sprague Dawley rats administrated with free triptolide and triptolide-loaded PLA nanoparticles at day 5, 10 and 15. The relative concentrations of biomarkers for renal lesion caused by triptolide were determined by 1H-NMR. The disorder of metabolism was characterized by the exceptional changes of the relative concentrations of succinate, 2-oxoglutarate and citrate. Similarly, the renal lesion was characterized by an increase of the relative concentrations of trimethylamine N-oxide and dimethylglycine, and a decrease of that of urea and allantoin. These results revealed that triptolide-loaded PLA nanoparticles might abate the renal toxicity of triptolide in comparison with identical doses of the free drug. The higher the dose (0.6 mg/kg), the more pronounced was this trend during long-term application. These results were further confirmed by histopathological changes. These results indicated that PLA nanoparticles provided a promising new formulation to abate the renal toxicity caused by triptolide.
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PMID:Effect of poly(D, L-lactic acid) nanoparticles as triptolide carrier on abating rats renal toxicity by NMR-based metabolic analysis. 1905 1


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