Inhibitory Effect Of EDTA Research Articles

Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

Blood antimicrobial activity varies against different Mycobacterium spp.

In vitro analysis of mycobacterial pathogenicity or host susceptibility has traditionally relied on the infection of macrophages, the target cell of mycobacteria, despite difficulties reproducing their antimycobacterial activity. We have employed alternative models, namely whole blood and leukocytes in plasma, from QuantiFERON negative individuals, and performed infections with the pathogenic M. tuberculosis, the less pathogenic M. avium, M. kansasii and M. chelonae and the occasionally pathogenic M. gordonae and M. bovis. The anticoagulant used in blood extraction, heparin or EDTA, had a major influence in the outcome of the infection. Thus, while in the heparinized models a similar number of bacteria were enumerated in the inoculum and after seven days, in the presence of EDTA a killing effect was observed, despite the inhibitory effect of EDTA on cellular functions like the production of cytokines or reactive oxygen species (ROS). A special case was the rapidly growing mycobacteria M. chelonae, that multiplied in heparinized models but was eliminated in models with EDTA. We verified that EDTA is not responsible for the bactericidal effect, but acts as a bacteriostatic agent. Further work will determine whether blood derived models are a better alternative to the classical macrophage.

Purification and optimization of conditions for DNA polymerase isolated from thermophile bacteria Bacillus caldolyticus

Тhermophilic bacteria Bacillus caldolyticus isolated from the hot spring in Bansko, Republic of Macedonia, were used for the isolation of DNA polymerase. Bacterial cells were disrupted by sonication and the first step in the purification of DNA polymerase was 40% ammonium sulfate precipitation. This was followed by chromatographic procedures on Sephadex G-50, DE-52 and CM-52 cellulose. DNA polymerase activity was analyzed at each step of purification using the incorporation of 3H dATP in activated calf thymus DNA. The purity of the DNA polymerase was analyzed on SDS PAGE. Optimal conditions of activity were determined for temperature, pH, dNTP and Mg++ concentration. In addition, the effect of ethanol and EDTA as possible inhibitors of polymerase activity was also analyzed. The optimal temperature of DNA polymerase was 66 ºC; the optimal pH was 7.2, optimal MgCl2 concentration was 2.5 mM, and the optimal substrate concentration was 2.5 × 10–6 M dNTP. The inhibitory effect of EDTA and ethanol on DNA polymerase was above 10 mM and 10%, respectively.

Catalytic, kinetic and thermodynamic properties of Bacillus pumilus FH9 keratinase conjugated with activated pectin

Bacillus pumilus FH9 keratinase was covalently coupled to several oxidized polysaccharides. The conjugates were evaluated for the retained activity, kinetic and thermodynamic stability. Among all preparations, the conjugated enzyme with oxidized pectin had the highest recovered activity (71.75%) and the highest thermal stability at 60°C (t1/2=333min). Compared to the native enzyme, the conjugated preparation exhibited higher optimum temperature, lower activation energy (Ea), lower deactivation constant rate (kd), higher t1/2, and higher decimal reduction time values (D) within the temperature range of 50–80°C. The thermodynamic parameters (ΔH*, ΔG*, ΔS*) of irreversible thermal denaturation for the native and conjugated keratinase were also evaluated. The values of enthalpy of activation (ΔH*), free energy of activation (ΔG*), and free energy of transition state binding (ΔG*E−T) for keratin hydrolysis were lower for the conjugated enzyme. Moreover, there was highly significant impact on improving the values of Vmax/Km, kcat, kcat/Km, and ΔG*E−S for the modified enzyme. Both native and conjugated enzymes were slightly activated by CaCl2 and MgCl2. However, the inhibitory effects of EDTA, HgCl2 and ZnSO4 were more pronounced with the native enzyme.

The Platelet Integrin alphaIIbbeta3 Differentially Interacts with Fibrin and Fibrinogen

Fibrinogen binding to the integrin αIIbβ3 can mediate platelet aggregation and platelet spreading on fibrinogen-coated surfaces. However, in vivo, where blood clotting is catalyzed by thrombin, αIIbβ3 activation and fibrinogen conversion to fibrin occur simultaneously, although the relative contributions of fibrinogen versus monomeric and polymerized fibrin to αIIbβ3-mediated platelet aggregation and adhesion are unknown. The interaction of αIIbβ3 with fibrin is responsible for clot retraction. Nonetheless, differences in the efficacy of αIIbβ3 antagonists with regard to clot retraction versus platelet aggregation suggest that the interaction of αIIbβ3 with fibrinogen and fibrin is different. Here, we have compared nanomechanical measurements of the interaction of αIIbβ3 with fibrin and fibrinogen to explain these differential effects. Briefly, a microscopic bead covalently coated with purified αIIbβ3 was captured by a focused laser beamand repeatedly brought into contact with surface-attached fibrinogen, monomeric fibrin produced by treating the immobilized fibrinogen with thrombin, or a naturally-formed hydrated fibrin fiber at the edge of a plasma clot. When an αIIbβ3-ligand complex was detected, the force required to dissociate the complex was measured at piconewton resolution. By analyzing the distribution of these forces, we determined the overall reactivity and the binding strength of the interacting protein pairs. Besides native fibrinogen, experiments were performed with recombinant fibrinogen variants. Rupture force histograms for αIIbβ3-fibrinogen interactions displayed forces ranging from 20 pN to 140 pN and could be segregated into moderate (20-60 pN) and strong (>60 pN) interactions. The inhibitory effects of EDTA and αIIbβ3antagonists such as abciximab and eptifibatide confirmed that fully functional αIIbβ3 was required to obtain these force signals. Monomeric fibrin displayed a higher cumulative probability of interacting with αIIbβ3 and a greater binding strength. αIIbβ3-fibrin interactions were also less sensitive to inhibition by abciximab and eptifibatide, suggesting that they had a different binding specificity. Both fibrinogen- and fibrin- interactions with αIIbβ3 were partially inhibited by RGD-containing peptides, suggesting the existence of common RGD-containing binding motifs. This assumption was supported using the fibrin variants αD97E or αD574E whose mutated RGD motifs were less reactive with αIIbβ3 than those of wild type fibrin. Monomeric fibrin made from a homodimeric fibrinogen γ chain splice variant lacking the γC αIIbβ3 binding motif was more reactive with αIIbβ3 than the parent fibrinogen, suggesting that this binding motif is less important in fibrin. Polymeric fibrin displayed a rupture force profile similar to fibrinogen and monomeric fibrin with moderate (20-60 pN) and strong (>60 pN) forces that peaked at 70-80 pN. The αIIbβ3 antagonist eptifibatide, the αVβ3-specific cyclic RGD-containing peptide XJ735, a non-selective cyclic RGD-containing peptide, and a dodecapeptide corresponding to the C-terminus of the fibrinogen γ chain each produced a sharp drop in the medium-to-high force range. Interaction forces >60 pN were more effectively reduced by EDTA than by any of the inhibitory peptides, indicating their dependence on the structural integrity and functionality of αIIbβ3. The free γC dodecapeptide was also less efficient than the cRGD peptides or eptifibatide in preventing the stronger interactions. Taken together, these results demonstrate that surface-bound fibrinogen and monomeric, as well as polymeric fibrin, are highly reactive with the αIIbβ3. Fibrin is more reactive than fibrinogen in terms of its binding probability and it has greater binding strength. Fibrin binding to αIIbβ3 is also less sensitive to αIIbβ3 inhibitors, suggesting that fibrin and fibrinogen have distinct binding requirements. In particular, the maintenance of αIIbβ3-binding activity when the C-terminus of the fibrinogen γ chain and the α chain RGD sequences are mutated suggests that the αIIbβ3-binding sites in fibrin are not confined to its known γ chain and RGD motifs. DisclosuresNo relevant conflicts of interest to declare.

Rapid purification of a new P-I class metalloproteinase from Bothrops moojeni venom with antiplatelet activity.

The present study aimed to evaluate the proteolytic and biological activities of a new metalloproteinase from B. moojeni venom. The purification of BmooMPα-II was carried out through two chromatographic steps (ion-exchange and affinity). BmooMPα-II is a monomeric protein with an apparent molecular mass of 22.5 kDa on SDS-PAGE 14% under nonreducing conditions. The N-terminal sequence (FSPRYIELVVVADHGMFTKYKSNLN) revealed homology with other snake venom metalloproteinases, mainly among P-I class. BmooMPα-II cleaves Aα-chain of fibrinogen followed by Bβ-chain, and does not show any effect on the γ-chain. Its optimum temperature and pH for the fibrinogenolytic activity were 30–50°C and pH 8, respectively. The inhibitory effects of EDTA and 1,10-phenantroline on the fibrinogenolytic activity suggest that BmooMPα-II is a metalloproteinase. This proteinase was devoid of haemorrhagic, coagulant, or anticoagulant activities. BmooMPα-II caused morphological alterations in liver, lung, kidney, and muscle of Swiss mice. The enzymatically active protein yet inhibited collagen, ADP, and ristocetin-induced platelet aggregation in a concentration-dependent manner. Our results suggest that BmooMPα-II contributes to the toxic effect of the envenomation and that more investigations to elucidate the mechanisms of inhibition of platelet aggregation may contribute to the studies of snake venom on thrombotic disorders.

Screening, Characterization, and Cloning of a Solvent-Tolerant Protease from Serratia marcescens MH6

A solvent-tolerant bacterium strain MH6 was isolated by hydrophilic organic solvent DMSO enrichment in medium and identified as Serratia marcescens. The extracellular protease with novel organic-solvent-stable properties from strain MH6 was purified and characterized. The molecular weight of the purified protease was estimated to be 52 kDa on SDS-PAGE. The open reading frame (ORF) of the MH6 protease encoded 504 amino acids with 471 amino acid residues in the mature protease. Based on the inhibitory effects of EDTA and 1, 10-phenathroline, the MH6 protease was characterized as a metalloproteinase. The enzyme activity was increased in the presence of Ni2+, Mg2+, and Ca2+. The protease could also be activated by the nonionic surfactants Tween 80 (1.0%) and Triton X-100 (1.0%). The protease showed remarkable solvent stability in the presence of 50% (v/v) solutions of long-chain alkanes and long-chain alcohols. It was also fairly stable in the presence of 25% solutions of hydrophilic organic solvents. Due to its high stability in solvents and surfactants, the MH6 protease is an ideal candidate for applications in organic catalysis and other related fields.

EDTA Inhibits the Binding of Clone 96.2C1, an Anti-CD41a Monoclonal Antibody, to the Platelets and Addition of Heparin and CaCl2 to the Antibody Neutralizes the EDTA-induced Inhibitory Effect

Background: The binding of some monoclonal antibodies platelet glycoprotein (GP) IIb/IIIa, which is frequently used for flow cytometric immnophenotyping, is known to be inhibited by EDTA. To select the ideal antibodies to be included in the 'Acute Leukemia Panel' for immunophenotyping of acute leukemia, we compared the inhibitory effect of EDTA on the binding of 5 different clones of monoclonal antibodies to platelet GP IIb/IIIa. We also discovered a simple method to neutralize this inhibitory effect. Methods: Flow cytometric measurement of the number of platelet GP IIb/IIIa binding sites with different anticoagulants was performed using a panel of 5 clones of monoclonal antibodies against CD41 (clone PM6/248), CD41a (clone 96.2C1 & clone HIP8), CD41b (clone HIP2) and CD61 (clone VI-PL2), and the results are expressed as the mean equivalent soluble fluorochrome (MESF) values. Results: The MESF value of the EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody showed a significantly lower value than the MESF of platelets anticoagulated with heparin or citrate (P＜0.001). The inhibitory effect of EDTA on the binding of anti-CD41a, clone 96.2C1 antibody to the plateletswas neutralized by addition of heparin and . The mean MESF value of EDTA platelets stained with anti-CD41a, clone 96.2C1 antibody was significantly increased by the addition of heparin and (P=0.0001). Conclusion: The false-negative results of the binding of anti-CD41a, clone 96.2C1 antibody to the platelets seem to be due to the calcium chelating property of EDTA, and the addition of and heparin could be used as an easy compensatory measure for the inhibitory effect of EDTA on other antibodies as well.

Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

A proteinase, named BmooMP α-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS–PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the A α-chain of fibrinogen first, followed by the B β-chain, and shows no effects on the γ-chain. On fibrin, the enzyme hydrolyzed only the β-chain, leaving the γ-dimer apparently untouched. It was devoid of phospholipase A 2, hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 °C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMP α-I is a metalloproteinase and inhibition by β-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMP α-I activity. Since the BmooMP α-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

Purification and characterization of a keratinolytic metalloprotease from Chryseobacterium sp. kr6

The Chryseobacterium sp. kr6 strain has been described as a highly keratinolytic bacterium showing effective feather-degrading and de-hairing activities. A keratinase Q1 enzyme was purified from Chryseobacterium sp. kr6 culture by Phenyl Sepharose and Superose 12HR chromatography. This enzyme showed a specific activity of 967U/mg for keratin azure. Electrophoresis under denaturing conditions showed a monomeric protein with approximately 64kDa. The enzyme showed pH and temperature optima of 8.5 and 50 degrees C, respectively. The inhibitory effect of EDTA, EGTA and 1,10-phenanthroline characterized Q1 enzyme as a Zn-metalloprotease. Its activity was increased by three-fold in the presence of Ca(2+). ESI-MS/MS analysis of peptides generated from a tryptic digestion revealed sequence homology which may characterize the Q1 keratinase as a member of the M14 metalloprotease family, with a consensus glycosylation region similar to proteins from Chryseobacerium meningosepticum.

Metal ion modulation of cystinyl aminopeptidase

Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.

Enhanced Inhibition of Escherichia coli O157:H7 by Lysozyme and Chelators

This study examined the effects of three chelating agents (EDTA, disodium pyrophosphate [DSPP], and pentasodium tripolyphosphate [PSTPP]) on the inhibition of the growth of Escherichia coli O157:H7 by lysozyme. The objective of this study was to identify replacement chelators that exhibit synergistic properties similar to those of EDTA. The inhibitory effects of EDTA at 300 to 1,500 μg/ml and of DSPP and PSTPP at 3,000 to 15,000 μg/ml in combination with lysozyme at 200 to 600 μg/ml for up to 48 h at pHs of 6.0, 7.0, and 8.0 on four strains of E. coli O157:H7 was studied with the use of a microbroth dilution assay. The addition of EDTA enhanced lysozyme's inhibitory effect on strains of E. coli O157:H7. EDTA at ≥300 μg/ml combined with lysozyme at 200 to 600 μg/ml was sufficient to inhibit the growth of the strains at pHs of 6.0 and 8.0. At pH 7.0, lysozyme at 200 to 600 μg/ml and EDTA concentrations of ≥1,000 μg/ml were effective in inhibiting three of the four strains. DSPP at pH 6.0 was inhibitory at ≥10,000 μg/ml when combined with lysozyme at 200 to 300 μg/ml. In contrast, PSTPP increased the inhibitory activity of lysozyme more effectively at pH 8.0. Lysozyme at 200 to 600 μg/ml was effective against two strains of E. coli O157:H7 when used in conjunction with PSTPP at ≥5,000 μg/ml. The remaining strains were inhibited by PSTPP at ≥10,000 μg/ml. Our results indicate that inhibition occurred with each lysozyme-chelator combination, but the concentrations of phosphates required to increase the antimicrobial spectrum of lysozyme against E. coli O157:H7 were higher than the EDTA concentrations required to achieve the same effect.

Development of nonradioactive microtiter plate assays for nuclease activity

We have developed two microtiter plate assays for the detection of DNA cleavage by nucleases, using 3 ′-biotinylated oligonucleotide substrates. In the covalently linked oligonucleotide nuclease assay (CLONA), the biotinylated substrates are phosphorylated at the 5 ′ end to facilitate their covalent immobilization on CovaLink NH plates. The cleavage of the covalently immobilized substrate by nucleases results in biotin release. The uncleaved substrate molecules are detected with an enzyme-avidin conjugate. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5 ′ end of the 3 ′-biotinylated DNA strand. The substrate binds specifically to the wells of streptavidin-coated microtiter plates, in which the nuclease reaction takes place. Uncleaved substrate retains the digoxigenin label, which is detected with an enzyme-labeled anti-digoxigenin antibody. We assessed the efficiency of these two assays by measuring S1 nuclease and DNase I activities, and the inhibitory effect of EDTA and aurintricarboxylic acid on the reaction. Both methods are more convenient than the standard radioactive nuclease assay and are suitable for high-throughput screening of potential nuclease inhibitors, nucleases, and catalytic antibodies. The ALONA assay was found to be more sensitive than the CLONA assay, with a performance similar to that of the standard nuclease assay.

A method for the detection and screening of catalytic anti-DNA antibodies

We have developed a microtiter plate assay for the detection and screening of anti-DNA hydrolytic antibodies. The affinity-linked oligonucleotide nuclease assay (ALONA) makes use of substrates with a digoxigenin on the 5′-end of the 3′-biotinylated DNA strands. The substrate binds specifically to the wells of streptavidin-coated microtiter plates where the reaction takes place. Uncleaved substrate retains the digoxigenin label, which is then detected with an enzyme-labeled anti-digoxigenin antibody. We first assessed the efficiency of this assay by measuring S1 nuclease and DNase I activities and the inhibitory effect of EDTA on the reaction. The ALONA procedure was then successfully applied to the screening of a high number of hybridoma clones derived from nonimmunized (NZB×NZW)F1 mice with spontaneous lupus erythematosus. We detected three potential catalytic antibodies and investigated their substrate specificity. Overall, our findings demonstrate the value of the ALONA method for high throughput screening of potential nucleases and catalytic antibodies. Although this assay was designed for the selection of catalysts active in DNA hydrolysis, it can be adapted to detect most types of substrate cleavage reaction.

In Vitro Methylation of Arsenite by Rabbit Liver Cytosol: Effect of Metal Ions, Metal Chelating Agents, Methyltransferase Inhibitors and Uremic Toxins

ABSTRACTThe methylation of carrier-free 74As-arsenite by liver cytosol of Flemish Giant rabbits is highly susceptible to additions of trace elements. in vitro supplementation of essential trace elements like zinc (Zn2+), vanadium (V5+), iron (Fe2+), copper (Cu2+) and selenate was shown to increase the methylation efficiency. Trivalent metal ions (e.g. Al3+, Cr3+ and Fe3+), Hg2+, Tl+ and SeO32- had a deleterious effect. The inhibitory effect of EDTA, oxime and many divalent cations (Ca2+, Mg2+, Sr2+,.) suggest a co-factor role for a specific divalent metal ion, possibly Zn2+. Chelating agents used in clinical treatment of acute and chronic inorganic arsenic poisoning lower the methylation capacity of cytosol by rendering the trivalent arsenic unavailable for the methyltransferase enzymes. S-adenosylhomocysteine and periodate-oxidized adenosine, inhibitors of s-adenosylmethionine dependent methylation pathways, inhibit the methylation of arsenite. Pyrogallol, a catechol-O-methyltransferase inhibitor, blocks the action of arsenite- and monomethylarsonic methyltransferase enzymes, suggesting a close structural relationship between the active sites of the different enzymes. Some uraemic toxins, namely oxalate, p-cresol, hypoxanthine, homocysteine and myo-inositol, inhibit arsenic methylation.

Flow-injection/standard subtraction method for the determination of iron(III) based on its catalytic effect and inhibition of EDTA

A new approach for the flow-injection catalytic determination of 10 −6 M levels of iron(III) is proposed. This method is based on the catalytic effect of iron(III) on the oxidative coupling of p-anisidine with N,N-dimethylaniline to form a blue compound ( λ max=735 nm) in the presence of hydrogen peroxide and 1,10-phenanthroline as an activator, and the inhibitory effect of EDTA on the iron(III)-catalyzed reaction. The concentration of iron(III) can be determined from only two flow signals which are obtained by injecting the iron(III) and iron(III) plus EDTA solutions into the flow lines without a calibration graph for iron(III); this procedure may be called standard subtraction. The proposed method permits the analysis of 12 samples per hour (24 peaks per hour) and was successfully applied to the determination of iron in standard iron ore samples.

The aggregation of human platelets by Lactobacillus species.

The ability to aggregate human platelets was examined for five Lactobacillus rhamnosus strains and five Lactobacillus paracasei subsp. paracasei strains isolated from patients with infective endocarditis (IE), 25 laboratory isolates from the same two species, and 14 strains from five other oral species, namely Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salivarius. Amongst the L. rhamnosus strains, platelets were aggregated by all five IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains, the respective numbers were 2/5 and 2/9. Aggregation also occurred with 11/14 strains of the other five species; each species was represented. The optimal ratio of bacteria to platelets for aggregation was approximately 1:1, and there was considerable variation in the lag phase that preceded aggregation, depending on the source of the platelets. Overall, the lag phase varied between 0.25 +/- 0.1 and 20.4 +/- 3.2 min and the percentage aggregation ranged between 70 +/- 2.6 and 104 +/- 13.5%. Confirmation that aggregation was being observed came from studies with five strains on the inhibitory effects of EDTA, dipyridamole, apyrase, imipramine, acetylsalicylic acid and quinacrine. Inhibition of aggregation by L. rhamnosus strains by the peptide arginine-glycine-aspartic acid-serine (RGDS) further indicated a role for fibronectin and/or fibrinogen. Pronase treatment of cells for 1 h and extraction of bacterial surface components with 0.1 M-Tris/HCl (pH 8.5) at 37 degrees C for 1 h stopped aggregation in 8/9 IE strains. Extracted surface proteins (200 micrograms) completely inhibited platelet aggregation by 8/9 of the homologous strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of EDTA · Ca 2+ on the hydrolysis of synaptosomal phospholipids by phospholipase A 2 toxins and enzymes

Phospholipases A 2 (PLA 2) are Ca 2+-dependent enzymes that are inhibited by EDTA; this inhibition would be expected to be reversed by restoring the Ca 2+ concentration. By examining the hydrolysis of synaptosomal phospholipids by PLA 2 enzymes, Naja naja atra and Naja nigricollis, and by toxins with PLA 2 activity, β-bungarotoxin (β-BuTX) and notexin, we demonstrated a novel inhibitory action of EDTA manifested in the presence of excess Ca 2+. We postulate the formation of an EDTA·Ca 2+ complex which inhibits PLA 2 activity in connection-dependent manner. Synaptosomes in which phospholipids are hydrolyzed by PLA 2, have membranal damage expressed by increased acetylcholine (ACh) release and decreased osmotic activity. Addition of EDTA·Ca 2+, which inhibits phospholipid hydrolysis, also reversed the PLA 2 effect on ACh release, but not its effect on osmotic activity. The inhibition of PLA 2 was observed on membranal phospholipids as well as on an artificial substrate of phospholipid-Triton mixed micelles. Moreover, we found that another enzyme, lactate dehydrogenase, was also inhibited. Our results indicate a non-specific inhibition exerted on the enzyme rather than on the substrate.

Enhancement of hydroperoxide-dependent lipid peroxidation in rat liver microsomes by ascorbic acid

Simultaneous addition of ascorbic acid and organic hydroperoxides to rat liver microsomes resulted in enhanced lipid peroxidation (~threefold) relative to incubation of organic hydroperoxides with microsomes alone. No lipid peroxidation was evident in incubations of ascorbate alone with microsomes. The stimulatory effect of ascorbate on linoleic acid hydroperoxide (LAHP)-dependent peroxidation was evident at all times whereas stimulation of cumene hydroperoxide (CHP)-dependent peroxidation occurred after a lag phase of up to 20 min. EDTA did not inhibit CHP-dependent lipid peroxidation but completely abolished ascorbate enhancement of lipid peroxidation. Likewise, EDTA did not significantly inhibit peroxidation by LAHP but dramatically reduced ascorbate enhancement of lipid peroxidation. The results reveal a synergistic prooxidant effect of ascorbic acid on hydroperoxide-dependent lipid peroxidation. The inhibitory effect of EDTA on enhanced peroxidation suggests a possible role for endogenous metals mobilized by hydroperoxide-dependent oxidations of microsomal components.

Lipid peroxidation enzyme systen in rainbow trout ( Salmo gairdnerii) skeletal muscle microsomes

1. 1. A microsomal lipidperoxidation enzyme system in rainbow trout white skeletal muscle was identified and characterized. 2. 2. This enzyme system is very similar to that found in other fish such as red hake, winter flounder and herring. 3. 3. NADH-cytochrome b 5 reductase is suggested to be the enzyme involved, because of the specific 4. 4. The activating and inhibitory effects of EDTA and KCN on lipid peroxidation enzyme sytem activity at different concentration are discussed extensively in terms of the possible initiation reaction mechanism.