P-chloromercuribenzoate Research Articles

The effect of cytochrome c on Na,K-ATPase.

Na,K-ATPase is a crucial enzyme responsible for maintaining Na+, K+-gradients across the cell membrane, which is essential for numerous physiological processes within various organs and tissues. Due to its significance in cellular physiology, inhibiting Na,K-ATPase can have profound physiological consequences. This characteristic makes it a target for various pharmacological applications, and drugs that modulate the pump's activity are thus used in the treatment of several medical conditions. Cytochrome c (Cytc) is a protein with dual functions in the cell. In the mitochondria, it is essential for ATP synthesis and energy production. However, in response to apoptotic stimuli, it is released into the cytosol, where it triggers programmed cell death through the intrinsic apoptosis pathway. Aside from its role in canonical intrinsic apoptosis, Cytc also plays additional roles. For instance, Cytc participates in certain non-apoptotic functions -those which are less well-understood in comparison to its role in apoptosis. Within this in vitro study, we have shown the impact of Cytc on Na,K-ATPase for the first time. Cytc has a biphasic action on Na,K-ATPase, with activation at low concentrations (0.06ng/ml; 6ng/ml) and inhibition at high concentration (120ng/ml). Cytc moreover displays isoform/subunit specificity and regulates the Na+ form of the enzyme, while having no effect on the activity or kinetic parameters of the K+-dependent form of the enzyme. Changing the affinity of p-chloromercuribenzoic acid (PCMB) by Cytc is therefore both a required and sufficient condition for confirming that PCMB and Cytc share the same target, namely the thiol groups of cysteine in Na,K-ATPase.

Kinetics and thermodynamic stability of native and chemically modified acid invertase: Extracted from yellow pea (Lathyrus aphaca) Seedlings

In this work, an acid invertase isolated from Lathyrus aphaca seedlings is purified, and its thermal properties are investigated. Acid invertase was purified through salt fractionation, CM-cellulose, and Ultrogel ACA-44 chromatography. Using SDS-PAGE, the consistency of the isolated enzyme was verified. The enzyme weighs 29 kDa at the molecular level. Acid invertase functions best at a pH of 3.0 and a temperature of 45 °C. Activation energy of 29.5 kJ mol−1 and Km and Vmax values of 0.5 mM and 119.7 µmol. min−1. mg−1 of protein was found for the isolated enzyme. It was shown that the mercaptide-forming agent p-chloromercuribenzoic acid (PCMB; 0.5 mM) and the enzyme's activity were both modestly increased by Ca2+, Mn2+, and Mg2+ ions but inhibited by Hg2+, Cd2+, and Pb2+ ions. At elevated temperatures, the invertase exhibited an increase in thermostability due to an increase in activation entropies (∆Sº) and a decrease in activation enthalpies (∆Hº). The interactions between 4 M urea and α-chymotrypsin were tetraphasic, causing periodic increases and decreases in invertase activity. It also suggests a potential explanation for acid-invertase thermal inactivation at high temperatures. This enzyme might be a catabolite-resistant invertase to produce high-gravity ethanol or fructose syrup.

Purification, characterization, and enzyme kinetics of a glutathione S transferase from larvae of the camel tick Hyalomma dromedarii

BackgroundGlutathione s-transferases (GSTs) perform an essential role in detoxification of xenobiotics and endogenous compounds via their conjugation to reduce glutathione. ResultsA GST enzyme, designated tick larvae glutathione S transferase (TLGST), was purified from larvae of the camel tick Hyalomma dromedarii via ammonium sulfate precipitation, glutathione-Sepharose affinity column and Sephacryl S-300 chromatography. TLGST-specific activity was found to be 1.56 Umg−1 which represents 39 folds and 32.2% recovery. The molecular weight of TLGST purified from camel tick larvae was found as 42 kDa by gel filtration. TLGST has a pI value of 6.9 and was found a heterodimeric protein of 28 and 14 kDa subunits as detected on SDS-PAGE. The Lineweaver–Burk plot calculated the km for CDNB to be 0.43 mM with Vmax value of 9.2 Umg−1. TLGST exhibited its optimal activity at pH 7.9. Co2+, Ni2+ and Mn2+ increased the activity of TLGST while Ca2+, Cu2+, Fe2+ and Zn2+ inhibited it. TLGST was inhibited by cumene hydroperoxide, p-hydroxymercuribenzoate, lithocholic acid, hematin, triphenyltin chloride, p-chloromercuribenzoic acid (pCMB), N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetamide, EDTA and quercetin. pCMB inhibited TLGST competitively with Ki value of 0.3 mM. ConclusionsThese findings will help to understand the various physiologic conditions of ticks and targeting TLGST could be significant tool for development of prospective vaccines against ticks as a bio-control strategy to overcome the rapid grows in pesticide-resistant tick populations. Graphical Abstract▪

Targeted Covalent Inhibitors Allosterically Deactivate the DEDDh Lassa Fever Virus NP Exonuclease from Alternative Distal Sites.

For using targeted covalent inhibitors (TCIs) as anticancer and antiviral drugs, we establish that the model compounds PCMPS (p-chloromercuriphenyl sulfate) and PCMB (p-chloromercuribenzoate) are inhibitors of the DEDDh family of exonucleases. The underlying mechanism is analyzed by X-ray crystallography, activity/nucleic acid-binding assays, and all-atom molecular dynamics (MD) simulations. The first TCI-complexed structures of a DEDDh enzyme, the Lassa fever virus NP exonuclease (NPexo), are resolved to elucidate that the Cys409 binding site is away from the active site and the RNA-binding lid. The NPexo C409A structures indicate Cys461 as the alternative distal site for obstructing the equally active mutant. All-atom MD simulations of the wild type and mutant NPexos in explicit solvent uncover an allosteric inhibition mechanism that the local perturbation induced by PCMPS sulfonate propagates to impact the RNA-binding lid conformation. Binding assay studies confirm that PCMPS does affect the RNA binding of NPexo. The predicted relative potency between PCMPS and PCMB is also in line with experiments. The structural data and inhibition mechanism established in this work provide an important molecular basis for the drug development of TCIs.

Covid-19 vaccination should be mandatory for healthcare workers

Transport characteristics of benzylpenicillin in the central nervous system was examined using ATP-depleted rat choroid plexus. In the presence of an outwardly directed Cl- gradient, accumulation of benzylpenicillin was stimulated...

Purification and kinetic properties of a novel β-amylase from Penicillum citrinum AS-9

β-amylase-rich preparation produced by the soil fungal strain Penicillium citrinum AS-9 was completely purified within the succeeding steps; ultrafiltration, acetone fractionation and gel filtration on Sephadex G-150 column. Acetone fractionation recovered the highest β-amylase activity and the 85% acetone fraction exhibited 3.5-fold activity that of the crude enzyme. The column affected 8.6-fold purification for β-amylase, which produced maltose as the major product of starch hydrolysis. The pure enzyme showed a Km value of 17.5 mM and Vmax of 17.5 Umg-1 protein, applying Woolf plot and exhibited its maximum velocity at pH 7.1 and 50°C. In absence of substrate, thermal treatments of the enzyme solution at pH 5.2 and 5.5 had the most adverse effects on the enzyme activity. CaCl2 (1mM) activated the enzyme, while each of Ca2+, Fe3+, cysteine, cystine, I2 and p-chloromercuribenzoate (PCMB) had different inhibitory effects. Maltose as the enzyme product at final concentration of 80 mM strongly inhibited the enzyme.

Purification and characterization of thermo-alkaline stable lipase from Bacillus coagulans and its compatibility with commercially available detergents

Thermo-alkaline stable lipase producing B. coagulans was isolated and identified by 16S rRNA sequencing. The lipase production was optimized using different growth parameters. The enzyme was purified and characterized in terms of pH, temperature, solvents, heavy metal ions and inhibitors. Compatibility with commercially available detergents was also studied. The isolate showed maximum lipase production at 37 ⁰C; pH of 9 within 48 h. Addition of magnesium chloride increased lipase production. Sephadex G-100 chromatography was used to purify lipase. The enzyme showed maximum activity at pH 8 of 30 ⁰C. Lipase form B. coagulans was active at a wide range of temperature between 30-70 ⁰C. It was stable in most of the solvents at a concentration 5 and 10%, except dimethyl sulfoxide (DMSO) and methanol. Iodoacetic acid (IAA) and p-chloromercuribenzoic acid (pCMB) had an inhibitory effect on lipase. The lipase was compatible with commercially available detergents that increased the brightness and whiteness of the tested cotton fabrics. Lipase from B. coagulans with alkaline stability at a wide range of temperature has potential application in the detergent industry.

Characterization of a Thermally Stable β-galactosidase Produced by Thermophilic Anoxybacillus sp. AH1

Thermostable β-galactosidases from thermophilic bacteria have attracted increasing interest to have various advantages in industrial and biotechnological applications. In this study, a highly thermally stable β-galactosidase produced by Anoxybacillus sp. AH1was purified and characterized. The highest enzyme production was achieved after the bacterium was incubated for 24 hours. The enzyme was purified by precipitation with ammonium sulphate dialysis, gel filtration chromatography using Sephadex G-75. After the purification steps, β-galactosidase was found to be purified 10.2-fold and a yield of 13.9%. The molecular mass of the galactosidase was estimated to be 75 kDa by SDS-PAGE. The purified enzyme was highly stable and retained at 71% of the original activity at 60 °C and 53% at 70 oC within 120 minutes. The Km and Vmax values of purified β-galactosidase were calculated as 1.249 mM and 0.5 μmol minutes-1, respectively. Ca2+, Zn2+, and Mg2+ significantly activated β-galactosidase activity, whereas enzyme activity was inhibited significantly by Cu+2 as well as by the metal ion chelators1,10-phenanthroline (phen) and ethylenediaminetetraacetic acid (EDTA). The Purified β-galactosidase activity was increased by PMSF (phenylmethylsulfonyl fluoride), PCMB (p-chloromercuribenzoic acid), DTT (dithiothreitol), and β-ME (β-mercaptoethanol) at 2 mM, but inhibited completely by NEM (N-ethylmaleimide) at 1 mM.

Acyl-CoA thioesterase activity of peroxisomal ABC protein ABCD1 is required for the transport of very long-chain acyl-CoA into peroxisomes

The ABCD1 protein, one of the four ATP-binding cassette (ABC) proteins in subfamily D, is located on the peroxisomal membrane and is involved in the transport of very long chain fatty acid (VLCFA)-CoA into peroxisomes. Its mutation causes X-linked adrenoleukodystophy (X-ALD): an inborn error of peroxisomal β-oxidation of VLCFA. Whether ABCD1 transports VLCFA-CoA as a CoA ester or free fatty acid is controversial. Recently, Comatose (CTS), a plant homologue of human ABCD1, has been shown to possess acyl-CoA thioesterase (ACOT) activity, and it is suggested that this activity is required for transport of acyl-CoA into peroxisomes. However, the precise transport mechanism is unknown. Here, we expressed human His-tagged ABCD1 in methylotrophic yeast, and characterized its ACOT activity and transport mechanism. The expressed ABCD1 possessed both ATPase and ACOT activities. The ACOT activity of ABCD1 was inhibited by p-chloromercuribenzoic acid (pCMB), a cysteine-reactive compound. Furthermore, we performed a transport assay with ABCD1-containing liposomes using 7-nitro-2–1,3-benzoxadiazol-4-yl (NBD)-labeled acyl-CoA as the substrate. The results showed that the fatty acid produced from VLCFA-CoA by ABCD1 is transported into liposomes and that ACOT activity is essential during this transport process. We propose a detailed mechanism of VLCFA-CoA transport by ABCD1.

Heme-Thiolate Perturbation in Cystathionine β-Synthase by Mercury Compounds.

Cystathionine β-synthase (CBS) is an enzyme involved in sulfur metabolism that catalyzes the pyridoxal phosphate-dependent condensation of homocysteine with serine or cysteine to form cystathionine and water or hydrogen sulfide (H2S), respectively. CBS possesses a b-type heme coordinated by histidine and cysteine. Fe(III)-CBS is inert toward exogenous ligands, while Fe(II)-CBS is reactive. Both Fe(III)- and Fe(II)-CBS are sensitive to mercury compounds. In this study, we describe the kinetics of the reactions with mercuric chloride (HgCl2) and p-chloromercuribenzoic acid. These reactions were multiphasic and resulted in five-coordinate CBS lacking thiolate ligation, with six-coordinate species as intermediates. Computational QM/MM studies supported the feasibility of formation of species in which the thiolate is proximal to both the iron ion and the mercury compound. The reactions of Fe(II)-CBS were faster than those of Fe(III)-CBS. The observed rate constants of the first phase increased hyperbolically with concentration of the mercury compounds, with limiting values of 0.3–0.4 s–1 for Fe(III)-CBS and 40 ± 4 s–1 for Fe(II)-CBS. The data were interpreted in terms of alternative models of conformational selection or induced fit. Exposure of Fe(III)-CBS to HgCl2 led to heme release and activity loss. Our study reveals the complexity of the interactions between mercury compounds and CBS.

4-Chloromercuribenzoic acid enhances carbapenem sensitivity among pathogenic Gram negative bacteria by altering blaVIM, adeB and ompC expression

BackgroundRapid global dissemination of carbapenem resistant Gram negative bacteria (CRGNB) is supposed to be clinically most alarming. Since, p-chloromercuribenzoic acid (pCMB) is a well known metallo-beta-lactamase inhibitor; evaluation of its bactericidal and carbapenem resistance reversing potential would be important. MethodsIn this study, bactericidal and meropenem resistance reversing potential of pCMB was investigated against CRGNB by MIC determination, checkerboard assay, time-kill assay and cellular viability assay. Effect of pCMB on cellular morphology was visualized by Scanning Electron Microscopy. Further, quantitative Real Time-PCR was performed to evaluate effects of pCMB on clinically relevant metallo-beta-lactamases, major efflux pumps and outer membrane proteins expression. ResultspCMB exhibited at least four fold reduced MIC value (2–256μg/ml) than that of meropenem against CRGNB. Moreover, pCMB exhibited synergism with meropenem against 86.06% of CRGNB. MIC of pCMB (16−32μg/ml) could kill upto 99.96% bacteria within 6−8h of dosing. pCMB exerted bactericidal activity by severely disrupting cell wall integrity. Reversal of carbapenemase property of CRGNB by pCMB might have developed through alteration of blaVIM, acrB, mexB and ompk36 expression. ConclusionsHence, the current study identified pCMB as a potential bactericidal agent which enhanced meropenem sensitivity by altering blaVIM, acrB, mexB and ompk36 expression.

Purification and biochemical characterization of a new organic solvent-tolerant chitinase from Paenibacillus timonensis strain LK-DZ15 isolated from the Djurdjura Mountains in Kabylia, Algeria

A new extracellular chitinase (called ChiA-Pt70) was produced and purified from a newly isolated Paenibacillus timonensis strain LK-DZ15. The maximum chitinase activity recorded after 44-h of incubation at 30 °C was 11,500 U/mL. Pure enzyme was obtained after ammonium sulphate precipitation (40–70%) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 70,166.11 kDa. The sequence of the 25 NH2-terminal residues of the mature ChiA-70 showed high homology with Paenibacillus GH-18 chitinases family. Optimal activity was achieved at pH 4.5 and 80 °C. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB), 5,5′-dithio-bis-2-nitro benzoic acid (DTNB), and N-ethylmaleimide (NEM). Chitinase activity was high on colloidal chitin, chitin azure, glycol chitin, glycol chitosane, chitotriose, and chito-oligosaccharide while it did not hydrolyse chitibiose and amylose. Furthermore, thin-layer chromatography (TLC) analysis from enzymatic catalyzed hydrolysis of colloidal chitin showed that ChiA-Pt70 acted as an endo-splitting enzyme. Its Km and kcat values were 0.611 mg colloidal chitin/mL and 87,800 s−1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Mt45 from Melghiribacillus thermohalophilus strain Nari2AT, ChiA-Hh59 from Hydrogenophilus hirchii strain KB-DZ44, Chitodextrinase® from Streptomyces griseus, and N-acetyl-β-glucosaminidase® from Trichoderma viride. Therefore, ChiA-Pt70 exhibited remarkable biochemical properties suggesting that it is suitable for the enzymatic degradation of chitin.

Purification and biochemical characterization of a novel acido-halotolerant and thermostable endochitinase from Melghiribacillus thermohalophilus strain Nari2AT

An extracellular acido-thermostable endochitinase (called ChiA-Mt45) from thermohalophilic Melghiribacillus thermohalophilus strain Nari2AT gen. nov. sp. nov., was purified and biochemically characterized. The maximum chitinase activity recorded after 48-h of incubation at 55 °C was 9000 U/mL. Pure enzyme was obtained after heat treatment (20 min at 90 °C) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on MALDI–TOF/MS analysis, the purified enzyme is a monomer with a molecular mass of 45201.10 Da. The 27 residue NH2-terminal sequence of the enzyme showed high homology with Bacillus GH-18 chitinases family. The optimum pH and temperature values for chitinase activity were pH 3.5 and 90 °C, respectively. In addition, the enzyme was halotolerant and can be classified as an extremozyme. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). Its Km and kcat values were 0.253 mg colloidal chitin/mL and 47000 s−1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Hh59 from Hydrogenophilus hirchii KB-DZ44 and chitodextrinase from Streptomyces griseus, and N-acetyl-β-glucosaminidase from Trichoderma viride. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Additionally, thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Mt45 acted as an endosplitting enzyme. Overall, the chitinase ChiA-Mt45 may have great potential for the enzymatic degradation of chitin.

Toxicological and behavioral study of two potential antibacterial agents:4-chloromercuribenzoic acid and quercetin on Swiss-albino mice

Global dissemination of carbapenem resistant-Gram negative bacteria (CR-GNB) is supposed to be clinically alarming because it extremely delimits the treatment options against serious infections. 4-Chloromercuribenzoic acid (pCMB) is an efficient metallo-enzyme inhibitor, and quercetin is known for antioxidant, antiviral, anticancer, antimicrobial, and anti-inflammatory activities. These two compounds could be considered as potential candidates for the treatment of CR-GNB mediated infections. Hence, in this study, antibacterial activity of pCMB and quercetin was evaluated against CR-GNB through minimum inhibitory concentration (MIC) determination. Toxicity of pCMB and quercetin was evaluated by LC50 calculation through brine shrimp test (BST) and by investigating hematological, serum biochemical, and histopathological parameters in Swiss-albino mice. Moreover, aggressive–depressive–cognitive behavioral effects of pCMB and quercetin on murine model were evaluated. All the carbapenem resistant isolates (CR-GNB) exhibited MIC values in the range of 4–256 μg/ml, 16–256 μg/ml, and 64–1024 μg/ml for pCMB, quercetin, and meropenem, respectively. BST determined LC50 of pCMB and quercetin at 91.57 ± 0.35 mg/L and 448.45 ± 0.46 mg/L, respectively. Oral administration of low dose of pCMB and quercetin did not induce any significant changes in morphological, behavioral, hematological, serum biochemical, and histopathological parameters among Swiss-albino mice. But, a high dose of pCMB and quercetin exhibited slight toxicity. However, no death was reported for any dosage of pCMB and quercetin. Therefore, pCMB and quercetin might be considered for further investigations on alternative therapeutics to combat against CR-GNB.

Purification and characterisation of a protease (tamarillin) from tamarillo fruit

A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60 °C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'.

Biochemical characterization of a novel thermostable chitinase from Hydrogenophilus hirschii strain KB-DZ44

An extracellular acido-thermostable endo-chitinase (called ChiA-Hh59) from thermophilic Hydrogenophilus hirschii strain KB-DZ44, was purified and characterized. The maximum chitinase activity recorded after 36-h of incubation at 60°C was 3000U/ml. Pure enzyme was obtained after heat and acidic treatment, precipitation by ammonium sulphate and acetone, respectively, followed by sequential column chromatographies on Sephacryl S-200 and Mono Q-Sepharose. Based on Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 59103.12-Da. The 22 residue NH2-terminal sequence of the enzyme showed high homology with family-18 bacterial chitinases. The optimum pH and temperature values for chitinase activity were pH 5.0 and 85°C, respectively. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB) and N-ethylmaleimide (NEM). The obtained results suggest that ChiA-Hh59 might be an endo-chitinase. The studied chitinase exhibited high activity towards colloidal chitin, chitin azure, glycol chitin, while it did not hydrolyse chitibiose and amylose. Its Km and kcat values were 0.298mg colloidal chitin/ml and 14400s−1, respectively. Its catalytic efficiency was higher than those of chitodextrinase and ChiA-65. Additionally, Thin-layer chromatography (TLC) analysis from chitin-oligosaccharides showed that ChiA-Hh59 acted as an endo-splitting enzyme. In conclusion, this chitinase may have great potential for the enzymatic degradation of chitin.

Interaction of carbapenems and β-lactamase inhibitors towards CTX-M-15 and CTX-M-15G238C mutant.

The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various β-lactamase inhibitors. A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.

Effect of Peptidase Inhibitors on Dynorphin A (1-17) or (1-13)-Induced Antinociception and Toxicity at Spinal Level

Our group has earlier demonstrated that three enzymes sensitive to peptidase inhibitors (PIs), amastatin (A)-, captopril (C)-, and phosphoramidon (P), played an important role in inactivation of enkephalins at the spinal level. Dynorphin-converting enzyme (DCE) hydrolyzes dynorphin (Dyn) A (1-17) or Dyn A (1-13) mainly at the Arg6-Arg7 bond. Dynorphin A and its derived peptides interact with opioid and glutamate receptors at their N- and C-terminals, respectively. The purpose of the present study was to evaluate the antinociceptive potency and toxicity of intrathecal administered Dyn A (1-17), Dyn A (1-13), or Dyn A (1-6) under pretreatment with ACP and/or the DCE inhibitor p-hydroxymercuribenzoate (PHMB). The effect of these PIs on Dyn A (1-17)-induced inhibition of electrically-evoked contractions in mouse vas deferens was also investigated. The inhibitory potency of Dyn A (1-17) on electrically-evoked contractions in mouse vas deferens under pretreatment with ACP was higher than that with AC, AP, or CP. Pretreatment with ACP augmented Dyn A (1-17) or (1-13)-induced antinociception by approximately 50- or 30-fold with no sign of allodynia when administered intrathecally at low doses. Pretreatment with ACP and PHMB induced neuropathy. These findings showed that intrathecal administration of low-dose Dyn A (1-17) or DynA (1-13) increased antinociception under pretreatment with ACP, but without signs of allodynia in rat.

Purification and Characterization of Anti-cancer l-Glutaminase of Bacillus cereus Strain LC13

The present investigation was carried out in order to isolate a bacterial strain from marine soil which could produce l-glutaminase. The isolated organism identified as Bacillus cereus strain LC13 by 16s rRNA analysis was able to produce the enzyme under optimal conditions of pH 7.0, 37 °C and 0.3 % l-glutamine. Maltose (0.4 %, w/v) was best carbon source supplement. A 5.4-fold increase (from 6.1 to 69.1 U/mL) was achieved after optimization. Ammonium sulphate precipitation and ion exchange chromatography were used to purify the protein, and 12.7-purification fold (from 4.6 to 58.6 U/mg protein) was achieved from cell free extract with a recovery of 49.4 %. The molecular weight of the enzyme was found to be 35 ± 1 kDa as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and confirmed by liquid chromatography–mass spectrometry. The purified l-glutaminase was stable at physiological pH and temperature, activated by Na+, K+, slightly inhibited by Cu2+, Co2+, Fe2+, Fe3+, Zn2+, inhibited by Hg2+, and no effect was observed with Ca2+, Mg2+, Pb2+, and Mn2+. Phenylmethylsulphonyl fluoride, sodium azide, ethylenediaminetetraacetic acid and N-acetyl imidazole had no effect on enzyme whereas iodoacetamide, tosyl-l-lysylchloromethylketone, N-ethylmaleimide, p-Chloromercuribenzoic acid and N-bromosuccinimide strongly inhibited the enzyme, indicating the involvement of cysteine and histidine in the catalytic activity. The enzyme possesses low Km value of 0.4 mM (with Vmax of 61.34 U/ml) which defines higher affinity for its substrate. The l-glutaminase has also shown good radical scavenging activity which could have application in the medical field, like anti-tumour agent.

Purification and Characterization of Thermostable and Detergent-Stable α-Amylase from Anoxybacillus sp. AH1.

A thermostable and detergent-stable α-amylase from a newly isolated Anoxybacillus sp. AH1 was purified and characterized. Maximum enzyme production (1874.8 U/mL) was obtained at 24 h of incubation. The amylase was purified by using Sephadex G-75 gel filtration, after which an 18-fold increase in specific activity and a yield of 9% were achieved. The molecular mass of the purified enzyme was estimated at 85 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature values of the enzyme were 7.0 and 60 °C, respectively. The enzyme was highly stable in the presence of 30% glycerol, retaining 85% of its original activity at 60 °C within 120 min. Km and vmax values were 0.102 µmol and 0.929 µmol/min, respectively, using Lineweaver-Burk plot. The enzyme activity was increased by various detergents, but it was significantly inhibited in the presence of urea. Mg2+ and Ca2+ also significantly activated α-amylase, while Zn2+, Cu2+ and metal ion chelators ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (phen) greatly inhibited the enzyme activity. α-Amylase activity was enhanced by β-mercaptoethanol (β-ME) and dithiothreitol (DTT) to a great extent, but inhibited by p-chloromercuribenzoic acid (PCMB). Iodoacetamide (IAA) and N-ethylmaleimide (NEM) had a slight, whereas phenylmethylsulfonyl fluoride (PMSF) had a strong inhibitory effect on the amylase activity.