Polymerization of coniferyl alcohol by Mn3+‐mediated (enzymatic) oxidation: Effects of H2O2 concentration,aqueous organic solvents,and pH

The objective of this study was to evaluate the ability of one versatile peroxidase and the biocatalytically generated complex Mn(III)‐malonate to polymerize coniferyl alcohol (CA) to obtain dehydrogenation polymers (DHPs) and to characterize how closely the structures of the formed DHPs resemble native lignin. Hydrogen peroxide was used as oxidant and Mn²⁺ as mediator. Based on the yields of the polymerized product, it was concluded that the enzymatic reaction should be performed in aqueous solution without organic solvents at 4.5 ≤ pH ≤ 6.0 and with 0.75 ≤ H₂O₂:CA ratio ≤ 1. The results obtained from the Mn³⁺‐malonate‐mediated polymerization showed that the yield was almost 100%. Reaction conditions had, however, effect on the structures of the formed DHPs, as detected by size exclusion chromatography and pyrolysis‐GC/MS. It can be concluded that from the structural point of view, the optimal pH for DHP formation using the presently studied system was 3 or 4.5. Low H₂O₂/CA ratio was beneficial to avoid oxidative side reactions. However, the high frequency of β–β linkages in all cases points to dimer formation between monomeric CA rather than endwise polymerization. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:81–90, 2018     

Peroxidation of linoleic acid during the oxidation of phenols by fungal laccase

A system comprising laccase and a suitable phenol such as 4-hydroxybenzoic acid (HBA) or synthetic lignin (DHP) exhaustively peroxidized linoleic acid in acetate buffer. The presence of phenols in lignin was essential since an exhaustively methylated preparation of the same lignin did not support peroxidation. The peroxidation rate was greatly enhanced by Mn²⁺, which was oxidized to Mn³⁺ by laccase/HBA, whereas H₂O₂ inhibited strongly due to rapid reduction of Mn³⁺ by H₂O₂ with concomitant formation of O₂. When acetate was replaced by Mn³⁺–chelating oxalate or malonate, there was no change in peroxidation rates in the absence of Mn²⁺, whereas strong inhibition was observed in the presence of Mn²⁺. In case of malonate part of the inhibition was due to H₂O₂ formation as a result of Mn³⁺ reduction by malonate. These findings suggest that laccase may contribute to fungal lipid peroxidation in vivo thus expanding its role in the biodegradation of lignin and other recalcitrant aromatic compounds.     

Effects of structural analogues of nociceptin(1-13)NH₂ on brain antioxidant status in kainic acid-treated rats

The in vivo effects of nociceptin (N/OFQ(1–13)NH₂) and its structural analogues ([Dab⁹]N/OFQ(1–13)NH₂, [Dap⁹]N/OFQ(1–13)NH₂ and [Cav⁹]N/OFQ(1–13)NH₂) on the levels of lipid peroxidation and cell antioxidants (enzyme and non‐enzyme) in brain of control and kainic acid (KA)‐treated rats were studied. In control animals, [Dab⁹]N/OFQ(1–13)NH₂ and [Dap⁹]N/OFQ(1–13)NH₂, unlike N/OFQ(1–13)NH₂ and [Cav⁹]N/OFQ(1–13)NH₂, slightly increased the brain lipid peroxidation; the rest of the parameters were unchanged by all neuropeptides tested. KA (0.25 µg in 0.5 µl, i.c.v) increased the lipid peroxidation (4 and 24 h after KA‐injection) and decreased the glutathione level (1 h after KA‐administration). One hour after KA‐administration, the neuropeptides (2 µg in 0.5 µl, injected 30 min before KA) showed the following effects: a slight decrease in the KA‐induced lipid peroxidation by all nociceptin analogues and an enhancement of the KA‐decreased GSH level, but by [Cav⁹]N/OFQ(1–13)NH₂ only. The brain antioxidant enzyme activities were unchanged in all used experimental groups. In addition, the nociceptin analogues, especially [Can⁹]N/OFQ(1–13)NH₂, showed a good antioxidant capacity in chemical systems, generating reactive oxygen species. In conclusion, the substitution of lysin (Lys) in N/OFQ(1–13)NH₂ molecule with other amino acids might contribute to changes in its antioxidant properties. Copyright © 2011 John Wiley & Sons, Ltd.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.     

Chromium ions inactivate electron transport and enhance superoxide generation in vivo in pea (Pisum sativum L. cv. Azad) root mitochondria

The effect in vivo of hexavalent chromium (Cr⁶⁺) on the respiratory electron transport activity and production of superoxide (O₂^–) radicals, was studied in submitochondrial particles (SMPs) prepared from mitochondria isolated from roots of 15‐day‐old pea (Pisum sativum L. cv. Azad) plants exposed to environmentally relevant (20 µm ) and acute (200 µm ) concentrations of chromium for 7 d. A concentration ‐dependent inactivation of electron transport activity from both NADH to O₂ (NADH oxidase) and succinate to O₂ (succinate oxidase) was observed. The electron transport activity was more sensitive to Cr⁶⁺ with NADH as the substrate than with succinate as the substrate. Although NADH dehydrogenase and succinate dehydrogenase were less affected, NADH: cytochrome c oxidoreductase and succinate: cytochrome c oxidoreductase activities were prominently affected by Cr⁶⁺. Cytochrome oxidase was the most susceptible complex of mitochondrial membranes to Cr⁶⁺, exhibiting maximal inactivation of activity both at 20 and 200 µm chromium concentrations. Cr⁶⁺ increased the generation of O₂^– radicals. This effect was more evident at 200 than at 20 µm . A significant increase in lipid peroxidation of mitochondrial membranes at 200 µm Cr⁶⁺ was the physiological impact of the metal‐induced enhanced generation of O₂^– radicals. An increase in superoxide dismutase (SOD) activity at 20 µm Cr⁶⁺ towards enhanced production of O₂^– radicals appeared to be a defence response in pea root mitochondria that, however, could not be sustained at 200 µm Cr⁶⁺. The results obtained concerning inactivation of mitochondrial electron transport and subsequent enhancement in the generation of O₂^– radicals suggest that root mitochondria are an important target of Cr⁶⁺‐induced oxidative stress in pea.     

Laccase-Catalyzed Oxidation of Mn2+ in the Presence of Natural Mn3+ Chelators as a Novel Source of Extracellular H2O2 Production and Its Impact on Manganese Peroxidase

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn²⁺ to Mn³⁺, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn³⁺ complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H₂O₂ formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H₂O₂ production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn²⁺ oxidation and abiotic decomposition of these organic chelators by the resulting Mn³⁺, which leads to formation of superoxide and its subsequent reduction to H₂O₂. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn³⁺ complexes in assays containing 1 mM Mn²⁺ and 100 mM oxalate or malonate, but omitting an additional H₂O₂ source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn²⁺ oxidation in providing H₂O₂ for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.     

Rationally designed molecules for resurgence of cyanide mitigated cytochrome c oxidase activity

Cytochrome c oxidase (CcOX) containing binuclear heme a₃-Cu B centre (BNC) mechanises the process of electron transfer in the last phase of cellular respiration. The molecular modelling based structural analysis of CcOX – heme a₃-Cu B complex was performed and the disturbance to this complex under cyanide poisoning conditions was investigated. Taking into consideration the results of molecular docking studies, new chemical entities were developed for clipping cyanide from the enzyme and restoring its normal function. It was found that the molecules obtained by combining syringaldehyde, oxindole and chrysin moieties bearing propyl/butyl spacing groups occupy the BNC region and effectively remove cyanide bound to the enzyme. The binding constant of compound 2 with CN^– was 2.3 × 10⁵ M⁻¹ and its ED₅₀ for restoring the cyanide bound CcOX activity in 10 min was 16 µM. The compound interacted with CN^– over the pH range 5–10. The comparison of the loss of enzymatic activity in the presence of CN^– and resumption of enzymatic activity by compound 2 mediated removal of CN^– indicated the efficacy of the compound as antidote of cyanide.     

Trypanosoma cruzi phospho enol pyruvate carboxykinase (ATP-dependent) : transition metal ion requirement for activity and sulfhydryl group reactivity

We studied the transition metal ion requirements for activity and sulfhydryl group reactivity in phosphoenolpyruvate carboxykinase (PEP-carboxykinase; ATP:oxaloacetate carboxylase (transphosphorylating), EC 4.1.1.49), a key enzyme in the energy metabolism of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi. As for other PEP-carboxykinases this enzyme has a strict requirement of transition metal ions for activity, even in the presence of excess Mg²⁺ ions for the carboxylation reaction; the order of effectiveness of these ions as enzyme activators was: Co²⁺ > Mn²⁺ > Cd^u2+ > Ni²⁺ ⪢ Fe²⁺ > VO²⁺, while Zn²⁺ and Ca²⁺ had no activating effects. When we investigated the effect of varying the type or concentration of the transition metal ions on the kinetic parameters of the enzyme the results suggested that the stimulatory effects of the transition metal center were mostly associated with the activation of the relatively inert CO₂ substrate. The inhibitory effects of 3-mercaptopicolinic acid (3MP) on the enzyme were found to depend on the transition metal ion activator: for the Mn²⁺ activated enzyme the inhibition was purely non-competitive (K_ii = K_is) towards all substrates, while for the Co²⁺-activated enzyme the inhibitor was much less effective, produced a mixed-type inhibition and affected differentially the interaction of the enzyme with its substrates. The modification of a single, highly reactive, cysteine per enzyme molecule by 5,5′-dithiobis(2-nitro-benzoate) (DTNB) lead to an almost complete inhibition of Mn²⁺-activated T. cruzi PEP-carboxykinase; however, in contrast with the results of previous studies in vertebrate and yeast enzymes, the substrate ADP slowed the chemical modification and enzyme inactivation but did not prevent it. PEP and HCO₃⁻ had no significant effect on the rate or extent of the enzyme inactivation. The kinetics of the enzyme inactivation by DTNB was also dependent on the transition metal activator, being much slower for the Co²⁺-activated enzyme than for its Mn²⁺-activated counterpart. When the bulkier but more hydrophobic reagent N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) was used the enzyme was slowly and incompletely inactivated in the presence of Mn²⁺ and ADP afforded almost complete protection from inactivation; in the presence of Co²⁺ the enzyme was completely resistant to inactivation. Taken together, our results indicate that the parasite enzyme has a specific requirement of transition metal ions for activity and that they modulate the reactivity of a single, essential thiol group, different from the hyperreactive cysteines present in vertebrate or yeast enzymes.     

A novel system of galangin–potassium permanganate–polyphosphoric acid for the determination of tryptophan and its chemiluminescence mechanism

A novel galangin–potassium permanganate (KMnO₄)–polyphosphoric acid (PPA) system was found to have an outstanding response to tryptophan (Trp). Trp determination using this KMnO₄–PPA system was enhanced significantly in the presence of galangin. A highly sensitive flow‐injection chemiluminescence (CL) method to determine Trp was developed based on the CL reaction of galangin–KMnO₄–Trp in PPA media. The presence of galangin, a member of the flavonol class of flavonoid complexes, greatly increased the luminous intensity of Trp in KMnO₄–PPA systems. Under optimized conditions, Trp was determined in the 0.05–10 µg/mL range, with a detection limit (3σ) of 5.0 × 10^?3 µg/mL. The relative standard deviation (RSD) was 1.0% for 11 replicate determinations of 1.0 µg/mL Trp. Two synthetic samples were determined selectively with recoveries of 98.4–100.1% in the presence of other amino acids. The possible mechanism is summarized as follows: excited states of Mn(II)^* and Mn(III ^* types are the main means of generating chemical luminescent species, and Trp concentration and luminescence intensity have a linear relationship, which enables quantitative analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Luminescent and hydrophilic LaF3–polymer nanocomposite for DNA detection

Luminescent LaF₃–Ce³⁺/Tb³⁺ nanocrystals have been successfully prepared via a simple wet chemical technique. For the next bioapplication, these nanoparticles dispersed in cyclohexane have also been functionalized with poly(St‐co‐MAA), based on a designed oil‐in‐water microemulsion system. These polymer‐coated nanospheres are water‐soluble and bioconjugable. Unlike semiconductor quantum dots, the as‐prepared lanthanum fluoride nanocrystals possess non‐size‐dependent emissions and completely stable photocycles. With functionalized LaF₃ nanospheres as fluorescence probes, a fluorescence method was developed for the rapid quantitative analysis of DNA, due to the quenching effect of fluorescence by the DNA. Under optimum conditions, the fluorescence intensity was proportional to the concentration of the introduced DNA over the range 2.5–35 µg/mL for calf thymus DNA (ctDNA) and 2.5–30 µg/mL for fish sperm DNA (fsDNA), respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

An amperometric enzyme biosensor for real‐time measurements of cellobiohydrolase activity on insoluble cellulose

An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi‐crystalline and amorphous, can be monitored directly and in real‐time by an enzyme‐modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross‐linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current of the reduced mediator, hydroquinone, produced by the CDH‐catalyzed reaction with cellobiose, was recorded under constant‐potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH‐biosensors showed high sensitivity (87.7 µA mM^?1 cm^?2), low detection limit (25 nM), and fast response time (t_95% ～ 3 s) and this provided experimental access to the transient kinetics of cellobiohydrolases acting on insoluble cellulose. The response from the CDH‐biosensor during enzymatic hydrolysis was corrected for the specificity of PcCDH for the β‐anomer of cello‐oligosaccharides and the approach were validated against HPLC. It is suggested that quantitative, real‐time data on pure insoluble cellulose substrates will be useful in attempts to probe the molecular mechanism underlying enzymatic hydrolysis of cellulose. Biotechnol. Bioeng. 2012; 109: 3199–3204. © 2012 Wiley Periodicals, Inc.     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

The hamster adipocyte adenylate cyclase system I. Regulation of enzyme stimulation and inhibition by manganese and magnesium ions

In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E₁, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn²⁺ and Mg²⁺, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn²⁺ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E₁ (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn²⁺, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E_1^?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn²⁺, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn²⁺ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg²⁺, although about 100-fold higher concentrations of Mg²⁺ than of Mn²⁺ were required. The data indicate that Mn²⁺at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn²⁺ on the functions of the guanine nucleotide site(s) in the system.     

Characterization of glutamine synthetase in the apple

Glutamine synthetase (l -glutamate: ammonia ligase, ADP-forming, EC 6.3.1.2) in bark tissue of the apple (Malus domestica Borkh. cv. Golden Delicious) was partially purified and characterized. The Mn²⁺- and Mg²⁺-dependent activities were maximal at pH 7.2 and 7.5, respectively. The enzyme was almost completely inactivated within two weeks at 0°C. Both Mg²⁺ and β-mercaptoethanol were effective in stabilizing the enzyme during storage. The enzyme was protected from thermal inactivation at 60°C by the addition of Mg²⁺ and ATP. One-tenth mM phenylmercuric acetate inhibited the Mg²⁺-dependent activity by 50%. Equimolar dithiothreitol protected the enzyme from this inactivation. The K_m values of the enzyme were 0.27, 7.35, and 0.69 mM for ATP, glutamate, and NH₂OH, respectively. The constant for NH⁺₄ was an order of magnitude higher in the presence of Mn²⁺ than Mg²⁺. When the amino acids were externally added to the reaction mixtures, the measurement of P_i exhibited a higher degree of enzyme inhibition than the measurement of γ-glutamyl monohydroxamate (GHA). Ten mM histidine inhibited the Mg²⁺- and Mn²⁺-dependent activities by 26 and 45% respectively. Twenty mM aspartate (d,l -form) inhibited the enzyme 30% in the presence of either Mg²⁺ or Mn²⁺. Aspartate (Mg²⁺-dependent) and histidine (Mn²⁺-dependent) inhibited the enzyme competitively with respect to glutamate, the estimated inhibition constants being 17.6 and 1.6 mM, respectively. At 10 mM, amino acids such as tryptophan, arginine, alanine and citrulline inhibited enzyme activity from 1 to 18%. Glutamine stimulated the Mg²⁺-dependent activity 25% at 25 mM when GHA was measured. Glutamine above 32 mM inhibited the enzyme.     

Reversible inactivation of nitrate reductase in Chlorella vulgaris in vivo

Summary The NADH-nitrate oxidoreductase of Chlorella vulgaris has an inactive form which has previously been shown to be a cyanide complex of the reduced enzyme. This inactive enzyme can be reactivated by treatment with ferricyanide in vitro. In the present study, the activation state of the enzyme was determined after different prior in vivo programs involving environmental variations. Oxygen, nitrate, light and CO₂ all affect the in vivo inactivation of the enzyme in an interdependent manner. In general, the inactivation is stimulated by O₂ and inhibited by nitrate and CO₂. Light may stimulate or inhibit, depending on conditions. Thus, the effects of CO₂ and nitrate (inhibition of reversible inactivation) are clearly manifested only in the light. In contrast, light stimulates the inactivation in the presence of oxygen and the absence of CO₂ and nitrate. Since the inactivation of the enzyme requires HCN and NADH, and it is improbable that O₂ stimulates NADH formation, it is reasonable to conclude that HCN is formed as the result of an oxidation reaction (which is stimulated by light). The formation of HCN is probably stimulated by Mn²⁺, since the formation of reversibly-inactivated enzyme is impaired in Mn²⁺-deficient cells. The prevention of enzyme inactivation by nitrate in vivo is in keeping with previous in vitro results showing that nitrate prevents inactivation by maintaining the enzyme in the oxidized form. A stimulation of nitrate uptake by CO₂ and light could account for the effect of CO₂ (prevention of inactivation) which is seen mainly in the presence of nitrate and light. Ammonia added in the presence of nitrate has the same effect on the enzyme as removing nitrate (promotion of reversible inactivation). Ammonia added in the absence of nitrate has little extra effect. It is therefore likely that ammonia acts by preventing nitrate uptake. The uncoupler, carbonylcyanide-m-chloro-phenylhydrazone, causes enzyme inactivation because it acts as a good HCN precursor, particularly in the light. Nitrite, arsenate and dinitrophenol cause an enzyme inactivation which can not be reversed by ferricyanide in crude extracts. This suggests that there are at least two different ways in which the enzyme can be inactivated rather rapidly in vivo.     

Role of W181 in modulating kinetic properties of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase

Hypoxanthine‐guanine‐xanthine phosphoribosyltransference (HGXPRT), a key enzyme in the purine salvage pathway of the malarial parasite, Plasmodium falciparum (Pf), catalyses the conversion of hypoxanthine, guanine, and xanthine to their corresponding mononucleotides; IMP, GMP, and XMP, respectively. Out of the five active site loops (I, II, III, III', and IV) in PfHGXPRT, loop III' facilitates the closure of the hood over the core domain which is the penultimate step during enzymatic catalysis. PfHGXPRT mutants were constructed wherein Trp 181 in loop III' was substituted with Ser, Thr, Tyr, and Phe. The mutants (W181S, W181Y and W181F), when examined for xanthine phosphoribosylation activity, showed an increase in K_m for PRPP by 2.1‐3.4 fold under unactivated condition and a decrease in catalytic efficiency by more than 5‐fold under activated condition as compared to that of the wild‐type enzyme. The W181T mutant showed 10‐fold reduced xanthine phosphoribosylation activity. Furthermore, molecular dynamics simulations of WT and in silico W181S/Y/F/T PfHGXPRT mutants bound to IMP.PPi.Mg²⁺ have been carried out to address the effect of the mutation of W181 on the overall dynamics of the systems and identify local changes in loop III'. Dynamic cross‐correlation analyses show a communication between loop III' and the substrate binding site. Differential cross‐correlation maps indicate altered communication among different regions in the mutants. Changes in the local contacts and hydrogen bonding between residue 181 with the nearby residues cause altered substrate affinity and catalytic efficiency of the mutant enzymes. Proteins 2016; 84:1658–1669. © 2016 Wiley Periodicals, Inc.     

Manganese dependent production of 5′-nucleotidase by alkalophilic Bacillus No. C-3

Alkalophilic Bacillus no. C-3 isolated from soil produced 5′-nucleotidase (EC 3.1.3.5) extracellularly when cultured in a medium containing Mn²⁺. The unique point of enzyme production is that the enzyme was produced well in the medium containing a rather high concentration of Mn²⁺, in spite of a small difference in growth. The optimum concentration of Mn²⁺ for the enzyme production was 10 mM and over. Mn²⁺ could not be replaced by other divalent cations when added singly. In the presence of 10 mM Mn²⁺, the enzyme production was repressed by the addition of 0.5 mM phosphate to the medium. The course of the enzyme production closely paralleled the increase in growth. The optimum pH for the enzyme activity was 9.2–9.5, and KHCO₃-K₂CO₃ buffer was suitable for the enzyme.     

Determination of sulpiride in pharmaceutical preparations and biological fluids using a Cr (III) enhanced chemiluminescence method

A highly sensitive and simple method for identifying sulpiride in pharmaceutical formulations and biological fluids is presented. The method is based on increased chemiluminescence (CL) intensity of a luminol–H₂O₂ system in response to the addition of Cr (III) under alkaline conditions. The CL intensity of the luminol–H₂O₂–Cr (III) system was greatly enhanced by the addition of sulpiride and the CL intensity was proportional to the concentration of sulpiride in a sample solution. Various parameters affecting the CL intensity were systematically investigated and optimized for determination of the sulpiride in a sample. Under the optimum conditions, the CL intensity was proportional to the concentration of sulpiride in the range of 0.068–4.0 µg/mL, with a good correlation coefficient of 0.997. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 8.50 × 10^‐6 µg/mL and 2.83 × 10^‐5 µg/mL, respectively. The method presented here produced good reproducibility with a relative standard deviation (RSD) of 2.70% (n = 7). The effects of common excipients and metal ions were studied for their interference effect. The method was validated statistically through recovery studies and successfully applied for the determination of sulpiride in pure form, pharmaceutical preparations and spiked human plasma samples. The percentage recoveries were found to range from 99.10 to 100.05% for pure form, 98.12 to 100.18% for pharmaceutical preparations and 97.9 to 101.4% for spiked human plasma. Copyright © 2012 John Wiley & Sons, Ltd.     

Fluoride and beryllium interact with the (Na + K)-dependent ATPase as analogs of phosphate

Fluoride irreversibly inhibits the (Na + K)-ATPase, and this inactivation requires divalent cations (Mg²⁺, Mn²⁺, or Ca²⁺), is augmented by K⁺, but is diminished by Na⁺ and by ATP. Prior incubation with the aluminum chelator deferoxamine markedly slows inactivation, whereas adding 1 µM AlCl₃ speeds it, consistent with AlF _– ⁴ being the active species. Prior incubation of the enzyme with vanadate also blocks inactivation by fluoride added subsequently. Fluoride stimulates ouabain binding to the enzyme, and thus the analogy between AlF _– ⁴ and both orthophosphate and orthovanadate is reflected not only in the similar dependence on specific ligands for their enzyme interactions and their apparent competition for the same sites, but also in their common ability to promote ouabain binding. Beryllium also irreversibly inhibits the enzyme, and this inactivation again requires divalent cations, is augmented by K⁺, but is diminished by Na⁺ and by ATP. Similarly, prior incubation of the enzyme with vanadate blocks inactivation by beryllium added subsequently. Inactivation by beryllium, however, does not require a halide, and, unlike inactivation by fluoride, increases at basic pHs. These observations suggest that beryllium, as beryllium hydroxide complexes, acts as a phosphate analog, similar to AlF _– ⁴ and vanadate.Abbreviations EDTA ethylenediaminetetraacetate - EGTA Ethyleneglycol-bis(-aminoethylether)-N,N-tetraacetate - FITC fluorescein isothiocyanate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)1,4-piperazine diethanesulfonic acid