Synthesis of p-trifluoroacetamidophenyl O-α-D-galactopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→4)-α-L-rhamnopyranoside

The role of exposed tyrosine side-chains in enzyme-catalysed reactions has been studied for porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase using N-acetylimidazole as the specific protein reagent. The changes in activity binding affinity (Δk_?1/k₊₁), and kinetic parameters (K_m,k₂) due to acetylation of the phenolic hydroxyl groups have been determined. Acetylation of each enzyme occurred by an “apparent” first-order reaction with a rate constant of 0.72–1.4 x 10^?1min^?1. Acetylation increased the apparent K_m (soluble starch as the substrate) for each enzyme (appreciably for alpha-amylase and glucamylase), whereas k² remained unchanged. Similarly, for each enzyme, the binding affinity for immobilised cyclohexa-amylose decreased appreciably, whereas the catalytic activity was reduced only to a small degree (and remained unchanged for beta-amylase). It is concluded that the tyrosine groups located in the active centre of each enzyme have a substrate-binding function.     

A study by means of lactone inhibition of the role of a “half-chair” glycosyl conformation at the active centre of amylolytic enzymes

The kinetics of inhibition of porcine-pancreatic alpha amylase, sweet-potato beta amylase, and Aspergillus niger glucamylase enzymes have been studied by use of d-glucono-l,5-lactone and maltobiono-1,5-lactone as transition-state analogs. With d-glucono-1,5-lactone, alpha amylase can be inhibited, to a degree, non-competitively (K_i0.81mM,β≈e0.2), whereas with maltobionolactone, the inhibition is competitive (K_i10.31mM). The effect of beta amylase can be inhibited with maltobionolactone in a completely competitive way (K_iO.11mM), whereas with d-gluconolactone the inhibition is very poor (K_i21mM). Glucoamylase cannot be inhibited with maltobionolactone, whereas with d-gluconic acid, a completely mixed inhibition way be observed (K_i1.3mM). The ratio of the binding affinity of the lactones, products, and substrates, permits the conclusion that ring distortion takes place in the transition state with all three enzymes.     

Action of sweet-potato beta-amylase on phosphodextrin of potato starch

The specificity of sweet-potato beta-amylase in the vicinity of the phosphate ester groups was studied by determining the structures of the phosphorylated oligosaccharides (alpha-phosphodextrin and beta-limit-alpha-phosphodextrin) formed by its action on potato starch. The beta-limit-alpha-phosphodextrin was separated by chromatography on Dowex-1 (HCOO^?) resin into three fractions that were distinguishable by the d.p. and by the ratio of d-glucose 6-phosphate residues to total organic phosphate. Each fraction contained linear molecules having one phosphate ester group that was not located at the reducing or non-reducing terminals. The smallest phosphodextrin was 6²-phosphorylmaltotriose. It was deduced that beta-amylase hydrolysed (1→4)-α-d linkages from the non-reducing end until one or two d-glucosyl residues remained attached to the phosphorylated residue, depending on whether there was originally an odd or even number of glucosyl residues on the non-reducing side of the phosphorylated residue.     

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [¹⁴C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the ¹⁴C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.     

Affinity labeling of the active site of aspergillopeptidase B

Aspergillopeptidase B exhibits a strong preference for both the phenyl group and the carbobenzoxy moiety of carbobenzoxyphenylalanine methylester. The chloromethylketones of N⁶-tosyllysine and tosylphenylalanine do not inactivate the enzyme due to low binding affinities. Aspergillopeptidase B is slowly inactivated by the bromomethylketone of carbobenzoxyphenylalanine with an apparent second-order rate constant of 0.16 m^?1 sec^?1 at pH 7.0. A direct correspondence exists between the loss of activity, incorporation of tritiated carbobenzoxyphenylalanine, the disappearance of a histidine residue, and the appearance of a residue of N^r-carboxymethylhistidine. The rate of alkylation by carbobenzoxyphenylalanine is retarded by the competitive inhibitor N²-benzoylarginine.     

An aminopeptidase from Agave americana,chemical properties of the enzyme

Agave aminopeptidase, a new enzyme obtained from the plant Agave americana displayed activity towards a variety of substrates. A free alphaamino group on these substrates was essential, but the enzyme did not need any metal ions for optimal activity. Aliphatic, aromatic and basic amino acids situated at the amino terminal end of substrates could be hydrolysed by the enzyme. The enzyme had no endopeptidase or other proteolytic activity. Values of the apparent Michaelis constants for different amino acid substrates, all in the range from 0.1 to 0.6 × 10^?3 M, suggested a relative wide specificity. The pK-values of the two dissociating groups on the enzyme taking part in the catalytic process were pH 6.3 to 6.8 and pH 7.5 to 7.8. These and other studies suggested that histidine plays an active role in the catalytic process. The enzyme was inhibited competitively by free amino acids and this, together with other results, implied a compulsory order of product release.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

Enantioselective Surface Plasmon Resonance Sensor Based on C60 Fullerene‐Glutathione Self‐Assembled Monolayer (SAM)

A novel enantioselective surface plasmon resonance (SPR) sensor based on a self‐assembled monolayer of C₆₀ fullerene as the chiral selector is proposed. A binding assay, apparent affinity constant, and apparent dissociation binding constant were used to analyze and study the enantioselectivity of C₆₀ fullerene‐glutathione film for L‐histidine, which was chosen as the model analyte. The apparent affinity constant for the complex formed by L‐histidine with C₆₀ fullerene‐glutathione film was 5.2 x 10⁹ M^‐1. The proposed SPR sensor can be used for the assay of L‐histidine in the 10^‐10 – 10^‐7 mol/L concentration range. Chirality 26:129–131, 2014. © 2014 Wiley Periodicals, Inc.     

The binding of phloridzin to the isolated luminal membrane of the renal proximal tubule

The binding of [³H]ploridzin by isolated luminal membranes of the rabbit proximal tubule and by slices of rabbit kidney cortex was studied.Kinetic analyses of the relationship between the concentration of phloridizin in the incubation medium and the binding of phloridzin to the membrane indicated two distinct classes of receptors sites. One class, comprising high affinity sites, reached saturation at 20–25 μM phloridzin, had a K(phloridzin) of 8 μM, and 8·10⁺² nmoles interacted with 1 mg of brush border protein. The other class, comprising low affinity sites, had a K(phloridzin) of 2.5 mM, and the number of binding sites was 1.25 nmoles/mg Na⁺ was required for the binding of phloridzin at the high affinity sites. Na⁺ decreased the apparent K_i for phloridzin; the apparent V of binding was not altered. Binding at the low affinity sites was independent of Na⁺. Ca²⁺ was necessary for maximal binding at the high affinity sites. Binding of phloridzin at high affinity sites was more sensitive to N-ethylmalcimide and mersalyl than was binding at low affinity sites. Binding at high affinity sites, but not at low affinity sites, was temperature dependent.d-Glucose was a competitive inhibitor of the high affinity binding of phloridzin. The apparent K₁ was 1 mM. D-Glucoe inhibited non-competitively at the low affinity sites. l-Glucose had no influence on phloridzin binding. Phloretin was a competitive inhibitor of high affinity phloridzin binding with an apparent K_i of 16 μM. Phloretin inhibited low affinity bindings of phloridizin non-competitively. Binding of phloridzin at high affinity sites was completely reversible. Binding at low affinity sites was only partially reversed. Phloridzin bound at high affinity sites on the brush border was displaced by phloridzin and phloretin but not by d-glucose.The mechanism of the high affinity binding of phloridzin was distinguished from that of the initial interaction of d-glucose with the membrane. Binding of phloridzin required Na⁺, whereas the interaction of d-glucose with the membranes had a prominent Na⁺-independent component.Intact renal cells in cortical slices accumulated phloridzin. The uptake did not saturate, was Na⁺ independent, and was not competitively inhibited by sugars. These characteristics resemble those for the low affinity binding of phloridzin by isolated membranes. It is suggested that low affinity binding may represent an initial binding followed by uptake of the glycoside into membrane vesicles.     

On the molecular basis of the high affinity binding of basic amino acids to LAOBP,a periplasmic binding protein from Salmonella typhimurium

The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high‐affinity binding of ligands to proteins is still limited; such is the case for l ‐lysine–l ‐arginine–l ‐ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l ‐arginine, l ‐lysine, and l ‐ornithine with nanomolar affinity and to l ‐histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l ‐histidine and l ‐arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~?300 cal mol^?1 K^?1, most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000‐fold higher affinity of LAOBP for l ‐arginine as compared with l ‐histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy‐driven micromolar affinity toward l ‐arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd.     

Probing the equatorial groove of the hookworm protein and vaccine candidate antigen,Na-ASP-2

Hookworm activation-associated secreted proteins can be structurally classified into at least three different groups. The hallmark feature of Group 1 activation-associated secreted proteins is a prominent equatorial groove, which is inferred to form a ligand binding site. Furthermore, a conserved tandem histidine motif is located in the centre of the groove and believed to provide or support a yet to be determined catalytic activity.Here, we report three-dimensional crystal structures of Na-ASP-2, an L3-secreted activation-associated secreted protein from the human hookworm Necator americanus, which demonstrate transition metal binding ability of the conserved tandem histidine motif. We further identified moderate phosphohydrolase activity of recombinant Na-ASP-2, which relates to the tandem histidine motif. By panning a random 12-mer peptide phage library, we identified a peptide with high similarity to the human calcium-activated potassium channel SK3, and confirm binding of the synthetic peptide to recombinant Na-ASP-2 by differential scanning fluorimetry. Potential binding modes of the peptide to Na-ASP-2 were studied by molecular dynamics simulations which clearly identify a preferred topology of the Na-ASP-2:SK3 peptide complex.     

Interaction of the regulatory gene product with the operator site in the l-arabinose operon of Escherichia coli

The DNA binding properties of the araC protein in the absence of l-arabinose have been studied in Escherichia coli using the nitrocellulose membrane filter technique. Equilibrium competition experiments demonstrate that the araC protein binds specifically to the ara operator. The apparent K_m of the interaction is 1 × 10^?12m at 20 °C. The rates of association and dissociation of the complex have also been determined. A k_a of 2 × 10⁹m^?1 s^?1at 20 °C is calculated assuming binding to a single site. The half-life of the complex is three minutes. The equilibrium constant calculated from the ratio of k_a to k_d is 2.8 × 10^?12m at 20 °C. The good agreement between the equilibrium and kinetic determinations of the equilibrium constant suggest that the kinetic studies are providing true rate constants. It is calculated that about 1% of the purified araC protein is active with respect to operator binding activity.     

Metal ion requirement by glutamine synthetase of Escherichia coli in catalysis of gamma-glutamyl transfer.

The requirement for metal ions by glutamine synthetase of Escherichia coli in catalyzing the γ-glutamyl transfer reaction has been investigated. In order of decreasing V at pH 7.0, Cd²⁺, Mn²⁺, Mg²⁺, Ca²⁺, Co²⁺, or Zn²⁺ will support the activity of the unadenylylated enzyme in the presence of ADP. With AMP substituted for ADP to satisfy the nucleotide requirement, only Mn²⁺ or Cd²⁺ will support the activity of the unadenylylated enzyme. Kinetic and equilibrium binding measurements show a 1:1 interaction between the nonconsumable substrate ADP and each enzyme subunit of the dodecamer. (To obtain this result, each enzyme subunit must be active in catalyzing γ-glutamyl transfer.) The stability constant of the unadenylylated subunit for ADP-Mn is 3.5 × 10⁵m^?1, or ～2.86 × 10⁷m^?1 under assay conditions, with arsenate, Mn²⁺, and glutamine being responsible for this large affinity increase. Saturation of two Mn²⁺ ion-binding sites per enzyme subunit is absolutely required for activity expression. While apparently not affecting the affinity of the first Mn²⁺ bound (K′ = 1.89 × 10⁶ M^?1), glutamine increases the stability constant for the second Mn²⁺ bound from 2 × 10⁴ to 5.9 × 10⁵m^?1. Reciprocally, increasing Mn²⁺ concentrations decreases the apparent K_m′ value for glutamine. Glutamine (by producing a net uptake of protons in binding to the enzyme) is responsible for changing the proton release from 3 to about 1 for 2 Mn²⁺ bound per enzyme subunit, with ～0.5 H⁺ displaced in both fast and slow processes. The uv spectral change induced by the binding of the first Mn²⁺ to each enzyme subunit remains unchanged by the presence of glutamine. However, glutamine reduces the half-time of the spectral change or slow proton release from ～30 to ～20 sec at 37 °C. Binding and kinetic results indicate a mechanism involving a random addition of Mn²⁺ to two subunit sites. Saturation of the high-affinity site with Mn²⁺ induces a conformational change to an active configuration, while activity expression depends also on the saturation of a second Mn²⁺ binding site (at or near the catalytic site). Once the first Mn²⁺ binding site of the subunit is saturated, an active enzyme complex can be formed either by the sequential binding of Mn²⁺ and ADP at the second site or by the binding of ADP-Mn complex directly to this site if the concentration of ADP-Mn is greater than 10^?8m in the assay. Some additional observations on the binding of Mg²⁺, Ba²⁺, Ca²⁺, and Zn²⁺ to the enzyme are presented.     

Probing the fatty acid binding site of β-lactoglobulins

The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10^?7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10^?7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpH's lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.     

Cytosol androgen receptor from kidney of normal and testicular feminized (Tfm) mice

Steroid binding has been studied in cytoplasmic extracts of normal mouse kidney, an androgen sensitive organ, and of kidney from mice affected with testicular feminization (Tfm mutant) that have inherited androgen resistance. Macromolecules that bind ³H-5α-dihydrotestosterone (DHT, the presumed active androgen in most testosterone target organs) and sediment in glycerol gradient at 8–9S can be observed in cytosol from kidney of mice of different sex, age, and hormonal history. The 8–9S component from normal females is heat labile, pronase sensitive, and dissociated by high salt to a lower molecular weight entity. The apparent equilibrium dissociation constant (K_d) for the DHT-receptor complex is 1.4 × 10^?9M, and there are about 1500 binding sites per testosterone-sensitive kidney proximal tubule cell. Cytosol from Tfm/Y ^ animals also shows a sharp peak of ³H-DHT-binding activity at 8–9S. The Tfm protein, however, has reduced affinity for DHT and binds only 10–25% as much ³H-DHT as wild-type receptor at ³H-DHT concentrations from 5 × 10^?11M to 1.2 × 10^?8M. Scatchard analysis, and studies involving competition with unlabeled steroids, relative binding of various androgens, and dissociation of the ³H-DHT-binding protein complex after extensive dialysis have led to the conclusion that Tfm kidney contains very little, if any, androgen receptor with properties similar to that found in normal kidney.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.     

pH Effects on the Activity and Regulation of the NAD Malic Enzyme

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg²⁺ and NAD⁺ but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate⁻². This is characteristic of an induced conformational change. The slope of the V_max or V_max/K_m profiles is steeper on the alkaline side of the pH optimum. The K_m for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K_{m pH6.7} = 9.2 millimolar, K_{m pH7.7} = 28.3 millimolar). Similar changes in K_m have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V_max/K_m profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.     

E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

The plasma membrane Ca²⁺?ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeF_x), aluminium fluoride (AlF_x) and magnesium fluoride (MgF_x) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·P_i product states. Our results show that the phosphate analogues BeF_x, AlF_x and MgF_x inhibit PMCA Ca²⁺?ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca²⁺?ATPase inhibition by AlF_x and BeF_x depended on Mg²⁺ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlF_x and BeF_x but not MgF_x apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E?→?E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA.In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.     

Production of Antifungal Chitinase by Aspergillus niger LOCK 62 and Its Potential Role in the Biological Control

Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6?days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43?kDa. The highest activity was obtained at 40?°C for both crude and purified enzymes. The crude chitinase activity was stable during 180?min incubation at 40?°C, but purified chitinase lost about 25?% of its activity under these conditions. Optimal pH for chitinase activity was pH 6–6.5. The activity of crude and purified enzyme was stabilized by Mg²⁺ and Ca²⁺ ions, but inhibited by Hg²⁺ and Pb²⁺ ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected.