A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 ± 0.1 Å. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete α-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an α-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.

Conformational coupling in the chemotaxis response regulator CheY

CheY, a response regulator protein in bacterial chemotaxis, serves as a prototype for the analysis of response regulator function in two-component signal transduction. Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ. The objective of this study was to probe the conformational coupling between the phosphorylation site and the signaling surface of CheY in the reverse direction by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatically impacted autophosphorylation of CheY by small molecule phosphodonors, which is indicative of reverse signal propagation in CheY. Autodephosphorylation and substrate affinity, however, were not significantly affected. Kinetic characterization of several CheY mutants suggested that conserved residues Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, are not essential for conformational coupling. These findings provide structural and conceptual insights into the mechanism of CheY activation. Our results are consistent with a multistate thermodynamic model of response regulator activation.

In vitro characterization of rosiglitazone metabolites and determination of the kinetic parameters employing rat liver microsomal fraction

Rosiglitazone (RSG), a thiazolidinedione antidiabetic drug, is metabolized by CYP450 enzymes into two main metabolites: N-desmethyl rosiglitazone (N-Dm-R) and rho-hydroxy rosiglitazone (rho-OH-R). In humans, CYP2C8 appears to have a major role in RSG metabolism. On the other hand, the in vitro metabolism of RSG in animals has not been described in literature yet. Based on these concerns, the kinetic metabolism study of RSG using rat liver microsomal fraction is described for the first time. Maximum velocity (V (max)) values of 87.29 and 51.09 nmol/min/mg protein were observed for N-Dm-R and rho-OH-R, respectively. Michaelis-Menten constant (K (m)) values were of 58.12 and 78.52 mu M for N-Dm-R and rho-OH-R, respectively. Therefore, these results demonstrated that this in vitro metabolism model presents the capacity of forming higher levels of N-Dm-R than of rho-OH-R, which also happens in humans. Three other metabolites were identified employing mass spectrometry detection under positive electrospray ionization: ortho-hydroxy-rosiglitazone (omicron-OH-R) and two isomers of N-desmethyl hydroxy-rosiglitazone. These metabolites have also been observed in humans. The results observed in this study indicate that rats could be a satisfactory model for RSG metabolism.

A new approach to evaluate immobilization of β-galactosidase on Eupergit® C: structural, kinetic, and thermal characterization

This study aimed to evaluate β-galactosidase immobilization. For this purpose, the ionic strength of the buffer, reaction time, amount of the immobilization support, and pH were evaluated by a central composite design. Assay 8, which consisted of 1.5 mol L-1 phosphate buffer (pH 7.5) and a reaction time of 2 h, produced the maximum yield. Eupergit® C (400 mg) was subsequently used as an immobilization support. Immobilization kinetics wereinvestigated, and a significant increase in the yield was obtained after immobilization compared with that obtained from assay 8 (22.0 U mL-1 vs. 15.6 U mL-1). The enzyme efficiency of actuation was evaluated using o-nitrophenyl-β-D-galactopyranoside and lactose, with lactose providing better results. The reuse of β-galactosidase was evaluated, and more than 50% of the initial enzyme activity was maintained after five cycles of use. Enzyme characterization revealed that immobilization improved some aspects of the thermostability of β-galactosidase.

Synthesis, characterization and kinetic studies on complex formed between amantadine hydrochloride and sodium molybdate at physiological pH

The title reaction was undertaken to establish the interaction between amantadine and molybdate at physiological pH. Identical FTIR spectra, TG-DTA curves and CHN data of the complexes formed from three solutions at pH 1.5, 7.4 and 8.0 indicate that the same complex was formed at all the three pHs. The FTIR spectrum shows shift in peaks corresponding to primary amino group of the drug due to coordination to molybdate. An octahedral geometry is assigned to the complex. The kinetics of the complexation has been studied at low concentrations of the reactants using UV-visible spectrophotometry. At pH 7.4, the initial rate varies linearly with [molybdate]. A plot of initial rate versus [drug] is linear passing through origin. These results indicate that the drug and molybdate react at pH 7.4 even at low concentrations. At pH 1.5, the rate increases linearly with increase in [drug] but decreases with [molybdate]. The effect of pH and ionic strength on the rate of the reaction has also been studied. A suitable mechanism has been proposed for the reaction. Reaction between the drug and molybdate even at low concentrations and the fact that the amino group of amantadine required to be free for its function as antiviral, is bound to molybdate in the complex suggests that simultaneous administration of the drug and molybdate supplements should be avoided.

Pozzolanic behavior of bamboo leaf ash: Characterization and determination of the kinetic parameters

The paper presents a characterization and study of the pozzolanic behavior between calcium hydroxide (CH) and bamboo leaf ash (BLAsh), which was obtained by calcining bamboo leaves at 600 degrees C for 2 h in a laboratory electric furnace. To evaluate the pozzolanic behavior the conductometric method was used, which is based on the measurement of the electrical conductivity in a BLAsh/CH solution with the reaction time. Later, the kinetic parameters are quantified by applying a kinetic-diffusive model. The kinetic parameters that characterize the process (in particular, the reaction rate constant and free energy of activation) were determined with relative accuracy in the fitting process of the model. The pozzolanic activity is quantitatively evaluated according to the values obtained of the kinetic parameters. Other experimental techniques, such as X-ray diffraction (XRD) and scanning electron microscopy (SEM), were also employed. The results show that this kind of ash is formed by silica with a completely amorphous nature and a high pozzolanic activity. The correlation between the values of free energy of activation (Delta G(#)) and the reaction rate constants (K) are in correspondence with the theoretical studies about the rate processes reported in the literature. (C) 2010 Elsevier Ltd. All rights reserved.

Kinetic and mechanistic characterization of the Sphingomyelinases D from Loxosceles intermedia spider venom

Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide-phosphate and choline as well as the cleavage of lysophosphatidyl choline generating the lipid mediator lysophosphatidic acid. We have, previously, cloned and expressed two functional SMase D isoforms, named P1 and P2, from Loxosceles intertnedia venom and comparative protein sequence analysis revealed that they are highly homologous to SMase I from Loxosceles laeta which folds to form an (alpha/beta)(8) barrel. In order to further characterize these proteins, pH dependence kinetic experiments and chemical modification of the two active SMases D isoforms were performed. We show here that the amino acids involved in catalysis and in the metal ion binding sites are strictly conserved in the SMase D isoforms from L. intermedia. However, the kinetic studies indicate that SMase P1 hydrolyzes sphingomyelin less efficiently than P2, which can be attributed to a substitution at position 203 (Pro-Leu) and local amino acid substitutions in the hydrophobic channel that could probably play a role in the substrate recognition and binding. (c) 2005 Elsevier Ltd. All rights reserved.

Synthesis and characterization of poly-o-toluidine: Kinetic and structural aspects

Poly(o-methylaniline) (poly-o-toluidine, PTOL) was synthesized by chemical oxidation of o-toluidine with ammonium peroxydisulfate in an aqueous 1.0 mol L -1 HCl solution. The progress of polymerization was followed by measuring the open-circuit potential (OCP) of a Pt electrode immersed in the reaction medium with the polymerization time. The chemical synthesis of PTOL was carried out at different monomer:oxidant (M:O) molar ratios (4:1, 2:1, 1.5:1, 1:1, and 0.66:1), and the products obtained were characterized by infrared spectroscopy, gel permeation chromatography, and X-ray diffraction. The molecular weight and percentage of crystallinity of PTOL are higher for samples synthesized in an excess of the monomer, i.e. at higher M:O ratios. However, the yield of PTOL prepared at higher M:O ratios is considerably low, in particular at a 4:1 M:O ratio, which is the M:O ratio most commonly used in the literature to synthesize polyaniline and its derivatives.

Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community

The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 1 6S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16SrDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like 'Dehalococcoides ethenogenes' or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56(.)6%) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18(.)5%), the Chloroflexi phylum (16(.)4%), the Bacteroidetes phylum (6(.)3%), the Synergistes genus (11(.)1%) and a lineage that could not be affiliated with existing phyla (11(.)1%). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured microorganisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.

Isopeptidase activity of human transglutaminase 2:disconnection from transamidation and characterization by kinetic parameters

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca2+-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins or γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the Km and the Vmax kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2 -driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of cross-linked proteins correlates with the manifestation of degenerative disorders.

Cluster identification and characterization in the riser of a circulating fluidized bed from numerical simulation results

A methodology of identification and characterization of coherent structures mostly known as clusters is applied to hydrodynamic results of numerical simulation generated for the riser of a circulating fluidized bed. The numerical simulation is performed using the MICEFLOW code, which includes the two-fluids IIT`s hydrodynamic model B. The methodology for cluster characterization that is used is based in the determination of four characteristics, related to average life time, average volumetric fraction of solid, existing time fraction and frequency of occurrence. The identification of clusters is performed by applying a criterion related to the time average value of the volumetric solid fraction. A qualitative rather than quantitative analysis is performed mainly owing to the unavailability of operational data used in the considered experiments. Concerning qualitative analysis, the simulation results are in good agreement with literature. Some quantitative comparisons between predictions and experiment were also presented to emphasize the capability of the modeling procedure regarding the analysis of macroscopic scale coherent structures. (c) 2007 Elsevier Inc. All rights reserved.

Characterization and kinetics of sulfide-oxidizing autotrophic denitrification in batch reactors containing suspended and immobilized cells

Sulfide-oxidizing autotrophic denitrification is an advantageous alternative over heterotrophic denitrification, and may have potential for nitrogen removal of low-strength wastewaters, such as anaerobically pre-treated domestic sewage. This study evaluated the fundamentals and kinetics of this process in batch reactors containing suspended and immobilized cells. Batch tests were performed for different NO(x)(-)/S(2-) ratios and using nitrate and nitrite as electron acceptors. Autotrophic denitrification was observed for both electron acceptors, and NO(x)(-)/S(2-) ratios defined whether sulfide oxidation was complete or not. Kinetic parameter values obtained for nitrate were higher than for nitrite as electron acceptor. Zero-order models were better adjusted to profiles obtained for suspended cell reactors, whereas first-order models were more adequate for immobilized cell reactors. However, in the latter, mass transfer physical phenomena had a significant effect on kinetics based on biochemical reactions. Results showed that sulfide-oxidizing autotrophic denitrification can be successfully established for low-strength wastewaters and have potential for nitrogen removal from anaerobically pre-treated domestic sewage.

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Development and characterization of novel potent and stable inhibitors of endopeptidase EC 3.4.24.15

Solid-phase synthesis was used to prepare a series of modifications to the selective and potent inhibitor of endopeptidase EC 3.4.24.15 (EP24.15), N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), which is degraded at the Ala-Tyr bond, thus severely limiting its utility in vivo. Reducing the amide bond between the Ala and Tyr decreased the potency of the inhibitor to 1/1000. However, the replacement of the second alanine residue immediately adjacent to the tyrosine with alpha-aminoisobutyric acid gave a compound (JA-2) that was equipotent with cFP, with a K-i of 23 nM. Like cFP, JA-2 inhibited the closely related endopeptidase EC 3.4.24.16 1/20 to 1/30 as potently as it did EP24.15, and did not inhibit the other thermolysin-like endopeptidases angiotensin-converting enzyme, endothelin-converting enzyme and neutral endopeptidase. The biological stability of JA-2 was investigated by incubation with a number of membrane and soluble sheep tissue extracts. In contrast with cFP, JA-2 remained intact after 48 h of incubation with all tissues examined. Further modifications to the JA-2 compound failed to improve the potency of this inhibitor. Hence JA-2 is a potent, EP24.15-preferential and biologically stable inhibitor, therefore providing a valuable tool for further assessing the biological functions of EP24.15.

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.