The duration of effectiveness of root canal medicaments.
Explore the source record for details and available documents.
Camphor↗
SEARCH · Search PubMed
Results for “CAMPHOR”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Analysis of camphorquinone in visible light-cured composite resins.
Explore the source record for details and available documents.
Camphor↗
Hyposmia following laryngectomy.
Explore the source record for details and available documents.
Acetates↗
Autocatalytic nitration of P450CAM by peroxynitrite.
Peroxynitrite (PN) gains high selectivity as a physiological oxidizing and nitrating agent through catalysis by metal ions. This was established for the heme-thiolate (P450) enzyme prostacyclin synthase which was tyrosine nitrated and inhibited at low PN levels [FEBS Lett. 382 (1996) 101]. Other P450 proteins reacted in a similar manner and a ferryl species (Compound II) has been identified as an intermediate during reactions with PN [Nitric Oxide 3 (1999) 142]. Here we investigated cytochrome P450CAM and found that it catalyzes the decomposition of PN as well as an increased nitration of phenol. The latter at the expense of phenol hydroxylation is characteristic for the proton-assisted PN action. PN also caused self-nitration of P450CAM at several tyrosine residues. Two of them, Y96 and Y305 were largely protected in the presence of the ligand metyrapone. Unlike other heme-thiolate proteins P450CAM did not form distinct spectral intermediates characteristic for Compound II. We conclude that P450CAM serves as a model for the nitration of prostacyclin synthase with respect to its autocatalytic tyrosine nitration and its prevention by blocking the active site.
Binding Sites↗
Enantiomeric discrimination of Ru-substrates by cytochrome P450cam.
Molecules with photosensitizers attached to substrates (Wilker et al., Angew. Chem. Int. Ed. 38 (1999) 90-92) or cofactors (Hamachi et al., J. Am. Chem. Soc. 121 (1999) 5500-5506) can rapidly deliver redox equivalents to buried active sites. The structure of cytochrome P450cam (P450) co-crystallized with a prototypal sensitizer-substrate, [Ru-C9-Ad]Cl2, has been determined (Dmochowski et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12987-12990); and, in separate UV-vis absorption and time-resolved luminescence experiments, the binding of the lambda and delta enantiomers of Ru-C9-Ad to P450 has been measured. The results, KD(delta/lambda) approximately 2, indicate that the bipyridyl ligands of the lambda isomer interact more favorably with hydrophobic residues at the entrance to the substrate channel. We conclude that enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates.
Binding Sites↗
Synthetic active site analogues of heme-thiolate proteins. Characterization and identification of intermediates of the catalytic cycles of cytochrome P450cam and chloroperoxidase.
In view of recent results from different sources, the reaction mechanisms of two heme-thiolate proteins, cytochrome P450cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H2O2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl+ donor chlorinating activated C-H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.
Binding Sites↗
A scanning tunneling microscopy (STM) investigation of complex formation between cytochrome P450(cam) and putidaredoxin.
We have previously reported the scanning tunnelling microscopy (STM) imaging under buffer of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida [Faraday Discuss. 116 (2000) 1]. We describe here the adsorption and STM imaging under buffer of complexes of a mutant of cytochrome P450(cam), K344C, and wild-type putidaredoxin (Pdx) on gold(111). The images of Pdx on its own on gold(111) are not uniform, presumably due to multiple orientations of protein adsorption because of the presence of five or more cysteines on the protein surface. STM imaging of a 1:1 mixture of P450(cam)-K344C/Pdx showed a regular array of pairs of different-sized proteins 20-25 A apart arranged in rows across the gold(111) surface which we attribute to the P450(cam)/Pdx complex. The images of the pairs are more regular than those of Pdx on its own, probably as a result of complex formation with P450(cam) partly overcoming the heterogeneity of Pdx adsorption. As far as we are aware this is the first report of STM imaging of a protein/protein complex, and the first direct observation of P450(cam)/Pdx complex formation which is a key step in the catalytic cycle of P450(cam) catalysis. The redox centers of the two proteins are ca. 20 A apart, too far for rapid intracomplex electron transfer. Whether the observed complex is competent for electron transfer or physiologically relevant is not known, and further work is in progress to elucidate the protein-protein interaction.
Camphor 5-Monooxygenase↗
The involvement of glutamate in recall of the conditioned NK cell response.
The molecular mechanisms responsible for the conditioned enhancement of natural killer (NK) cell activity were investigated. The primary goal of the study was to examine the roles of glutamate and gamma-aminobutyric acid (GABA) in recall of the conditioned NK cell response. Both neurochemical blocking assay and high performance liquid chromatography (HPLC) technique were used in the study. Results from the neurochemical blocking assay demonstrated that glutamate but not GABA was required in recall of the conditioned NK cell response. NMDA but not the kainate/AMPA receptors, are believed to be involved. The levels of glutamate that were released and/or taken up also appeared to be critical in that interruption of glutamate release and/or uptake blocked the conditioned NK cell response. Results from the HPLC analysis, however, did not show any significant difference in the glutamate content between the conditioned and control brains.
Administration, Inhalation↗
Absorption spectral studies on heme ligand interactions of P-450nor.
Heme-external ligand interactions of P-450nor were examined spectrophotometrically and compared with those of other P-450s. Most nitrogenous ligands induced type II spectral changes on binding to ferric P-450nor, as did other P-450s. In contrast with other P-450s, 2-methylpyridine and 1-butanol induced type I changes in the spectrum of P-450nor. No spectral interaction of ferrous P-450nor with these ligands was observed. The absorption spectra of the alkyl isocyanide complexes of ferrous P-450nor exhibited the Soret peak at 427 nm with a slight shoulder at around 455 nm at neutral pH, and this shoulder was intensified as the pH was increased, suggesting that the isocyanide complexes of P-450nor existed in two states (the 430 and 455 nm states) which were in pH-dependent equilibrium in a similar manner to microsomal P-450s. However, the equilibrium was shifted mostly to the 430 nm state in the complexes of P-450nor. The findings suggest that P-450nor, especially its ferrous form, has some distinct features from P-450cam and microsomal P-450s in the distal heme environment.
Binding, Competitive↗
Isothermal titration calorimetric studies on the associations of putidaredoxin to NADH-putidaredoxin reductase and P450cam.
Putidaredoxin (Pdx), an iron-sulfur protein containing a 2Fe-2S cluster, serves as a physiological electron mediator from NADH-putidaredoxin reductase (PdR) to P450cam in the P450cam monooxygenation reaction cycle. Previous studies have revealed that the associations of Pdx with P450cam and PdR are not strongly dominated by electrostatic interactions, although such interactions stabilize most electron-transfer complexes [A.R. De Pascalis, I. Jelesarov, F. Ackermann, W.H. Koppenol, M. Hiroasawa, D.B. Knaff, H.R. Bosshard, Protein Sci. 2 (1993) 1126-1135]. In the present study, to elucidate the interactions dominating the specific associations in the electron-transfer reaction mediated by Pdx, the thermodynamic properties--entropy (delta S), enthalpy (delta H), and heat capacity changes (delta Cp)--for PdR/Pdx and P450cam/Pdx association reactions have been examined by isothermal titration calorimetry (ITC). Although the binding enthalpy change, delta Hbind, for the PdR/Pdx association is positive at 10 degrees C, it declines linearly with temperature in the range 10-22 degrees C and becomes negative above 11 degrees C. On the other hand, the binding entropy change, delta Sbind, is positive at all temperatures examined in this study, indicating that the association of Pdx to PdR is entropically driven. On the basis of the temperature dependence of delta Hbind, delta Cpbind for the association of Pdx to PdR was estimated as -1.24 kJ mol-1 K-1. This value is larger than those reported for other electron-transfer protein systems (e.g., -0.68 kJ mol-1 K-1 for ferredoxin/ferredoxin: NADP+ reductase), suggesting that the PdR/Pdx association may be dominated by hydrophobic rather than electrostatic components. For the P450cam/Pdx association, the negative delta Sbind and highly favorable delta Hbind were observed, behavior that stands in sharp contrast to the association reactions in other electron-transfer proteins. The energetics of the P450cam/Pdx association are similar to those of binding reaction of antibody to antigen in which van der Waals and hydrogen bonding interactions are dominant, resulting in high specificity in the association of Pdx with P450cam.
Animals↗
NMR studies of putidaredoxin: associations of putidaredoxin with NADH-putidaredoxin reductase and cytochrome p450cam.
To characterize the electron-transfer reaction in the P450cam monooxygenation system, the binding regions of putidaredoxin (Pdx) to NADH-putidaredoxin reductase (PdR) and P450cam were investigated using isotope-filtered NMR experiments in which uniformly 15N-labeled Pdx ([U-15N]Pdx) is mixed with unlabeled PdR and P450cam. By addition of PdR to Pdx, site specific signal broadening was observed for the N-H correlation peaks from Val-28, Glu-72, Ile-88, and Gln-105. Although previous studies have suggested the contribution from acidic amino acid residues on the G-helix of Pdx to the binding with PdR, no site specific broadening was observed for the resonances from these residues except for Glu-72. The lesser contribution of electrostatic interactions to the Pdx/PdR complex formation was also suggested by our previous study (M. Aoki, K. Ishimori, H. Fukada, K. Takahashi, I. Morishima, Biochim. Biophys. Acta 1384 (1998) 180-188), which is in sharp contrast to the complex formation between adrenodoxin and adrenodoxin reductase. Upon the complex formation between Pdx and P450cam, the site specific NMR line broadening was observed for several amino acid residues distributed near the iron-sulfur cluster, corresponding to the large binding site in the complex formation with P450cam. Since some of the amino acid residues included in the binding site are not conserved for the electron-transfer iron-sulfur proteins such as ferredoxin and adrenodoxin, the interactions formed by these amino acid residues would be highly specific to the binding with P450cam, consistent with very low cross-reactivity to other iron-sulfur proteins in the P450cam monooxygenation system.
Amino Acid Sequence↗
Roles of negatively charged surface residues of putidaredoxin in interactions with redox partners in p450cam monooxygenase system.
To investigate the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam system, we focused on the role of negatively charged surface amino acid residues. The amino acid residues we examined in this mutational study are Asp-58, Glu-65, Glu-72, and Glu-77, which are located on the alpha-helical segment to form a negatively charged region on the surface of Pdx and have been supposed to play key roles in the association with the redox partners, NADH-putidaredoxin reductase (PdR) and P450cam. The neutralization of the single negative charge on these amino acid residues did not significantly inhibit the electron-transfer reaction with the redox partners, except for the mutation at Glu-72. Together with the previous results, we can conclude that the negatively charged cluster on the alpha-helical segment is not so crucial for the electron transfer of the Pdx/PdR complex, and, instead of the negative charges, the steric hindrance is essential for the binding of Pdx with PdR. In the electron transfer from Pdx to P450cam, the alpha-helical region would not be included in the binding site with P450cam and some specific hydrogen bonds on the surface loop near the Fe-S center contribute to the electron transfer to P450cam. Such different binding sites and interactions for Pdx will shed light on the electron-transfer mechanism mediated by Pdx, the shuttle mechanism.
Aspartic Acid↗
Broad-spectrum protection in sunscreen products.
Explore the source record for details and available documents.
Benzoates↗
Determination of leachable components from four commercial dental composites by gas and liquid chromatography/mass spectrometry.
OBJECTIVES: The purpose of our study was to determine the quality and quantity of leachable residual (co)monomers and additives eluted from various commercial dental composite resins after polymerization. METHODS: Polymerized specimens from four universal hybrid-type composite resins were eluted for 3 days with methanol resp, water. Then all extracts were analysed by gas chromatography/mass spectrometry or liquid chromatography/mass spectrometry using a particle beam interface. RESULTS: In all polymerized composite resin specimens, (co)monomers and various additives as well as contaminants from manufacturing processes were identified. Almost every compound detected in the unpolymerized resins could also be identified in the methanol extracts, but only a few of them were found in the water extracts. From these the co-monomer TEGDMA was extracted in quantities higher than those reported to be cytotoxic in primary human oral fibroblast cultures. CONCLUSIONS: From our results we conclude that the extractable quantities of composite resin components should be minimized, either by reducing the mobility of leachable substances within the set material or by applying less water-soluble components. Furthermore, all ingredients of a dental composite should be declared by the manufacturers, in order to identify those substances in a product which may cause adverse side effects in patients and dental personnel.
4-Aminobenzoic Acid↗
Rational approach to improving reductive catalysis by cytochrome P450cam.
Although halogenated hydrocarbons are noted for low chemical reactivity, small amounts are toxic to humans. Cytochromes P450 have been implicated in transforming these compounds to more reactive species, under anaerobic conditions, through reduction at the heme. A significant amount of effort has been directed toward turning this catalytic ability to our advantage by engineering P450 variants than can efficiently remediate these compounds in situ, before they come in contact with the human population. We have taken a 'rational' approach to this problem, in which a combination of theory and molecular modeling is applied to identify which properties of the enzyme have the greatest influence over reductive dehalogenation. Recent progress in this area is briefly reviewed. Two novel mutants, incorporating tryptophan (positions 87 and 396) and histidine (position 96, neutral and protonated) amino acid substitutions in the active site, are proposed and evaluated using molecular dynamics simulations. The upper bound on rate enhancement relative to wild-type is estimated in each mutant using electron transfer theory. The most significant rate enhancement is predicted for the His 96 mutant in the protonated state; while some His residues of certain proteins exhibit a pKa high enough to support a large protonated population, such information is not presently available for this proposed mutant.
Camphor 5-Monooxygenase↗
A structure-based model for cytochrome P450cam-putidaredoxin interactions.
Putidaredoxin (Pdx) is a Fe2S2 ferredoxin which acts as the physiological reductant of cytochrome P-450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined using NMR methods (Pochapsky et al (1994) Biochemistry 33, 6424-6432). 1H-15N correlations and redox-dependent amide exchange rates have also been described (Lyons et al (1996) Protein Sci 5, 627-639). Data obtained from mutagenesis and kinetic measurements concerning the interactions of Pdx and CYP101 are summarized. A model for the structure of the homologous ferredoxin adrenodoxin (Adx) is also described, and data concerning Adx activity are discussed in relation to this structure. The structures of Pdx and CYP101 were used as starting points for molecular modeling and molecular dynamics simulations. Close approach between the metal centers of the two proteins and interaction between aromatic residues on the surfaces of the proteins are premised. The resulting complex exhibits three intermolecular salt bridges, five intermolecular hydrogen bonds and a 12 A distance between the metal centers. The first direct observations of interaction between Pdx and CYP101 (by two-dimensional NMR of 15N-labeled Pdx in solution with CYP101) are described. The results of the NMR experiments indicate that conformational gating of the electron transfer complex between CYP101 and Pdx may be important.
Adrenodoxin↗
Antigenic mapping of bacterial and animal cytochromes P-450.
A peptide scanning (PEPSCAN) approach was used for antigenic mapping of two hepatic microsomal cytochromes P450 (rab1A2 and rab2B4) and the microbial cytochrome from Pseudomonas putida (P450 101 or P450cam). This approach includes simultaneous synthesis of pin-linked overlapping hexapeptides covering the whole sequences of three P450s and testing them by ELISA with corresponding polyclonal antisera. Microsomal cytochrome P450 maps were shown to vary depending on an antiserum used for testing the peptides, however, the most active linear B-epitopes were revealed with antisera from two animal species used. P450 linear B-epitopes were classified into individual and group-specific epitopes. While almost all P450 101 linear antigenic determinants are unique for this protein, rab1A2 and rab2B4 contain epitopes both individual for each protein, and subfamily- or even family-specific epitopes. These results point out the possibility of producing both monospecific and group-specific antipeptide antibodies against different P450s. The antigenic map of P450 101 was superimposed on the structural-functional map of this protein. Its linear B-epitopes were shown to coincide with boundaries of secondary structure elements, with surface-located, water accessible regions and with sites responsible for intermolecular interactions in the Pseudomonas putida monooxygenase system. Several known or predicted functionally active sites in microsomal cytochrome P450 rab1A2 and rab2B4 were also shown to coincide with linear B-epitopes. The peculiarities of epitope locations in the protein tertiary structure will allow to predict antigenic regions starting from protein structural information and vice versa, to structural protein models in accordance with antigenic mapping results. Antigenic regions which coincide with sites responsible for intermolecular interactions in monooxygenase systems may be synthesized as separate peptides and used as blockers of such interactions.
Animals↗