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81.
A three-dimensional structural model of the dichloromethane dehalogenase (DCMD) from Methylophilus sp. DM11 is constructed based on sequence similarities to the glutathione S-transferases (GSTs). To maximize sequence identity and minimize gaps in the alignment, a hybrid approach is used that takes advantage of the increased homology found between DM11 and domain I of the sheep blowfly θ class GST (residues 1–79) and domain II of the human α class GST (residues 81–222). The resulting structure has Cα root mean square deviations of 1.16 Å in domain I and 1.83 Å in domain II from the template GSTs, which compare well to those seen in other GST interclass comparisons. The model is further applied to explore the structural basis for substrate binding and catalysis. A conserved network of hydrogen bonds is described that binds glutathione to the G site, placing the thiol group in a suitable location for nucleophilic attack of dichloromethane. A mechanism is proposed that involves activation through a hydrogen bond interaction between Ser12 and glutathione, similar to that found in the θ-GSTs. The model also demonstrates how aromatic residues in the hydrophobic site (H site) could play a role in promoting catalysis: His116 and Trp117 are ideally situated to accept a growing negative charge on a chlorine of dichloromethane, stabilizing displacement. This scheme is consistent with experimental results of single-point mutations and comparisons with other GST structures and mechanisms. Proteins 28:217–226, 1997. © 1997 Wiley-Liss Inc. 相似文献
82.
The antibiotic resistant faecal flora of a domestic dog suffering from an acute enteric infection was examined. The flora exhibited overall resistance to a wide variety of antibiotics. However, following restoration of the animal to normal health, overall resistance to ampicillin (Ap), tetracycline (Tc), chloramphenicol (Cm) and streptomycin (Sm) was lost, although low numbers of bacteria resistant to these four antimicrobial agents could still be isolated up to one year later. A total of 11 strains were purified for further study. All 11 were positively identified as Escherichia coli and shown to be resistant to various combinations of the above antibiotics, and additionally to kanamycin (Km). Each strain harboured from one to five plasmids, although only four proved capable of transferring antibiotic resistance to Escherichia coli K-12. One of the strains was found to harbour two conjugal plasmids pNJ101 (60 Md) and pNJ102 (133 Md) which coded for resistance to Cm, Tc, Ap and Cm, Tc, Km respectively. A third plasmid pNJ103 (29 Md) remains cryptic. The possession of the two plasmids pNJ101 and pNJ102 appears to be an unstable situation as variants arose harbouring one or other of the plasmids. 相似文献
83.
Analysis of the bilayer phase transition temperatures of phosphatidylcholines with mixed chains 下载免费PDF全文
Derek Marsh 《Biophysical journal》1992,61(4):1036-1040
The analysis of the chain-length dependence of the chain-melting transition temperatures of bilayers composed of lipids with identical chains (Marsh, D. 1991. Biochim. Biophys. Acta. 1062: 1-6) is extended to include lipids with chains of unequal length. The bilayer transition temperatures of saturated asymmetrical phosphatidylcholines are interpreted by assuming that the transition enthalpy and transition entropy are linearly related to the absolute value of the difference in chain length between the sn-1 and sn-2 chains, with constant end contributions. Such an assumption is supported by calorimetric data on phosphatidylcholines of constant mean chainlength and varying chain asymmetry. In particular, a symmetrical linear dependence is observed on the chain asymmetry, Δn, which is centered around a value Δn° that corresponds to the conformational inequivalence of the sn-1 and sn-2 chains. The transition temperature then takes the form: Tt = Tt∞(n - nH - h′ | Δn + Δn° |)/(n - ns - s′ | Δn + Δn°) where nH, ns are the end contributions, and h′, s′ are fractional deficits in the incremental transition enthalpy and entropy, respectively, arising from the overlapping regions of the longer chains. Optimization on the transition temperature data for the dependence on chain asymmetry of three series of phosphatidylcholines with constant mean chainlength, n, yields parameters that are capable of predicting the dependence of the transition temperatures on chain asymmetry for other mean chainlengths. The dependence of the transition temperature on mean chainlength for phosphatidylcholines in which the chain asymmetry is maintained constant, as well as the dependence on both mean chain length and chain asymmetry for phosphatidylcholines in which one of the two chains is maintained of constant length, are also described with high accuracy by using the same parameters. 相似文献
84.
Methylmalonyl-CoA mutase from Propionibacterium shermanii. Evidence for the presence of two masked cysteine residues. 总被引:1,自引:0,他引:1 下载免费PDF全文
Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii contains no intramolecular disulphide bridges, but two of the six thiol groups in the heterodimer are only revealed after reduction of the denatured enzyme with dithiothreitol. The available evidence suggests that they are present in disulphide linkages to unknown thiols of low Mr. The two specifically masked cysteine residues are Cys-535 in the alpha-subunit and Cys-517 in the beta-subunit, which occupy exactly homologous positions in each chain. 相似文献
85.
Differential scanning calorimetry of thermotropic phase transitions in vitaminylated lipids: aqueous dispersions of N-biotinyl phosphatidylethanolamines. 下载免费PDF全文
The thermotropic phase behavior of a homologous series of saturated diacyl phosphatidylethanolamines in which the headgroup is N-derivatized with biotin has been investigated by differential scanning calorimetry. In 1 M NaCl, derivatives with acyl chainlengths from C(12:0) to C(20:0) all exhibit sharp chain-melting phase transitions, which are reversible with a hysteresis of 1.5 degrees or less, except for the C(12:0) lipid which has a transition temperature below 0 degree C. The transition enthalpy and the transition entropy depend approximately linearly on the lipid chainlength, with incremental values per CH2 group that are very similar to those obtained for the corresponding underivatized phosphatidylethanolamines in aqueous dispersion. The chainlength-independent contribution to the transition enthalpy is significantly smaller than that for the underivatized phosphatidylethanolamines, and that for the transition entropy is much smaller; the latter suggesting that the N-biotinylated phosphatidylethanolamine headgroups are differently hydrated from those of the underivatized lipids. The gel-to-fluid phase transition temperatures of the N-biotinylated lipids are lower than those of the parent phosphatidylethanolamines, and their chainlength dependence conforms well with that predicted by assuming that the transition enthalpy and entropy are linearly dependent on chainlength. Although the chain-melting phase behavior is generally similar to that of the parent phosphatidylethanolamines, the gel phases (and the fluid phases in the case of chainlengths C(12:0) to C(16:0)) have a different lyotropic structure in the two cases, and this is reflected in the chainlength-independent contributions to the thermodynamic parameters. In the absence of salt, the thermotropic phase behavior of aqueous dispersions of the N-biotinyl phosphatidylethanolamines is considerably more complex.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
86.
P. J. Klasse Mette M. Rosenkilde Nathalie Signoret Annegret Pelchen-Matthews Thue W. Schwartz Mark Marsh 《Journal of virology》1999,73(9):7453-7466
Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SU(LAI) protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1(LAI) infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)-CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions with the chemokine receptor moiety, this did not overcome the specific coreceptor requirement of different HIV-1 strains: the X4 virus HIV-1(LAI) and the X4R5 virus HIV-1(89. 6), unlike the R5 strain HIV-1(SF162), infected Mv-1-lu cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of CD4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule hybrid constructs that mimic receptor-coreceptor complexes can be used to dissect coreceptor function and its inhibition. 相似文献
87.
Black LA Nersesian DL Sharma P Ku YY Bennani YL Marsh KC Miller TR Esbenshade TA Hancock AA Cowart M 《Bioorganic & medicinal chemistry letters》2007,17(5):1443-1446
4-[6-(2-Tertiaryaminoethyl)naphthalen-2-yl]benzonitriles are conformationally constrained histamine H3 receptor antagonists with high potency and selectivity. The analogs were designed around a naphthalene core, with the goal of enhancing lipophilicity and CNS penetration, as compared to a previously reported benzofuran series. The SAR of the tertiary amine moiety is similar to that reported for the benzofuran series, with analogs bearing a 2-methylpyrrolidine substituent possessing the greatest rat and human H3 receptor binding affinities. 相似文献
88.
Epifluorescent and histochemical aspects of shoot anatomy of Typha latifolia L., Typha angustifolia L. and Typha glauca Godr 总被引:1,自引:0,他引:1
Using epifluorescent and histochemical techniques, we examined anatomical differences in the shoot organs of Typha latifolia, T. angustifolia and T. glauca. The leaf lamina of T. latifolia and T. glauca had enlarged epidermal cells and a thickened cuticle above the subepidermal vascular bundles; that of T. angustifolia lacked these characteristics. Leaf sheaths were similar among the species and all lacked the epidermal thickenings found in the lamina. The fertile stems had typical scattered vascular bundles with a band of fibres that was most prominent in T. glauca. The sterile stems were only 1 cm in length and contained a multiseriate hypodermis and a uniseriate endodermis over part of their length. The rhizomes were similar except for a pronounced band of fibres surrounding the central core in T. angustifolia. The rhizome was also characterized by an outer cortical region with a large multiseriate hypodermis/exodermis and a uniseriate endodermis with Casparian bands, suberin lamellae and secondarily thickened walls. 相似文献
89.
Amanda I. Bradley Nicole M. Marsh Heather R. Borror Kaitlyn E. Mostoller Amber I. Gama Richard G. Gardner 《Molecular biology of the cell》2021,32(11):1121
Stress is ubiquitous to life and can irreparably damage essential biomolecules and organelles in cells. To survive, organisms must sense and adapt to stressful conditions. One highly conserved adaptive stress response is through the posttranslational modification of proteins by the small ubiquitin-like modifier (SUMO). Here, we examine the effects of acute ethanol stress on protein sumoylation in the budding yeast Saccharomyces cerevisiae. We found that cells exhibit a transient sumoylation response after acute exposure to ≤7.5% vol/vol ethanol. By contrast, the sumoylation response becomes chronic at 10% ethanol exposure. Mass spectrometry analyses identified 18 proteins that are sumoylated after acute ethanol exposure, with 15 known to associate with chromatin. Upon further analysis, we found that the chromatin structural proteins Smc5 and Smc6 undergo ethanol-induced sumoylation that depends on the activity of the E3 SUMO ligase Mms21. Using cell-cycle arrest assays, we observed that Smc5 and Smc6 ethanol-induced sumoylation occurs during G1 and G2/M phases but not S phase. Acute ethanol exposure also resulted in the formation of Rad52 foci at levels comparable to Rad52 foci formation after exposure to the DNA alkylating agent methyl methanesulfonate (MMS). MMS exposure is known to induce the intra-S-phase DNA damage checkpoint via Rad53 phosphorylation, but ethanol exposure did not induce Rad53 phosphorylation. Ethanol abrogated the effect of MMS on Rad53 phosphorylation when added simultaneously. From these studies, we propose that acute ethanol exposure induces a change in chromatin leading to sumoylation of specific chromatin structural proteins. 相似文献
90.
The incorporation of concanamycin A, a potent inhibitor of vacuolar ATPases, into membranes of dimyristoyl phosphatidylcholine has been studied by using EPR of spin-labelled lipid chains. At an inhibitor/lipid ratio of 1:1 mol/mol, concanamycin A broadens the chain-melting transition of the phospholipid bilayer membrane, and effects the lipid chain motion in the fluid phase. The outer hyperfine splitting of a spin label at the C-5 position and the line widths of a spin label at the C-14 position of the lipid chain are increased by concanamycin A. Considerably larger membrane perturbations are caused by equimolar admixture of a designed synthetic 5-(5,6-dichloro-2-indolyl)-2,4-pentadienoyl V-ATPase inhibitor. These results indicate that concanamycin A intercalates readily between the lipid chains in biological membranes, with minimal perturbation of the bilayer structure. Essentially identical results are obtained with concanamycin A added to preformed membranes as a concentrated solution in DMSO, or mixed with lipid in organic solvent prior to membrane formation. Therefore, the common mode of addition in V-ATPase inhibition assays ensures incorporation of concanamycin into the lipid bilayer milieu, which provides an efficient channel of access to the transmembrane domains of the V-ATPase. 相似文献