Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Lipid-gramicidin interactions: dynamic structure of the boundary lipid by 2D-ELDOR

The use of 2D-electron-electron double resonance (2D-ELDOR) for the characterization of the boundary lipid in membrane vesicles of DPPC and gramicidin A' (GA) is reported. We show that 2D-ELDOR, with its enhanced spectral resolution to dynamic structure as compared with continuous-wave electron spin resonance, provides a reliable and useful way of studying lipid-protein interactions. The 2D-ELDOR spectra of the end-chain spin label 16-PC in DPPC/GA vesicles is composed of two components, which are assigned to the bulk lipids (with sharp auto peaks and crosspeaks) and to the boundary lipids (with broad auto peaks). Their distinction is clearest for higher temperatures and higher GA concentrations. The quantitative analysis of these spectra shows relatively faster motions and very low ordering for the end chain of the bulk lipids, whereas the boundary lipids show very high "y-ordering" and slower motions. The y-ordering represents a dynamic bending at the end of the boundary lipid acyl chain, which can then coat the GA molecules. These results are consistent with the previous studies by Ge and Freed (1999) using continuous-wave electron spin resonance, thereby supporting their model for GA aggregation and H(II) phase formation for high GA concentrations. Improved instrumental and simulation methods have been employed.     

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Background  

Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum.     

Spin-labeled gramicidin a: channel formation and dissociation

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.     

The Lipid-binding Domain of Wild Type and Mutant α-Synuclein: COMPACTNESS AND INTERCONVERSION BETWEEN THE BROKEN AND EXTENDED HELIX FORMS*

α-Synuclein (αS) is linked to Parkinson disease through its deposition in an amyloid fibril form within Lewy Body deposits, and by the existence of three αS point mutations that lead to early onset autosomal dominant Parkinsonism. The normal function of αS is thought to be linked to the ability of the protein to bind to the surface of synaptic vesicles. Upon binding to vesicles, αS undergoes a structural reorganization from a dynamic and disordered ensemble to a conformation consisting of a long extended helix. In the presence of small spheroidal detergent micelles, however, this extended helix conformation can convert into a broken helix state, in which a region near the middle of the helix unwinds to form a linker between the two resulting separated helices. Membrane-bound conformations of αS likely mediate the function of the protein, but may also play a role in the aggregation and toxicity of the protein. Here we have undertaken a study of the effects of the three known PD-linked mutations on the detergent- and membrane-bound conformations of αS, as well as factors that govern the transition of the protein between the extended helix and broken helix states. Using pulsed dipolar ESR measurements of distances up to 8.7 nm, we show that all three PD-linked αS mutants retain the ability to transition from the broken helix to the extended helix conformation. In addition, we find that the ratio of protein to detergent, rather than just the absolute detergent concentration, determines whether the protein adopts the broken or extended helix conformation.     

Reconstruction of the chemotaxis receptor-kinase assembly

In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes.     

Determination of the oligomeric states of human and rat monoamine oxidases in the outer mitochondrial membrane and octyl beta-D-glucopyranoside micelles using pulsed dipolar electron spin resonance spectroscopy

Human monoamine oxidase A (hMAOA) is considered to be unique among mammalian MAOs in having a non-conservative Glu-X-Lys mutation (X being 151 in MAOAs and 142 in MAOB's), which is suggested to be the reason for its monomeric structure. This hypothesis has been tested in this work. A pargyline based nitroxide spin labeled irreversible inhibitor (ParSL) was used as a MAO active site specific spin probe to measure intersubunit distances in detergent (octyl beta-d-glucopyranoside, OGP) purified and OMM bound forms by a pulsed dipolar ESR spectroscopic (PDS) technique. In a parallel approach, the covalent flavin cofactor present in the MAO active sites was reduced to its respective anionic flavin semiquinone and used for measuring inter-flavin distances in detergent purified samples. The measured interspin distances are within 0.1-0.3 nm of those estimated from the available dimeric crystal structures of human MAOB and rat MAOA and show that all human and rat MAOs exist as dimers in the OMM. In the OGP micelle, however, human and rat MAOAs exist only partially (相似文献   

Locating a Lipid at the Portal to the Lipoxygenase Active Site

Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.