Markers of protein oxidation: different oxidants give rise to variable yields of bound and released carbonyl products

Exposure of proteins to radicals in the presence of O2 gives both side-chain oxidation and backbone fragmentation. These processes can be interrelated, with initial side-chain oxidation giving rise to backbone damage via transfer reactions. We have shown previously that alkoxyl radicals formed on the C-3 carbons of Ala, Val, Leu, and Asp residues undergo beta-scission to give backbone alpha-carbon radicals, with the release of the side- chain as a carbonyl compound. We now show that this is a general mechanism that occurs with a wide range of oxidants. The quantitative significance of this process depends on the extent of oxidation at C-3 compared with other sites. HO*, generated by gamma radiolysis, gave the highest total carbonyl yield, with protein-bound carbonyls predominating over released. In contrast, metal ion/H2O2 systems, gave more released than bound carbonyls, with this ratio modulated by EDTA. This is ascribed to metal ion-protein interactions affecting the sites of initial oxidation. Hypochlorous acid gave low concentrations of released carbonyls, but high yields of protein-bound material. The peroxyl radical generator 2,2'-azobis(2-amidinopropane) hydrochloride, and a peroxynitrite generator, 3-morpholinosydnonimine hydrochloride, gave lower overall carbonyl yields, with released carbonyls predominating over protein-bound species similar to that observed with metal ion/H2O2 systems.     

The potential for analyses of monitoring scheme data to inform about the impacts of invasive on native species

Biological Invasions - UK animals and plants are surveyed annually by a wide range of long-term citizen science monitoring schemes, which are designed to detect species’ range, status and...     

Two proton translocation pathways in a secondary active multidrug transporter

LmrP is a secondary active multidrug transporter from Lactococcus lactis. The protein belongs to the major facilitator superfamily and utilizes the electrochemical proton gradient (inside negative and alkaline) to extrude a wide range of lipophilic cations from the cell. Previous work has indicated that ethidium, a monovalent cationic substrate, is exported by LmrP by electrogenic antiport with two (or more) protons. This observation raised the question whether these protons are translocated sequentially along the same pathway, or through different routes. To address this question, we constructed a 3-D homology model of LmrP based on the high-resolution structure of the glycerol-3P/Pi antiporter GlpT from Escherichia coli, and we tested by mutagenesis the possible proton conduction points suggested by this model. Similar to the template, LmrP is predicted to contain an internal cavity formed at the interface between the two halves of the transporter. On the surface of this cavity lie two clusters of polar, aromatic and carboxylate residues with potentially important function in proton shuttling. Cluster 1 in the C-terminal half contains D235 and E327 in immediate proximity of each other, and is located near the apex of the cavity. Cluster 2 in the N-terminal half contains D142. Analyses of LmrP mutants containing charge-conservative or carboxyl-to-amide replacements at positions 142, 235 and 327 suggest that D142 is part of a dedicated proton translocation pathway in the ethidium translocation reaction. In contrast, D235 and E327 are part of an independent pathway, in which D235 interacts with protons. E327 appears to modulate the pKa of D235 and plays a role in the interaction with ethidium. These results are consistent with the proposal that major facilitator superfamily proteins consist of two membrane domains, one of which is involved in substrate binding and the other in ion coupling, and they indicate that there are two proton conduction pathways at play in the transport mechanism.     

The histochemical localization of steroid binding sites in the pituitary gland of a teleost (the platyfish)

Summary Specific binding sites for estrogen, testosterone, and progesterone have been demonstrated in the pituitary gland of mature male and female platyfish (Xiphophorus maculatus). With a histochemical procedure, fluorescent-steroid-hormone conjugates were localized in the cytoplasm and nucleus of the gonadotrops of the caudal pars distalis (CPD) and in cells of the pars intermedia (PI) previously demonstrated to contain immunoreactive gonadotropin. The specificity of the response was confirmed by means of competitive binding analyses and by using fluoresceinated BSA not linked to steroids. The physiological significance of steroid binding in the PI, as well as in the CPD, is discussed in the light of other recent studies on the pituitary gland of the platyfish.     

Purification and properties of the Escherichia coli nucleoside transporter NupG,a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K_m values of ～20–30 μM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1′-¹³C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly α-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

Phylogenetic relationships of Cryphonectria and Endothia species, based on DNA sequence data and morphology

The fungal genera Endothia and Cryphonectria include some of the most important pathogens of forest trees. Despite available new technology, no comprehensive comparative study based on DNA sequence data and morphology has been done on the available isolates representing these two genera. The main objectives of this study were to assess the phylogenetic relationships among species of Cryphonectria and Endothia, for which cultures are available, and to establish a taxonomic framework based on DNA sequence and morphological data, which will aid future studies and identification of species in these and related genera. Comparisons were based on sequence variation found in the ITS region of the ribosomal RNA operon and two regions of the β-tu-bulin gene. In addition, the morphology of these species was examined. The phylogenetic data indicated that Endothia and Cryphonectria reside in two distinct phylogenetic clades. Cryphonectria parasitica, C. macrospora, C. nitschkei, C. eucalypti and C. radicalis represented the Cryphonectria clade. Endothia gyrosa and E. singularis were included in the Endothia clade. An isolate representing E. viridistroma grouped outside the Endothia clade and separately from other groups. Other clades outside the one encompassing Cryphonectria were those represented by the C. cubensis isolates and fungi isolated from Elaeocarpus dentatus originating from New Zealand. These clades could be distinguished from Endothia and Cryphonectria, based on anamorph morphology, stromatal structure and ascospore septation. Cryphonectria and Endothia, therefore, appear to be paraphyletic and taxonomic relationships for these fungi need to be revised.