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Structural and functional characterization of the dimerization region of soluble guanylyl cyclase
(2004)
Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. Despite the obligate heterodimeric nature of sGC, the sequence segments mediating subunit association have remained elusive. Our initial screening for relevant interaction site(s) in the most common sGC isoenzyme, α1 β1, identified two regions in each subunit, i.e. the regulatory domains and the central regions, contributing to heterodimer formation. To map the relevant segments in the β1 subunit precisely, we constructed multiple N- and C-terminal deletion variants and cotransfected them with full-length α1 in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204–408 mediates binding of β1 to α1 The same region of β1[204–408] was found to promote β /β1 homodimerization. Fusion of [204 β1–408] to enhanced green fluorescent protein conferred binding activity to the recipient protein. Coexpression of β1[204–408] with α1 or β1 targeted the sGC subunits for proteasomal degradation, suggesting that β1[204–408] forms structurally deficient complexes with α1 and β1. Analysis of deletion constructs lacking portions of the β1 dimerization region identified two distinct segments contributing to α1 binding, i.e. an N-terminal site covering positions 204–244 and a C-terminal site at 379–408. Both sites are crucial for sGC function because deletion of either site rendered sGC dimerization-deficient and thus functionally inactive. We conclude that the dimerization region of β1 extends over 205 residues of its regulatory and central domains and that two discontinuous sites of 41 and 30 residues, respectively, facilitate binding of β1 to the α1 subunit of sGC.
A small electrostatic storage ring is the central machine of the Frankfurt Ion Storage Experiments (FIRE) which will be built at the new Stern-Gerlach Center of Frankfurt University. As a true multiuser, multipurpose facility with ion energies up to 50 keV, it will allow new methods to analyze complex many-particle systems from atoms to very large biomolecules. With envisaged storage times of some seconds and beam emittances in the order of a few mm mrad, measurements with up to 6 orders of magnitude better resolutions as compared to single-pass experiments become possible. In comparison to earlier designs, the ring lattice was modified in many details: Problems in earlier designs were related to, e.g., the detection of light particles and highly charged ions with different charge states. Therefore, the deflectors were redesigned completely, allowing a more flexible positioning of the diagnostics. Here, after an introduction to the concept of electrostatic machines, an overview of the planned FIRE is given and the ring lattice and elements are described in detail.
Respiratory chain complex I contains 8-9 iron-sulfur clusters. In several cases, the assignment of these clusters to subunits and binding motifs is still ambiguous. To test the proposed ligation of the tetranuclear iron-sulfur cluster N5 of respiratory chain complex I, we replaced the conserved histidine 129 in the 75-kDa subunit from Yarrowia lipolytica with alanine. In the mutant strain, reduced amounts of fully assembled but destabilized complex I could be detected. Deamino-NADH: ubiquinone oxidoreductase activity was abolished completely by the mutation. However, EPR spectroscopic analysis of mutant complex I exhibited an unchanged cluster N5 signal, excluding histidine 129 as a cluster N5 ligand.
Stable supercomplexes of bacterial respiratory chain complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) have been isolated as early as 1985 (Berry, E. A., and Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458-2467). However, these assemblies did not comprise complex I (NADH:ubiquinone oxidoreductase). Using the mild detergent digitonin for solubilization of Paracoccus denitrificans membranes we could isolate NADH oxidase, assembled from complexes I, III, and IV in a 1:4:4 stoichiometry. This is the first chromatographic isolation of a complete “respirasome.” Inactivation of the gene for tightly bound cytochrome c552 did not prevent formation of this supercomplex, indicating that this electron carrier protein is not essential for structurally linking complexes III and IV. Complex I activity was also found in the membranes of mutant strains lacking complexes III or IV. However, no assembled complex I but only dissociated subunits were observed following the same protocols used for electrophoretic separation or chromatographic isolation of the supercomplex from the wild-type strain. This indicates that the P. denitrificans complex I is stabilized by assembly into the NADH oxidase supercomplex. In addition to substrate channeling, structural stabilization of a membrane protein complex thus appears as one of the major functions of respiratory chain supercomplexes.
"Solidarität" ist ein Wieselwort von gallertartiger Konsistenz. Es ist allgegenwärtig, gibt sich bedeutungsschwer und meist auch etwas vorwurfsvoll. Sein Kontext ist durchweg normativ. Es taucht dort auf, wo es um den Appell an Personen auf der gleichen Ebene geht. Typischerweise rufen Gruppen nach "Solidarität", deren innere Bindungen bröckeln oder die sich in Gefahrenlagen zusammenscharen. Als es noch Standesgenossen gab, erinnerten sie gerne an die Solidarität, wenn es darum ging, den Zusammenhalt derselben Schicht zu wahren. Die alten Zünfte, Gilden, Gaffeln, Einungen und Genossenschaften waren Solidaritätsverbände. Die Arbeiterbewegung übernahm hiervon nicht nur das Wort "Genossen", sondern auch den Appell an die Klassensolidarität. Und selbst wer heutzutage einen Krieg führen will, erinnert an alte Dankesschulden und appelliert an die Solidarität der Bundesgenossen. ...
The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. All three isoforms are highly conserved among different species, suggesting distinct functional roles. We produced the three LHC-II isoforms by heterologous expression of the polypeptide in Escherichia coli and in vitro refolding with purified pigments. Although Lhcb1 and Lhcb2 are very similar in polypeptide sequence and pigment content, Lhcb3 is clearly different because it lacks an N-terminal phosphorylation site and has a higher chlorophyll a/b ratio, suggesting the absence of one chlorophyll b. Low temperature absorption and fluorescence emission spectra of the pure isoforms revealed small but significant differences in pigment organization. The oligomeric state of the pure isoforms and of their permutations was investigated by native gel electrophoresis, sucrose density gradient centrifugation, and SDS-PAGE. Lhcb1 and Lhcb2 formed trimeric complexes by themselves and with one another, but Lhcb3 was able to do so only in combination with one or both of the other isoforms. We conclude that the main role of Lhcb1 and Lhcb2 is in the adaptation of photosynthesis to different light regimes. The most likely role of Lhcb3 is as an intermediary in light energy transfer from the main Lhcb1/Lhcb2 antenna to the photosystem II core.
Im Niemandsland
(2004)
Dass die Regierungen westlicher Staaten eine offene oder verdeckte Tendenz haben, die ihnen in einem langen historischen Prozess auferlegten rechtsstaatlichen Bindungen aus politischen Gründen wenn nicht abzustreifen, so doch zu lockern, ist ein Thema, das Juristen, Ökonomen, Politologen und Soziologen gleichermaßen beschäftigt. Giorgio Agamben, italienischer Philosoph und Ästhetiker, spitzt die Thematik mit der These zu, die "Normalität" staatlichen Lebens existiere nicht mehr, es sei das "Niemandsland" des Ausnahmezustandes, "in dem wir leben" ("lo stato di eccezione 'in cui viviamo'"). Er sei die "fundamentale politische Struktur" unserer Zeit; er habe inzwischen die "größte planetarische Entfaltung" erreicht, die Menschen auf die "nuda vita", das nackte Leben, reduziert, das dem Kalkül der Macht unterworfen sei und in eine "beispiellose biopolitische Katastrophe" auszuarten drohe. Seine "Materialisierung" hat der Ausnahmezustand in den nationalsozialistischen Konzentrationslagern wie in allen Lagern gefunden, in denen Menschen von der "normalen Ordnung" ausgeschlossen werden; Guantánamo ist für Agamben ein aktuelles Beispiel. Die "Lager" in allen ihren Erscheinungsformen sind die "verborgene Matrix der Politik". Der Ausnahmezustand ist ein arcanum imperii, dessen "Demaskierung" die einzige Möglichkeit ist, den durch ihn bewirkten "Bann" zu brechen. ...
Quantitative analysis of the cardiac fibroblast transcriptome implications for NO/cGMP signaling
(2004)
Cardiac fibroblasts regulate tissue repair and remodeling in the heart. To quantify transcript levels in these cells we performed a comprehensive gene expression study using serial analysis of gene expression (SAGE). Among 110,169 sequenced tags we could identify 30,507 unique transcripts. A comparison of SAGE data from cardiac fibroblasts with data derived from total mouse heart revealed a number of fibroblast-specific genes. Cardiac fibroblasts expressed a specific collection of collagens, matrix proteins and metalloproteinases, growth factors, and components of signaling pathways. The NO/cGMP signaling pathway was represented by the mRNAs for α1 and β1 subunits of guanylyl cyclase, cGMP-dependent protein kinase type I (cGK I), and, interestingly, the G-kinase-anchoring protein GKAP42. The expression of cGK I was verified by RT-PCR and Western blot. To establish a functional role for cGK I in cardiac fibroblasts we studied its effect on cell proliferation. Selective activation of cGK I with a cGMP analog inhibited the proliferation of serum-stimulated cardiac fibroblasts, which express cGK I, but not higher passage fibroblasts, which contain no detectable cGK I. Currently, our data suggest that cGK I mediates the inhibitory effects of the NO/cGMP pathway on cardiac fibroblast growth. Furthermore the SAGE library of transcripts expressed in cardiac fibroblasts provides a basis for future investigations into the pathological regulatory mechanisms underlying cardiac fibrosis.
"Ein jegliches hat seine Zeit und alles Vornehmen unter dem Himmel hat seine Stunde". Wir wissen das. Wir wussten es vermutlich schon lange bevor der "Prediger" Salomo (3.1), der ein Philosoph war, uns vor nunmehr gut 2200 Jahren darüber belehrte. Aber wir handeln nur selten nach unseren Einsichten. Trennungen fallen schwer und Gewohnheiten verkleiden sich bereitwillig als Notwendigkeit. Verschwendung ist den Reichen keine Kategorie, und wer vom Verschwender lebt, erhebt keine Vorwürfe. Schließlich das Wichtigste: Wer außer Gott hat Kraft und Befugnis "Zeit" und "Stunde" zu bestimmen? ...