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The European Journal of Biochemistry is an international journal devoted to the rapid publication of full length papers describing original research in the areas of biochemistry, molecular and cell biology, and molecular biophysics.
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Phenotypic difference between Bcg(r) and Bcg(s) macrophages is related to differences in protein-kinase-C-dependent signalling.
Mice of diverse genetic backgrounds may be classified as being either resistant or susceptible to infection with Mycobacteria. These phenotypes appear to be determined by a single gene on chromosome 1, the Bcg gene, and are expressed at the level of the macrophage in vitro. When compared to macrophages from mice of the susceptible phenotype (Bcg[s]), macrophages from mice of the resistant phenotype (Bcg[r]) show enhanced functional properties including increased expression of MHC class II molecules, increased nitric oxide production, and greater capacity to inhibit the growth of several intracellular pathogens. The bacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide produced by the macrophages. Since protein kinase C (PKC) has been shown to be involved in the induction of a range of macrophage functional activities, experiments were conducted to examine the possibility that phenotypic differences between Bcg(r) and Bcg(s) macrophages may be related to differences in PKC-dependent signalling. Macrophage cell lines were derived from mice congenic at the Bcg locus that are either resistant (B10R) or susceptible (B10S) to infection with Mycobacteria. In the basal state, PKC-specific activity was significantly increased in the cytosolic fractions of B10R cells when compared to B10S cells. Following phorbol myristate acetate (PMA) treatment and following the stimulation with Mycobacteria bovis BCG, PKC-specific activity increased significantly in membrane fractions of both B10R and B10S cells, but the absolute level was significantly greater in particulate fractions from B10R macrophages. Furthermore, B10R cells had a superior ability to phosphorylate endogenous substrates compared to B10S macrophages. Scatchard analysis of phorbol ester receptors revealed no differences between B10R and B10S cells. In contrast, the sensitivity of partially purified PKC from B10S cells to activation in vitro by diacylglycerol was decreased by approximately 50% when compared to enzyme from B10R cells. Western-blotting analysis using antibodies specific for PKC isoforms (alpha, beta, delta, epsilon, zeta and eta) showed similar levels of PKC isoforms present in B10R and B10S cells. To examine whether differences in PKC activity of B10R and B10S cells had functional consequences, the induction of c-fos gene expression was compared in the two cell lines. In response either to infection with M. bovis BCG or to stimulation with PMA, c-fos mRNA levels in B10R macrophages were increased 2-4-fold in comparison to B10S macrophages. Since we have previously found that the bacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide produced by the macrophages, we have tested if the enhancement of PKC activity in these macrophages affects their ability to produce nitric oxide. We have found that interferon-gamma-(IFNgamma)-induced secretion of nitric oxide by B10R macrophages could be augmented a few fold by the activation of PKC whereas, in B10S macrophages stimulated with IFNgamma, nitric oxide release could be augmented by only about 10-20%. These results indicate that the differences in PKC activity between B10R and B10S macrophages may contribute to altered responsiveness to IFNgamma that results in different production of effector molecules crucial for bacteriostatic activity against M. bovis BCG.
被引量：7 发表：1998
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Comparative biochemical characterization of the iron-only nitrogenase and the molybdenum nitrogenase from Rhodobacter capsulatus.
The component proteins of the iron-only nitrogenase were isolated from Rhodobacter capsulatus (delta nifHDK, delta modABCD strain) and purified in a one-day procedure that included only one column-chromatography step (DEAE-Sephacel). This procedure yielded component 1 (FeFe protein, Rc1Fe), which was more than 95% pure, and an approximately 80% pure component 2 (Fe protein, Rc2Fe). The highest specific activities, which were achieved at an Rc2Fe/Rc1Fe molar ratio of 40:1, were 260 (C2H4 from C2H2), 350 (NH3 formation), and 2400 (H2 evolution) nmol product formed x min(-1) x mg protein(-1). The purified FeFe protein contained 26 +/- 4 Fe atoms; it did not contain Mo, V, or any other heterometal atom. The most significant catalytic property of the iron-only nitrogenase is its high H2-producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N2 reduction, the activity ratios (mol N2 reduced/mol H2 produced) obtained were 1:1 (molybdenum nitrogenase) and 1:7.5 (iron nitrogenase). The Rc1Fe protein has only a very low affinity for C2H2. The Km value determined (12.5 kPa), was about ninefold higher than the Km for Rc1Mo (1.4 kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5. In complementation experiments, component 1 proteins coupled very poorly with the 'wrong' component 2. Rc1Fe, if complemented with Rc2Mo, showed only 10-15% of the maximally possible activity. Cross-reaction experiments with isolated polyclonal antibodies revealed that Rc1Fe and Rc1Mo are immunologically not related. The most active Rc1Fe samples appeared to be EPR-silent in the Na2S2O4-reduced state. However, on partial oxidation with K3[Fe(CN)6] or thionine several signals occurred. The most significant signal appears to be the one at g = 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between -100 mV and +150 mV. Another novel EPR signal (g = 1.96, 1.92, 1.77) occurred on further reduction of Rc1Fe by using turnover conditions in the presence of a substrate (N2, C2H2, H+).
被引量：32 发表：1997
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Detection of the in vivo incorporation of a metal cluster into a protein. The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus.
The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. The MoO4(2-) concentration causing 50% repression of the alternative nitrogenase in nifHDK- cells was 6 nM. If MoO4(2-) was added to a growing nifHDK- culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H4 formation decreased continuously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yield of C2H6 increased from 2% (C2H6/C2H4 x 100) in the absence of MoO4(2-), to a maximal value of 69% in the presence of MoO4(2-) (1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronger the higher the MoO4(2-) concentration in the medium and the longer the incubation time. In the presence of ReO4-, WO4(2-) or VO4(3-), a similar effect did not occur. The 'Mo effect' was not observed in a nifHDK- nifE- double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK- nifQ- mutant. Crude extracts from nifHDK- cells cultivated in the presence of MoO4(2-), also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase. The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo protein synthesis. The analysis of nifE-lacZ and nifN-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO4(2-) deficiency. The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.
被引量：9 发表：1993
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A Rhizobium meliloti ferredoxin (FdxN) purified from Escherichia coli donates electrons to Rhodobacter capsulatus nitrogenase.
The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coli under the control of the lac promoter. The fdxN gene product was purified under anaerobic conditions by ion-exchange chromatography and gel-filtration steps using an antiserum raised against an FdxN-LacZ fusion protein as a detection system. The purified ferredoxin was shown to be identical to the predicted R. meliloti FdxN protein in its amino acid composition and N-terminal amino acid sequence. Chemical determination of the iron content revealed 8.6 +/- 0.6 mol Fe/mol FdxN. The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A378/A284 ratio of 0.7. EPR spectroscopy revealed a rhombic signal when FdxN was partially reduced, and a broad signal indicative of spin-spin interaction when fully reduced, suggesting the presence of two Fe-S cluster/ferredoxin polypeptide. Our data suggest that FdxN contains two [4Fe-4S] clusters. Purified FdxN was able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro.
被引量：1 发表：1995
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Characterization of a 2[4Fe-4S] ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus.
The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.
被引量：6 发表：1995
