Untersuchungen über Methangärung und die dabei wirksamen Bakterien

Ohne Zusammenfassung     

The G157C mutation in the Escherichia coli sliding clamp specifically affects initiation of replication

Escherichia coli cells with a point mutation in the dnaN gene causing the amino acid change Gly157 to Cys, were found to underinitiate replication and grow with a reduced origin and DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA to ADP-DnaA. The DnaA protein was, however, not the element limiting initiation of replication. Overproduction of DnaA protein, which in wild-type cells leads to over-replication, had no effect in the dnaN(G157C) mutant. Origins already opened by DnaA seemed to remain open for a prolonged period, with a stage of initiation involving β clamp loading, presumably limiting the initiation process. The existence of opened origins led to a moderate SOS response. Lagging strand synthesis, which also requires loading of the β clamp, was apparently unaffected. The result indicates that some aspects of β clamp activity are specific to the origin. It is possible that the origin specific activities of β contribute to regulation of initiation frequency.     

Does human attractin have DP4 activity?

Mutations in the mouse ATRN gene, which encodes attractin, offer links between this protein and pigmentation, metabolism, immune status and neurodegeneration. However, the mechanisms of attractin action are not understood. The protein was first identified in humans in a circulating form in serum. A protease activity was postulated similar to the membrane-bound ectoenzyme DP4/CD26. In the last decade, both DP4/CD26 and attractin were controversially described to be the major source of human serum DP4 activity. We purified attractin from human plasma, and found that the DP4-like activity of the preparation shows nearly identical kinetic properties to that of recombinant human DP4. In contrast, the native electrophoretic behavior of this activity is clearly different from human and porcine DP4, but co-migrates with the protein band identified as attractin by Western blotting and N-terminal sequencing. Nevertheless, a DP4 impurity could be demonstrated in purified plasma attractin and the activity could be removed by ADA affinity chromatography, resulting in a homogenous attractin preparation without DP4 activity. These results are substantiated by expression of different attractin isoforms, in which no DP4 activity was found either. This indicates that the multidomain protein attractin acts as a receptor or adhesion protein rather than a protease.     

Regulation and function of xanthophyll cycle-dependent photoprotection in algae

The xanthophyll cycle represents one of the important photoprotection mechanisms in plant cells. In the present review, we summarize current knowledge about the violaxanthin cycle of vascular plants, green and brown algae, and the diadinoxanthin cycle of the algal classes Bacillariophyceae, Xanthophyceae, Haptophyceae, and Dinophyceae. We address the biochemistry of the xanthophyll cycle enzymes with a special focus on protein structure, co-substrate requirements and regulation of enzyme activity. We present recent ideas regarding the structural basis of xanthophyll cycle-dependent photoprotection, including different models for the mechanism of non-photochemical quenching of chlorophyll a fluorescence. In a dedicated chapter, we also describe the unique violaxanthin antheraxanthin cycle of the Prasinophyceae, together with its implication for the mechanism of xanthophyll cycle-dependent heat dissipation. The interaction between the diadinoxanthin cycle and alternative electron flow pathways in the chloroplasts of diatoms is an additional topic of this review, and in the last chapter we cover aspects of the importance of xanthophyll cycle-dependent photoprotection for different algal species in their natural environments.     

Discrepancy between GLUT4 translocation and glucose uptake after ischemia

Objective: Low-flow ischemia results in glucose transporter translocation and in increased glucose uptake. After total ischemia in rat heart, we found no increase in glucose uptake. Here we test the hypothesis that total ischemia is associated with decreased activation of GLUT4 despite translocation. Methods: Isolated working hearts (n=70, Sprague–Dawley rats) were perfused for 70 min at physiological workload with Krebs–Henseleit buffer containing [2-³H]glucose (5 mmol/l, 0.05 μCi/ml) with either oleate (0.4 mmol/l, 1%BSA) or pyruvate (5 mmol/l, 1%BSA). After 20 min, hearts were subjected to 15 min of total ischemia followed by 35 min of reperfusion. We measured glucose uptake and intracellular free glucose (IFG) using [2-³H]glucose and [¹⁴C]sucrose, and determined the distribution of GLUT4 by colocalization immunofluorescence with Na–K ATP-ase. Results: Cardiac power was 10.1 ± 0.90 mW before ischemia and did not differ between groups. Recovery was the same in both groups (55.7 ± 24.8$%). Glucose uptake did not differ between groups before ischemia, and did not increase during reperfusion. Despite evidence of GLUT4 translocation after reperfusion in both groups, IFG did not increase compared with before ischemia. Conclusion: We conclude that there is a discrepancy between glucose transporter availability and glucose uptake after ischemia, which may be due to inhibition of GLUT4 in the plasma membrane. (Mol Cell Biochem 278: 129–137, 2005)     

Veratrol-<Emphasis Type="Italic">O</Emphasis>-demethylase of <Emphasis Type="Italic">Acetobacterium dehalogenans</Emphasis>: ATP-dependent reduction of the corrinoid protein

The anaerobic veratrol O-demethylase mediates the transfer of the methyl group of the phenyl methyl ether veratrol to tetrahydrofolate. The primary methyl group acceptor is the cobalt of a corrinoid protein, which has to be in the +1 oxidation state to bind the methyl group. Due to the negative redox potential of the cob(II)/cob(I)alamin couple, autoxidation of the cobalt may accidentally occur. In this study, the reduction of the corrinoid to the superreduced [Co^I] state was investigated. The ATP-dependent reduction of the corrinoid protein of the veratrol O-demethylase was shown to be dependent on titanium(III) citrate as electron donor and on an activating enzyme. In the presence of ATP, activating enzyme, and Ti(III), the redox potential versus the standard hydrogen electrode (E _SHE) of the cob(II)alamin/cob(I)alamin couple in the corrinoid protein was determined to be −290 mV (pH 7.5), whereas E _SHE at pH 7.5 was lower than −450 mV in the absence of either activating enzyme or ATP. ADP, AMP, or GTP could not replace ATP in the activation reaction. The ATP analogue adenosine-5′-(β,γ-imido)triphosphate (AMP-PNP, 2–4 mM) completely inhibited the corrinoid reduction in the presence of ATP (2 mM).