Mitteilungen der Schweizer Informatik Gesellschaft · 6/2003

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Mitteilungen der Schweizer Informatik Gesellschaft · 4/2004

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Untersuchung zur Enzyminaktivierung in Hefe nach Einwirkung von Di?thyldicarbonat

Zusammenfassung Diäthyldicarbonat (PKE) wurde hinsichtlich seiner Inaktivierungswirkung gegenüber Enzymen in Backhefe untersucht. Dazu wurde die Abhängigkeit der Inaktivierung von ADH, GAPDH und MDH nach PIKE-Einwirkung auf die Hefe quantitativ bestimmt.Es wurde festgestellt, daß unter gleichen Bedingungen zu einer weitgehenden Inaktivierung der untersuchten Enzyme sehr unterschiedliche PKE-Mengen notwendig sind. Eine 90%ige Inaktivierung der ADH in Hefe erfolgt unter den gewählten Bedingungen mit etwa 0,01 ml PKE, eine 90%ige Inaktivierung der GAPDH erst mit mindestens 0,1 ml PKE und eine 90%ige Inaktivierung der MDH sogar erst mit 0,5 ml PKE. Diese Ergebnisse zeigen, daß bezüglich der Wirkungsspezifität von PKE gegenüber den verschiedenen Enzymen in Mikroorganismen mit großen Unterschieden gerechnet werden muß. Die hierfür möglichen Grönde werden diskutiert.Bei einer fünffachen Erhöhung der Hefemenge, aber gleicher PKE-Konzentration, wurde eine fünfmal größere ADH-Aktivität, aber nur eine zweimal größere GAPDH-Aktivität, dagegen keine Veränderung der MDH-Aktivität festgestellt.

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Investigations about enzyme inactivation in yeasts after application of diethyl pyrocarbonate

Summary The in vivo inactivation action of diethyl pyrocarbonate (DEP) on enzymes in bakers yeast was studied. The decrease in activity of ADH (Alcohol dehydrogenase, 1.1.1.1.), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase 1.2.1.12.), MDH (Malate dehydrogenase 1.1.1.37.) in relation to the DEP concentration was determined quantitatively.Various amounts of DEP are needed to inactivate the enzymes investigated. To accomplish a 90% inactivation of ADH 0.01 ml DEP are needed. For the same inactivation of GAPDH and MDH 0.1 ml and 0.5 ml DEP are needed respectively. Possible reasons for the inactivation specifity of DEP are discussed.An increase in the yeast concentration gives a corresponding lower inactivation by DEP for ADH. The effect of a higher yeast concentration on GAPDH is lower than that of ADH, while no change in the inactivation of MDH was observed.

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Der Autor dankt dem C. S. I. R. Südafrika für die Gewährung eines Auslands-Stipendiums.     

Influence of organic carbon decomposition on calcite dissolution in surficial sediments of a freshwater lake

A multi-sensor approach was used to determine high-resolution porewater gradients of Ca(2+), CO(3)(2-), H(+) and O(2) in sediment cores along a depth transect from eutrophic Lake Sempach (Switzerland). We quantified the reproducibility of measurements and analyzed concentration profiles with a one-dimensional diffusion-reaction model to quantify benthic fluxes. Calculation of oxic respiration in the sediment showed that almost all settled organic carbon was degraded with O(2) at shallow depths while only 28% was decomposed aerobically at the deepest location. Fluxes were highest at 8 m depth (24.4 mM m(-2) d(-1)) and lowest at the deepest site of 85 m (7.1 mM m(-2) d(-1)). Dissolution of calcite from the sediment was also depth dependent. A total of 1.1 mM m(-2) d(-1) was found for the shallowest site, and values decreased with depth to 0.6 mM m(-2) d(-1) at the deepest site.     

Single molecule fluorescence imaging of the photoinduced conversion and bleaching behavior of the fluorescent protein Kaede

Photoconversion and photobleaching behavior of the fluorescent protein Kaede immobilized in polyacrylamide gel matrix at room temperature was studied by single molecule wide-field fluorescence microscopy. Photobleaching kinetics of Kaede molecules upon excitation at 488 nm showed slight heterogeneity, suggesting the presence of different protein conformations and/or the distribution of local environments in the gel matrix. Statistical analysis of intensity trajectories of single molecules revealed four major types of fluorescence dynamics behavior upon short illumination by a violet light pulse (405 nm). In particular, two types of photoswitching behavior were observed: the green-to-red photoconversion (4% of Kaede molecules) and the photoactivation of green fluorescence without emission of red fluorescence (13%). Two other major groups show neither photoconversion nor red emission and demonstrate photoinduced partial deactivation (43%) and partial revival (30%) of green fluorescence. The significantly lower green-to-red conversion ratio as compared with bulk measurements in aqueous solution might be induced by the immobilization of the protein molecules within a polyacrylamide gel. Contrary to Ando et al. (Proc Natl Acad Sci 2002;99:12651-12656), we found a significant increase in green fluorescence emission upon illumination with 405-nm light, which is typical for GFP and related proteins.