Saccharomyces cerevisiae is able to use a wide variety of nitrogen sources for growth. Not all nitrogen sources support growth equally well. In order to select the best out of a large diversity of available nitrogen sources, the yeast has developed molecular mechanisms. These mechanisms consist of a sensing mechanism and a regulatory mechanism which includes induction of needed systems, and repression of systems that are not beneficial. The first step in use of most nitrogen sources is its uptake via more or less specific permeases. Hence the first level of regulation is encountered at this level. The next step is the degradation of the nitrogen source to useful building blocks via the nitrogen metabolic pathways. These pathways can be divided into routes that lead to the degradation of the nitrogen source to ammonia and glutamate, and routes that lead to the synthesis of nitrogen containing compounds in which glutamate and glutamine are used as nitrogen donor. Glutamine is synthesized out of ammonia and glutamate. The expression of the specific degradation routes is also regulated depending on the availability of a particular nitrogen source. Ammonia plays a central role as intermediate between degradative and biosynthetic pathways. It not only functions as a metabolite in metabolic reactions but is also involved in regulation of metabolic pathways at several levels. This review describes the central role of ammonia in nitrogen metabolism. This role is illustrated at the level of enzyme activity, translation and transcription.

The main objective of the present study was to determine alterations of calcium handling in the diabetic rat heart during the transition from adaptive to maladaptive phase of cardiomyopathy. By inhibiting the nuclear enzyme poly(ADP-ribose) polymerase (PARP), we also investigated the possible role of this enzyme in the sequence of pathological events. Six weeks after induction of type I diabetes by injection of streptozotocin in rats, the hearts were perfused according to Langendorff. Intracellular-free calcium (Ca 2+ i) levels were measured by surface fluorometry using Indo-1 AM. Cyclic changes in Ca 2+ i concentrations and hemodynamic parameters were measured simultaneously. The hearts were challenged by infusion of isoproterenol. Six weeks of diabetes resulted in reduced inotropy and lusitropy. The diabetic hearts (DM) expressed a significantly elevated end-diastolic Ca 2+ i level (control, 111±20 vs DM, 221±35 nM). The maximal transport capacity of SERCA2a and conductance of RyR2 were reduced. These changes were not accompanied by major alterations in the tissue content of SERCA2a, RyR2, phospholamban and Na + /Ca 2+ exchanger. In response to ß-adrenergic activation, SERCA2a transport capacity and RyR2 conductance were stunted in the DM hearts. Inhibition of PARP induced minor changes in the mechanical function and calcium handling of the DM hearts. In conclusion, the observed changes in contractility and in Ca 2+ i handling are most likely attributable to functional disturbances of SERCA2a and RyR2 in this transitional phase of diabetes. At this stage of diabetes, PARP does not appear to play a significant pathogenetic role in the alterations in contractile function and calcium handling.

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Under carbon source transitions, the intracellular pH of Saccharomyces cerevisiae is subject to change. Dynamics in pH modulate the activity of the glycolytic enzymes, resulting in a change in glycolytic flux and ultimately cell growth. To understand how pH affects the global behavior of glycolysis and ethanol fermentation, we measured the activity of the glycolytic and fermentative enzymes in S. cerevisiae under in vivo-like conditions at different pH. We demonstrate that glycolytic enzymes exhibit differential pH dependencies, and optima, in the pH range observed during carbon source transitions. The forward reaction of GAPDH shows the highest decrease in activity, 83%, during a simulated feast/famine regime upon glucose removal (cytosolic pH drop from 7.1 to 6.4). We complement our biochemical characterization of the glycolytic enzymes by fitting the V max to the progression curves of product formation or decay over time. The fitting analysis shows that the observed changes in enzyme activities require changes in V max , but changes in K m cannot be excluded. Our study highlights the relevance of pH as a key player in metabolic regulation and provides a large set of quantitative data that can be explored to improve our understanding of metabolism in dynamic environments.