Prediction of Oscillations in Glycolysis in Ethanol-Consuming Erythrocyte-Bioreactors.

A mathematical model of energy metabolism in erythrocyte-bioreactors loaded with alcohol dehydrogenase and acetaldehyde dehydrogenase was constructed and analyzed. Such erythrocytes can convert ethanol to acetate using intracellular NAD and can therefore be used to treat alcohol intoxication. Analysis of the model revealed that the rate of ethanol consumption by the erythrocyte-bioreactors increases proportionally to the activity of incorporated ethanol-consuming enzymes until their activity reaches a specific threshold level. When the ethanol-consuming enzyme activity exceeds this threshold, the steady state in the model becomes unstable and the model switches to an oscillation mode caused by the competition between glyceraldehyde phosphate dehydrogenase and ethanol-consuming enzymes for NAD. The amplitude and period of metabolite oscillations first increase with the increase in the activity of the encapsulated enzymes. A further increase in these activities leads to a loss of the glycolysis steady state, and a permanent accumulation of glycolytic intermediates. The oscillation mode and the loss of the steady state can lead to the osmotic destruction of erythrocyte-bioreactors due to an accumulation of intracellular metabolites. Our results demonstrate that the interaction of enzymes encapsulated in erythrocyte-bioreactors with erythrocyte metabolism should be taken into account in order to achieve the optimal efficacy of these bioreactors.

Biosynthesis and Reactivity of Cysteine Persulfides in Signaling.

Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys-SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys-SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys-SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys-SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys-SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys-SSH product is short and decomposition leads to a mixture of polysulfides (Cys-S-(S)n-S-Cys). These in vitro data, together with the intrinsic reactivity of Cys-SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.

Organization of the human mitochondrial hydrogen sulfide oxidation pathway.

Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.

Distribution of methionine between cells and incubation medium in suspension of rat hepatocytes.

Methionine is an essential amino acid involved in many significant intracellular processes. Aberrations in methionine metabolism are associated with a number of complex pathologies. Liver plays a key role in regulation of blood methionine level. Investigation of methionine distribution between hepatocytes and medium is crucial for understanding the mechanisms of this regulation. For the first time, we analyzed the distribution of methionine between hepatocytes and incubation medium using direct measurements of methionine concentrations. Our results revealed a fast and reversible transport of methionine through the cell membrane that provides almost uniform distribution of methionine between hepatocytes and incubation medium. The steady-state ratio between intracellular and extracellular methionine concentrations was established within a few minutes. This ratio was found to be 1.06±0.38, 0.89±0.26, 0.67±0.16 and 0.82±0.06 at methionine concentrations in the medium of 64±19, 152±39, 413±55, and 1,204±104 µmol/L, respectively. The fast and uniform distribution of methionine between hepatocytes and extracellular compartments provides a possibility for effective regulation of blood methionine levels due to methionine metabolism in hepatocytes.

An allosteric mechanism for switching between parallel tracks in mammalian sulfur metabolism.

Methionine (Met) is an essential amino acid that is needed for the synthesis of S-adenosylmethionine (AdoMet), the major biological methylating agent. Methionine used for AdoMet synthesis can be replenished via remethylation of homocysteine. Alternatively, homocysteine can be converted to cysteine via the transsulfuration pathway. Aberrations in methionine metabolism are associated with a number of complex diseases, including cancer, anemia, and neurodegenerative diseases. The concentration of methionine in blood and in organs is tightly regulated. Liver plays a key role in buffering blood methionine levels, and an interesting feature of its metabolism is that parallel tracks exist for the synthesis and utilization of AdoMet. To elucidate the molecular mechanism that controls metabolic fluxes in liver methionine metabolism, we have studied the dependencies of AdoMet concentration and methionine consumption rate on methionine concentration in native murine hepatocytes at physiologically relevant concentrations (40-400 microM). We find that both [AdoMet] and methionine consumption rates do not change gradually with an increase in [Met] but rise sharply (approximately 10-fold) in the narrow Met interval from 50 to 100 microM. Analysis of our experimental data using a mathematical model reveals that the sharp increase in [AdoMet] and the methionine consumption rate observed within the trigger zone are associated with metabolic switching from methionine conservation to disposal, regulated allosterically by switching between parallel pathways. This regulatory switch is triggered by [Met] and provides a mechanism for stabilization of methionine levels in blood over wide variations in dietary methionine intake.

Rat liver folate metabolism can provide an independent functioning of associated metabolic pathways.

Folate metabolism in mammalian cells is essential for multiple vital processes, including purine and pyrimidine synthesis, histidine catabolism, methionine recycling, and utilization of formic acid. It remains unknown, however, whether these processes affect each other via folate metabolism or can function independently based on cellular needs. We addressed this question using a quantitative mathematical model of folate metabolism in rat liver cytoplasm. Variation in the rates of metabolic processes associated with folate metabolism (i.e., purine and pyrimidine synthesis, histidine catabolism, and influxes of formate and methionine) in the model revealed that folate metabolism is organized in a striking manner that enables activation or inhibition of each individual process independently of the metabolic fluxes in others. In mechanistic terms, this independence is based on the high activities of a group of enzymes involved in folate metabolism, which efficiently maintain close-to-equilibrium ratios between substrates and products of enzymatic reactions.

Sulfur-based redox alterations in long-lived Snell dwarf mice.

Changes in sulfur-based redox metabolite profiles in multiple tissues of long-lived Snell dwarf mice were compared with age- and sex-matched controls. Plasma methionine and its oxidation products, hypotaurine and taurine, were increased in Snell dwarfs while cystine and glutathione levels were decreased, leading to an oxidative shift in the redox potential. Sexual dimorphism in renal cystathionine ß-synthase (CBS) activity was observed in control mice but not in Snell dwarfs. Instead, female Snell mice exhibited ~2-fold higher CBS activity, comparable to levels seen in male Snell dwarf and in control mice. Taurine levels were significantly higher in kidney and brain of Snell dwarf versus control mice. Methionine adenosyltransferase (MAT) was higher in liver of Snell dwarfs, and the higher concentration of its product, S-adenosylmethionine, was correlated with elevated global DNA methylation status. Application of a mathematical model for methionine metabolism revealed that the metabolite perturbations in Snell dwarfs could be explained by decreased methionine transport, increased MAT and increased methyltransferase activity. Our study provides a comprehensive map of systemic differences in the sulfur network between Snell dwarfs and controls, providing the necessary foundation for assessment of nutrition-linked metabolic status in long-lived versus control animals.