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M M Domach

Publications and source records attributed to M M Domach.

At least 19 recordsLinked to original sources
PubMed2003

A metabolic network analysis & NMR experiment design tool with user interface-driven model construction for depth-first search analysis.

A Windows program for metabolic engineering analysis and experimental design has been developed. A graphical user interface enables the pictorial, "on-screen" construction of a metabolic network. Once a model is composed, balance equations are automatically generated. Model construction, modification and information exchange between different users is thus considerably simplified. For a given model, the program can then be used to predict all the extreme point flux distributions that optimize an objective function while satisfying balances and constraints by using a depth-first search strategy. One can also find the minimum reaction set that satisfies different conditions. Based on the identified flux distributions or linear combinations, the user can simulate the NMR and GC/MS spectra of selected signal molecules. Alternately, spectra vectorization allows for the automated optimization of labeling experiments that are intended to distinguish between different, yet plausible flux extreme point distributions. The example provided entails predicting the flux distributions associated with deleting pyruvate kinase and designing 13C NMR experiments that can maximally discriminate between the flux distributions.

Computer Simulation↗
PubMed2001

A MILP-based flux alternative generation and NMR experimental design strategy for metabolic engineering.

A mixed-integer linear program (MILP) is described that can enumerate all the ways fluxes can distribute in a metabolic network while still satisfying the same constraints and objective function. The multiple solutions can be used to (1) generate alternative flux scenarios that can account for limited experimental observations, (2) forecast the potential responses to mutation (e.g., new reaction pathways may be used), and (3) (as illustrated) design (13)C NMR experiments such that different potential flux patterns in a mutant can be distinguished. The experimental design is enabled by using the MILP results as an input to an isotopomer mapping matrices (IMM)-based program, which accounts for the network circulation of (13)C from a precursor such as glucose. The IMM-based program can interface to common plotting programs with the result that the user is provided with predicted NMR spectra that are complete with splittings and Lorentzian line-shape features. The example considered is the trafficking of carbon in an Escherichia coli mutant, which has pyruvate kinase activity deleted for the purpose of eliminating acetate production. Similar yields and extracellular measurements would be manifested by the flux alternatives. The MILP-IMM results suggest how NMR experiments can be designed such that the spectra of glutamate for two flux distribution scenarios differ significantly.

Algorithms↗
PubMed2000

Computer model for glucose-limited growth of a single cell of Escherichia coli B/r-A. Reprinted from Biotechnology and Bioengineering, Vol. 26, Issue 3, Pp 203-216 (1984).

A computer model is described which is capable of predicting changes in cell composition, cell size, cell shape, and the timing of chromosome synthesis in response to changes in external glucose limitation. The model is constructed primarily from information on unrestricted growth in glucose minimal medium. The ability of the model to make reasonable quantitative predictions under glucose-limitation is a test of the plausibility of the basic biochemical mechanisms included in the model. Such a model should be of use in differentiating among competing hypotheses for biological mechanisms and in suggesting as yet unobserved phenomena. The last two points are illustrated with the testing of a mechanism for the control of the initiation of DNA synthesis and predictions on cell-width variations during the division cycle.

Computer Simulation↗
PubMed2000

Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant.

Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO(2) production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.

Bacillus subtilis↗
PubMed1999

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.

Bacillus subtilis↗
PubMed1995

In situ 31P nuclear magnetic resonance for observation of polyphosphate and catabolite responses of chemostat-cultivated Saccharomyces cerevisiae after alkalinization.

The proposed pH buffering and phosphagenic functions of polyphosphate were investigated by subjecting chemostat-cultivated Saccharomyces cerevisiae to alkalinization (NaOH addition) and anaerobiosis. The subsequent changes in intracellular phosphate-containing species were observed in situ by nuclear magnetic resonance (NMR) spectroscopy by using the NMR cultivator we developed. For the alkalinization experiments, changes in catabolite secretion were also measured in parallel experiments. Additionally, a range of potential neutralization capacity was investigated: a dilute culture and concentrated cultures with low or high polyphosphate content. The concentrated cultures displayed increased cytosolic pH and rapid polyphosphate degradation to small chains. The pH changes and extent of polyphosphate degradation depended inversely on initial polyphosphate content. The dilute culture restored extracellular pH rapidly and secreted acetate. The concentrated culture with low polyphosphate reserves also secreted acetate. In contrast to the alkalinization-induced polyphosphate dynamics, anaerobiosis resulted in the complete hydrolysis of polyphosphate to P(i), as opposed to small chains, and reduced cytosolic pH. The results and calculations suggest that the bulk of NMR-observable polyphosphate (vacuolar) degradation to short polymers conceivably contributes to neutralizing added alkalinity. In other circumstances, such as anaerobiosis, degradation serves other functions, such as phosphorylation potential regulation.

Anaerobiosis↗
PubMed1995

Using differential scanning calorimetry to elucidate metal-protein binding sites in alpha- and gamma-chymotrypsin.

Crystalline alpha-chymotrypsin preparations are contaminated by the post translational variant, gamma-chymotrypsin. The contaminant can account for 5-50 weight percent of the preparation based on thermal analysis. Such contamination can be problematic because this serine protease has both commercial and deactivation model system utility, and the presence of the contaminant may not be detectable by activity assays. Prior work has shown that simple pH gradient elution can separate the two chymotrypsins when loaded to a Cu(2+)-IMAC column; gamma-chymotrypsin eluted first indicating that its interaction with immobilized Cu2+ is weaker. The molecular features that endow these serine proteases with metal affinity has been investigated further by performing differential scanning calorimetry (DSC) studies in the presence and absence of Cu2+, and at different pH values. The dependence of thermostability on pH for fixed metal concentration reveals an interplay between stabilizing and destabilizing metal binding events. The results are consistent with Cu(2+)-chymotrypsin interaction occurring, in part, through binding to a glutamate- or aspartate-containing chelation site. The strength of this site may differ in the two chymotrypsins.

Binding Sites↗
PubMed1994

Flux adaptations of citrate synthase-deficient Escherichia coli.

The results from the experiments performed with a mutant deficient in citrate synthase activity can be summarized as follows. (1) Totally blocking entry into the TCA cycle did not appreciably alter the cellular ATP yield. The unchanged yield suggests that for growth on abundant glucose, the sensitivity of ATP yield to TCA cycle flux is low. ATP production in the mutant is altered, in part, by modulating the relative amounts of formate and acetate produced. (2) The in vivo operation of pyruvate-formate lyase and malic enzyme corresponds to proposals developed from in vitro studies. Namely, pyruvate activates the former, and acetyl CoA inhibits the latter. Overall, the diversion of pyruvate to formate under aerobic conditions constitutes an adaptation of the mutant to the enzymatic lesion. The low alpha-ketoglutarate dehydrogenase flux estimated for the mutant indicates that the enzyme is highly repressed in cells growing rapidly on glucose, which is in accord with prior induction-repression studies. Moreover, the lack of a change in uptake flux during the bulk of batch growth is consistent with prior induction-repression studies. (3) The mutant exhibits a heightened sensitivity to CO2 as compared to wild-type counterparts. Growth rate is increased, and the production of formate, malate, glycerate, and pyruvate is reduced. This sensitivity illustrates that citrate synthase is more than an expendable component in an amphibolic pathway. Its presence in wild-type cells "immunizes" against the effect of CO2 fluctuations. (4) The effects of CO2 can be tentatively explained by assuming that the PEP carboxylase-catalyzed reaction is stimulated.

Adenosine Triphosphate↗
PubMed1994

Residence time distributions of various tracers in tumors: implications for drug delivery and blood flow measurement.

BACKGROUND: The evaluation of rates of tumor blood flow with small, rapidly diffusing tracers requires an accurate model for mass transport within the tissue and tracer biodistribution. It is generally assumed that the whole tumor or several tumor regions act as well-mixed compartments, an assumption that has never been evaluated in tumors. PURPOSE: The purpose of this study was to assess the accuracy of compartmental flow models in tissue-isolated tumors. METHODS: We measured the residence time distributions of various tracers with the use of ex vivo perfusion of tissue-isolated rat R3230AC mammary tumors. This approach permits simultaneous, independent measurements of total blood flow and tracer concentrations in afferent and efferent vessels. The isolated tumors were perfused with Krebs-Henseleit solution, to which could be added D2O saline and either 3% by volume F44-E (a perfluorocarbon emulsion) or 1% by weight fluorescein isothiocyanate (FITC)-albumin. A pulse of D2O and one of the other tracers was added to the perfusing liquid, and the relative concentrations of both D2O and perfluorocarbon or FITC-albumin were measured in the tumor effluent. D2O and the perfluorocarbon were measured with an imaging spectrometer tuned to either 2H or 19F. FITC-albumin concentrations were measured by luminescence spectrometry. The results were analyzed using various compartmental models. RESULTS: The tracer residence time distribution deviated from that expected for a single well-mixed compartment. Only half of the D2O left the tumor with a time constant consistent with the known perfusate flow. The remainder exited the tumor more rapidly than expected, and neither vascular shunting nor macroscopic flow heterogeneity accounts for this component of the D2O flow. However, two-compartment models provide an improved fit to the data. CONCLUSIONS: Our experiments demonstrate that the simple compartmental model used to estimate blood flow with diffusible tracers is not accurate. IMPLICATIONS: The nonideal blood flow found in our experiments reflects phenomena that may have important effects in the development of pharmacokinetic models of drug delivery to tumors. The accuracy of blood flow measurements, made with such techniques as nuclear magnetic resonance, positron-emission tomography, and computed tomography, may also be affected when they rely on the assumption that the tumor is a collection of well-mixed compartments.

Adenocarcinoma↗
PubMed1994

Protein speciation and potential effects on membrane transport and immobilization illustrated by alpha-chymotrypsin.

Protein speciation via physiochemical and biochemical processes presents both challenges and opportunities for membrane applications and research. Different types of speciation and its potential effects are illustrated with the well-known enzyme, alpha-chymotrypsin. This enzyme is capable of self-association and undergoing post-translational modifications. Self-association is shown to alter transport fluxes and also be manipulable. The post-translational modifications generate catalytically-active species that are difficult to detect and different in stability. The presence of different species can conceivably impact on the fabrication, performance, and analysis of immobilized enzyme systems.

Biological Transport↗
PubMed1993

Role of oxygen vs. glucose in energy metabolism in a mammary carcinoma perfused ex vivo: direct measurement by 31P NMR.

The role of glycolysis vs. respiration in tumor energy metabolism has been studied, to date, primarily in vitro by using single cells, multicellular spheroids, or tissue slices. With the advent of in vivo NMR spectroscopy, several investigators have shown that tumor energy status depends on its blood flow. Since manipulation of blood flow alters both oxygen and glucose delivery to a solid tumor, these studies have not been able to separate the relative contribution of oxygen vs. glucose in energy metabolism in vivo. In the present study, we have overcome this problem by combining two methods: the tissue-isolated R3230AC mammary adenocarcinoma perfused ex vivo and 31P NMR spectroscopy. The isolated tumor permits one to control the perfusion pressure as well as the metabolite concentrations in the perfusate. NMR spectroscopy permits one to measure the ratio of nucleoside triphosphate to inorganic phosphate (NTP/Pi) and pH. Our results show that (i) the NTP/Pi ratio ex vivo is similar to that observed in vivo prior to surgery, (ii) the NTP/Pi ratio is insensitive to flow changes at high flow rates but is proportional to flow rate at flows comparable to those found in vivo, (iii) the NTP/Pi ratio of these tumors is resistant to hypoxia and is not maintained when glucose is removed or replaced with glutamine, and (iv) although both O2 and glucose are consumed by these tumors, the effect of perfusate flow rate appears to be mediated largely through glucose delivery. The current approach not only provides information about the role of glycolysis vs. respiration in a rodent tumor but also is general and versatile enough to provide similar data in human tumors perfused ex vivo.

Adenocarcinoma↗
PubMed1992

2H-nuclear magnetic resonance imaging of tumor blood flow: spatial and temporal heterogeneity in a tissue-isolated mammary adenocarcinoma.

2H-Nuclear magnetic resonance imaging of deuteron accumulation in tissue following an i.v. bolus of deuterium oxide provides a noninvasive means of constructing maps of tissue perfusion. With a measured arterial input function and a simple model for tissue-capillary exchange, these data can provide quantitative estimates of local flow. This technique was tested in rat brain and then applied to the study of spatial heterogeneity and temporal variation of blood flow in the tissue-isolated R3230AC mammary adenocarcinoma. Global flow from the brain averaged 0.96 ml/min.g, in good agreement with results obtained from other methods; the perfusion of brain was relatively homogeneous. Global tumor blood flow averaged 0.32 ml/min.g, ranging from 0.11 to 0.96 ml/min.g. Imaging revealed variations in perfusion both within and between the tumors that far exceeded those expected from brain flow heterogeneity and uncertainty in the flow estimates. By obtaining repeated flow images at 30-min intervals, it was possible to show that the regional blood flow shifted with time in single pixels and in multipixel regions. These experiments show that 2H-nuclear magnetic resonance may be useful in obtaining noninvasive and quantitative measurement of temporal blood flow changes in a solid tumor in vivo.

Adenocarcinoma↗
PubMed1987

Determining pathway structure-property relationships through experimentation and analytical frameworks.

A brief description of the information content of the experimental methods that are becoming increasingly useful for probing intracellular processes, a framework for interpreting observations, and an example that combines framework results and 13C NMR observations have been presented. Results in terms of structural criterion have been obtained that suggest that it may be possible to develop a glossary of structure-function heuristics. From the engineering point of view, such general work may also provide keys to system/subsystem modeling due to having some classic and nonclassic network properties mapped in advance. However, we note that approaches based solely on kinetics ignore physiochemical processes. A number of potential processes were mentioned earlier. Investigations of the importance of such processes, though, have been limited due to the dominance of in vitro enzyme kinetic and regulation work. Nonetheless, interesting proposals have been advanced by a limited number of workers, such as the suggestion that membrane-bound and soluble populations of enzymes with high and low activity, respectively, exist in eukaryotes (e.g., aldolase22) and the balanced attained between the two populations is an important regulatory mechanism. In an effort to contribute to the evaluation of physiochemical processes, our formalism was recently used to explore the logic of enzyme turnover number-enzyme amount distributions from the standpoint of minimizing excess enzymatic capacity (i.e., minimizing excess energy expenditure for protein biosynthesis) and the use of limited cytoplasmic solvation capacity (i.e., concentrated cytoplasm is water-limited; hence, maintaining the solubility of all constituents is difficult).

Biochemistry↗
PubMed1985

Consideration of the gain, enzymatic capacity utilization, and response time properties of metabolic networks as a function of operating point and structure.

Computer simulation is used to examine the gain, utilization of enzymatic capacity and response time properties of a simple metabolic network consisting of a sequence of irreversible enzyme reactions controlled by non-allosteric feedback inhibition. The latter properties are chosen because interpretation of a metabolic network's structure can be aided by envisioning the network to maximize a single objective such as flux control. However, by examining the properties as a function of the network's operating point (extent of saturation of the first enzymatic reaction) and structural characteristics, it is found that apparent trade-offs occur between definable objectives. It is suggested that greater consideration of the trade-offs that may occur could help generate metabolic network heuristics and facilitate the interpretation of metabolic networks in terms of the functional role they play in the intact organism.

Computers↗
PubMed1984

Testing of a potential mechanism for E. coli temporal cycle imprecision with a structural model.

A structured model for E. coli B/r-A was used to test a potential mechanism for cell cycle temporal imprecision. The mechanism assumes that variations in the activity of septation enzymes can occur. Such variations result in D period imprecision while the chromosome replication schedule is not directly affected. The main results of inserting the mechanism were the predicted ratio of cell doubling time to fission size coefficient of variations agreed with reported data as did the predicted negative correlation between parent-offspring cycle times. Consequently, the proposed mechanism, which suggests that D period fluctuations are the primary source of cell cycle variations, appears consistent with observed cell behavior.

Cell Cycle↗
PubMedDate not supplied

13C NMR evidence for pyruvate kinase flux attenuation underlying suppressed acid formation in Bacillus subtilis.

When batch and continuous Bacillus subtilis cultures are provided with a small amount of citrate, acid production ceases, carbon yield increases by more than 2-fold, and the productivity of recombinant protein increases. It has been hypothesized that pyruvate kinase activity is attenuated, which in turn lowers glucose flux and minimizes the acid overflow prompted by low Krebs cycle capacity. To complement existing enzyme activity, linear programming, and metabolite pool studies, (13)C NMR studies were performed. Atom mapping and isotopomer mapping matrix methods were used to select the best glucose label. "Best" was defined such that the NMR spectra of glutamate associated with metabolizing labeled glucose via the different candidate metabolic trafficking scenarios would differ considerably in fine structure (e.g., relative singlet intensities). When experiments were performed with 1-(13)C glucose, the observed NMR spectra corresponded well to the one predicted to arise when the metabolic trafficking occurs according to a pyruvate kinase attenuation scenario. This evidence further fortifies the prospects for successfully basing a metabolic engineering strategy on reducing pyruvate kinase activity to better match glycolytic and Krebs cycle capacities.

Bacillus subtilis↗
PubMedDate not supplied

Yeast alcohol dehydrogenase bound to membranes: surface and microenvironment effects on activity and stability.

The enzyme, yeast alcohol dehydrogenase, was adsorbed to porous nitrocellulose and nylon membranes. The two membranes provide different surface chemistries as indicated by the results of the streaming potential, enzyme adsorption, and fluorescein isothiocyanate adsorption experiments. The stability of the enzyme, as determined by continually measuring the extent of coenzyme reduction as a function of time, appeared to be much less for the enzyme adsorbed to the positively charged membrane surface. Moreover, the enzyme adsorbed to the positively charged membrane was the least responsive to pulses of the reducing agent, dithiothreitol, and appeared to exhibit the highest transition temperature when subjected to differential scanning calorimetry analysis. These results indicate that the entropically spreading process observed for other adsorbed proteins may be occurring and the process is more rapid and extensive when enzyme is adsorbed to the nylon than the nitrocellulose membrane. In addition to the relative stability of the enzyme on two different surfaces being examined, the effect of the microenvironment on modulating the activity of the enzyme was investigated by using the reversibility of the enzyme-catalyzed reaction as a probe of the average local environment of the enzyme. It was found that a threshold buffer concentration existed that, once exceeded, the effect of proton production by the reaction could be suppressed.

Alcohol Dehydrogenase↗