Biomedical subjects

M M Domach

Publications and source records attributed to M M Domach.

26 records · Page 2Linked to original sources

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↗

PubMedDate not supplied

Suppressed acid formation by cofeeding of glucose and citrate in Bacillus cultures: emergence of pyruvate kinase as a potential metabolic engineering site.

Microbial cultures typically produce acids when metabolizing the common carbon source, glucose. Acid production not only represents a waste of carbon, but its accumulation can limit cell concentration and culture stability, thereby reducing productivity. On the basis of prior work, acid production was attributed to be due to a mismatch between glycolytic and tricarboxylic acid (TCA) cycle capacities. To suppress acid production, a strategy entailing adding citrate to glucose minimal medium proved extremely effective. The effect of citrate on in-vivo flux distribution was quantified using a detailed flux-model. When the molar glucose-citrate ratio was varied between 3 and 6, a significant reduction in glycolytic flux and essentially complete suppression of acid formation was found as compared to chemostat cultures grown solely on glucose. Adding other biosynthetic precursors such as glutamine did not invoke the same suppression, thus indicating that citrate's effect is at the regulatory level. We hypothesized that the reduction of glycolytic flux in the presence of citrate results from its transport being coupled with the uptake of divalent metal ions. Citrate transport alters the intracellular balance of metal ions which in turn could trigger a sophisticated series of metabolic events leading to reduction of the activities of the pyruvate kinase and phosphofructokinase (PFK), the regulatory enzymes of glycolysis. On the basis of this scenario and other regulatory information, pyruvate kinase has emerged as a potential metabolic engineering site. It's deactivation in Bacillus subtilis or Escherichia coli strains is expected to yield constructs with a much lower tendency for making acid byproducts.

Bacillus↗

PubMedDate not supplied

Cell growth and by-product formation in a pyruvate kinase mutant of E. coli.

In this paper, we report on the analysis of acid formation in an E. coli pyk mutant. The results demonstrate that acid formation is insignificant for both the wild-type and the mutant at low glucose concentrations. However, at relatively high glucose concentrations, acid formation remains very low for the mutant but is significant for the wild-type. This substantial reduction in acids is accompanied by an increase in CO(2) production. Moreover, unlike the B. subtilis pyk mutant, the E. coli pyk mutant did not show a substantial increase in the PEP pool.

Acids↗

PubMedDate not supplied

NMR-Observed phosphate trafficking and polyphosphate dynamics in wild-type and vph1-1 mutant Saccharomyces cerevisae in response to stresses.

The phosphagenic, osmotic, and metabolic roles of polyphosphate in chemostat-cultivated yeast were investigated with a new NMR cultivator. Wild-type yeast and a vacuolar vph1-1 mutant, which lacks polyphosphate, were subjected to different stimuli. Starved wild-type yeast exclusively directed phosphate to vacuoles despite other competing sinks. After DNP or iodoacetate exposure, which significantly affected cytosolic pH or ATP metabolism, polyphosphate hydrolysis did not occur, which casts doubt on the phosphagen function of vacuolar polyphosphate. It took about 1 h for Mn2+ to traffic to vacuoles, and some evidence was obtained for polyphosphate responding to osmotic challenges. Fast NMR scans show that rapid polyphosphate hydrolysis to small polymers follows alkalinization. The small polymers then degrade to orthophosphate, which coincides with sugar phosphates increasing and subsequent reacidification. In contrast, when vph1-1 mutants were subjected to alkalinization, the absence of a vacuolar source of phosphate slowed reacidification. Based on known yeast physiology and observed sugar phosphate dynamics, polyphosphate degradation may enable rapid glycogen mobilization to glycolysis for considerable acid and ATP production. Overall, maintaining both polyphosphate and carbohydrate reserves may endow yeast with the ability to rapidly manage the extracellular environment.

Biological Transport↗

PubMedDate not supplied

Performance trade-offs in in situ chemostat NMR.

Investigating cell cultures with NMR requires high cell densities to provide adequate signal-to-noise, or scans must be summed over long time periods and short-term events are lost. The mixing within a chemostat can be used to shorten the time required to acquire informative in situ NMR spectra from cell cultures. However, performance trade-offs can occur between net signal, spectral resolution, and oxygenation due to sampling volume, conductivity, gas bubble, and fluid flow effects. These trade-offs and the effect of different mixing regimes were theoretically analyzed to quantify how device design decisions impact performance. The results were found to concur with data from cell-free NMR experiments performed in 18 mS/cm conductivity medium. The results also guided the redesign of an NMR bioreactor in terms of relative radio frequency (rf) coil and sample dimensions and other factors. The design, which entails using chemostat mixing to shunt sample through a rf coil in ca. 0.4 s, provides adequate oxygenation for the 4-16% (v/v) cell suspensions examined. Gains realized include lower conductive losses, better magnetic field homogeneity, and the exclusion of gas bubbles from the sampling zone. These gains enable the acquistion of spectra from dilute (3-4% v/v) Saccharomyces cerevisiae chemostat cultures in 6.9 min with high resolution in both the orthophosphate and the beta-NTP regions. Samples with 16% (v/v) cells also yield useful spectra within 0.5-1.0 min.

Bioreactors↗

PubMedDate not supplied

Metal-based affinity separation of alpha- and gamma-chymotrypsin and thermal stability analysis of isolates.

Chymotrypsin preparations are contaminated by autolysis products and other post-translational products derived from the zymogen. Some prevalent contaminants are difficult to detect with activity assays and molecular weight separation. However, our differential scanning calorimetry (DSC) studies of alpha-chymotrypsin have revealed that gamma-chymotrypsin is a common contaminant in commercial preparations, and the alpha- and gamma-species differ significantly in thermal stability. Thus, DSC analysis provides a sensitive and rapid means to assess the homogeneity of preparations. Moreover, free metal ion binding studies conducted indicate that the alpha- and gamma-species differ substantially in the number of metal binding sites and metal affinity. Therefore, to attempt to repurify a commercial preparation of alpha-chymotrypsin, a resolubilized sample of alpha-chymotrypsin was subjected to immobilized metal (Cu+2) affinity chromatography with pH elution and the fractions were subjected to DSC analysis. The process successfully removed the majority of the contaminating gamma-chymotrypsin.

Calorimetry, Differential Scanning↗