12th BTK meeting: 'Systems Biology: redefining BioThermoKinetics'.
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Cell Physiological Phenomena↗
Biomedical subjects
Johann M Rohwer
Publications and source records attributed to Johann M Rohwer.
Partial purification and characterisation of sucrose synthase in sugarcane.
Three sucrose synthase (SuSy) (EC 2.4.1.13) forms were isolated from sugarcane leaf roll tissue. During anion exchange chromatography, one peak of activity (SuSyA) eluted during the wash step and the other peak (SuSyB) during the salt gradient phase at 180mM KCl concentration. A third form of activity (SuSyC), which also eluted at 180mM KCl, was also present in the leaf roll and replaced SuSyB depending on the season of the year. Substrate Km values, as well as sucrose breakdown/synthesis ratios, differed between these forms. For SuSyA, SuSyB, and SuSyC, respectively, Km values+/-SE (mM) were: 41.8+/-3.4, 109+/-23, and 35.9+/-2.3 for sucrose, 1.07+/-0.08, 0.214+/-0.039, and 0.00191+/-0.00019 for UDP, 6.62+/-1.55, 11.7+/-2.6, and 6.49+/-0.61 for fructose, and 3.59+/-0.37, 0.530+/-0.142, and 0.234+/-0.025 for UDP-glucose. Sucrose breakdown/synthesis ratios+/-SE were 0.0791+/-0.0199, 0.330+/-0.180, and 0.426+/-0.069 for SuSyA, SuSyB, and SuSyC, respectively. The ratio of the area of peak 1 (low breakdown/synthesis ratio) to the area of peak 2 (high breakdown/synthesis ratio) in sucrose accumulating tissue (internode 9) was 0.88, while in non-accumulating (leaf roll) tissue it was 14.5 at the same time of year. The molecular mass of the denatured subunits of all three forms was 94kDa by SDS-PAGE. A polyclonal antiserum raised against SuSyB cross-reacted with all three forms on an immunoblot, but only SuSyA and SuSyB were immunoinactivated by this serum.
Fructose↗
Modelling cellular systems with PySCeS.
UNLABELLED: The Python Simulator for Cellular Systems (PySCeS) is an extendable research tool for the numerical analysis and investigation of cellular systems. AVAILABILITY: PySCeS is distributed as Open Source Software under the GNU General Public Licence and is available for download from http://pysces.sourceforge.net CONTACT: bgoli@sun.ac.za.
Cell Physiological Phenomena↗
A kinetic study of sugarcane sucrose synthase.
The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP-glucose: D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP-glucose dissociating last from the enzyme. Product inhibition studies showed that UDP-glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows: Km values (mm, +/- SE) were, for sucrose, 35.9 +/- 2.3; for UDP, 0.00191 +/- 0.00019; for UDP-glucose, 0.234 +/- 0.025 and for fructose, 6.49 +/- 0.61. values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP-glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5-fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady-state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrations in vivo, therefore impacting on metabolic regulation.
Binding, Competitive↗
ECA: control in ecosystems.
Although metabolic control analysis (MCA) cannot be applied directly to microbial ecological systems because of mass conservation and stoichiometric constraints, we demonstrate here that Hierarchical Control Analysis (HCA) can be applied to such systems. We illustrate the approach for a particular ecosystem example of the biological synthesis of acetic acid from glucose, and uncover some surprising aspects to the control of this miniature ecosystem.
Acetic Acid↗
Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae.
Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate that the NAD(+)-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (C(J)v1) of approximately 0.85 and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (R(J)DHAP= 0.48 to 0.69), followed by ATP concentration (R(J)ATP = -0.21 to -0.50). Interestingly, the pathway responds weakly to NADH concentration (R(J)NADH = 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.
Glycerol↗