Elizabeth Polidan Week3

Elizabeth Polidan

BIOL 398.03 / MATH 388

• Loyola Marymount University

• Los Angeles, CA, USA

• Email me through OpenWetWare

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Vocabulary

1. Permease
   1. (Science: enzyme) general term for a membrane protein that increases the permeability of the plasma membrane to a particular molecule, by a process not requiring metabolic energy. Reference: http://www.biology-online.org/dictionary/Permease
2. Allosteric inhibition
   1. See allosteric Enzyme. Inhibitors act as 'modulators' in enzyme execution as they can attach themselves to a molecule that will alter the binding Site for the enzyme, rendering it unusable and therefore rendering the enzyme inactive. Reference: http://www.biology-online.org/dictionary/Allosteric_Inhibitors
3. Northern analysis
   1. A procedure similar to the southern blot analysis, used mostly to separate and identify rNA fragments; typically via transferring RNA fragments from an agarose gel to a nitrocellulose filter followed by detection with a suitable probe. Reference: http://www.biology-online.org/dictionary/Northern_blot_analysis

Article Outline

• What is the main result presented in this paper?
• What is the importance or significance of this work?
• What were the limitations in previous studies that led them to perform this work?
• Methods
  • Saccharomyces cerevisiae was grown in cultures with continuous feed
    • Incoming concentrations of ammonia were varied from a low of 29 milimoles per liter (mM) to a high of 118 mM.
    • Incoming concentrations of glucose were all held at 100 mM
    • The dilution rate was 0.15/hour
  • Physiological parameters were monitor to determine the state of the yeast in the system
    • Biomass was measured in grams/liter
    • Residual ammonia was measured mM
    • Ammonia flux was calculated from resulting biomass, input ammonia concentration and residual ammonia concentration: NH4 flux = (diluation rate x (incoming ammonia concentration – residual ammonia concentration)/biomass)
    • Oxygen consumption was measured in mM per gram biomass per hour (mM/g/h)
    • Carbon Dioxide production was measured in mM/g/h
    • Respiratory quotient was calculated as (CO2 produced/O2 consumed)
    • Measured alpha-ketoglutarate at varying ammonia concentration
    • Measured glutamate at varying ammonia concentration
    • Measured glutamine at varying ammonia concentration
  • Northern analysis
    • Measured RNA to determine if levels varied with ammonia concentration in nitrogen regulated genes
      • GDH1, GDH2
      • GAP1, PUT4
      • ILV5, HIS4, GLN1
  • Enzyme activities
    • Levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were measured to determine if there was any change in enzyme activity as the ammonia concentration was varied.
• Discussion
  • What do the X and Y axes represent?
  • How were the measurements made?
  • What trends are shown by the plots and what conclusions can you draw from the data?
• Conclusion
  • It is the ammonia concentration that regulates nitrogen metabolism in S. cerevisiae, not the flux level.
    • The driver of the observed changes was either ammonia concentration itself (intra- and intercellular) or ammonia concentration-driven changes in levels of metabolites such as glutamate or glutamine.