Paul Magnano:Week 3 Individual Journal

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===Enzyme Activities===
===Enzyme Activities===
 +
*levels of NADPH-glutamate dehydrogenase(GDH), NAD-GDH and Gs activity were meaured to see if ammonia concentrations affected them
 +
*when input ammonia concentrationincreased, levels of NADPH-GDH decreased
 +
*Fig 3.
 +
**the decrease in NADPH-GDH was coupled with GDH1 decrease
 +
**NAD-GDH activity increased dratically as ammonia concentration increased from 29-61mM
 +
**decreases in the levels of GS transferase activity were seen as ammonia concentration levels increased
 +
*overall the data shows that the concentration of ammonia is thekey factor that controls how S. cerevisiae regulates its nitrogen metabolism
 +
*changes in nitrogen metabolism are controlled by the concentrations of ammonia inside or outside the cell
==Ten New Terms==
==Ten New Terms==

Revision as of 02:12, 31 January 2013

Contents

Outline

Experiment

  • sacchromyces cerevisiae was grown in continuous culture, with varying ammonia input concentrations, while ammonia assiilation rates remained constant
  • ammonia is a preffered nitrogen source for s. cerevisiae, because it results in faster growth than other sources
  • in this study, cultures were grown with same flux (relationship b/w external ammonia concentration and rte of assimilation) but different inflow ammonia concentrations
  • Physiological parameters:
    • S. cerevisiae SU32 grown in continuous cultures with ammonia inflow concentrations ranging from 29 to 118mM
    • fixed glucose concentration of 100mM
  • Fig 1A
    • increase of ammonia concentration from 29 to 61mM resulted in increase of biomass from 4.9 to 8.2 g/liter
    • ammonia concentration above 61mM biomass remained at 8.2 g/liter
    • over trhe entire range of ammonia concentrations the ammonia flux into biomass was 1.1 mmol/gh
  • Fig 1B
    • input ammonia concentration above 44mM resulted in (c02 production/02 consumption) remained constant
    • with ammonia limitation (ammonia input below 44mM) c02 production and 02 consumption values differed
    • no changes in the residual glucose concentration were seen
    • no significant changes in carbon metabolism occured when the culture was switched from ammonia excess to ammonia limitation
  • Fig 1C
    • ammonia within the cells reacts with ketoglutarate to produce glutamate which is converted to glutamine
    • ketoglutarate concentration decreased from 10 to 5 umol/g when the ammonia concentration changed from limited to excess
    • glutamate concentration increased within the cell, linearly
    • increaing ammonia concentrations caused the concentrations of glutamate and glutamine within the cell to increase

Northern Analyses

  • RNA analyses were done to see if RNA levels of nitrogen regulating genes changed with increased ammonia concentrations
  • genes GDH1, GDH2,GLN1 were determined to study responses to ammonia levels
  • GAP1 and PUT4 are amino acid permease genes
  • biosynthetic genes were ILV5 and HIS4
  • Fig 2
    • increasing ammonia levels resulted in GDH1 levels staying the same
    • ammonia concentrations of 29-44mM there was no GDH2, but with increase to 61mM GDH2 level increased
    • maximum GLN1 expression was observed at 61mM
    • overall it was seen that ammonia concentration both repressed GDH1 and caused the expression of GDH2
    • GAP1 was shown to be regulated in response to ammonia concentration
    • the influence of ammonia concentration on GAP1 and PUT4 was analzyed in this study
    • when ammonia concentration was above 44mM, GAP1 and PUT4 decreased
    • in ammonia limited culture a relationship between ammonia flux GAP1 expression wa observed
    • GAP1 and PUT4 expression are regulated by ammonia flux not by ammonia concentration
    • genes ILV5 and HIS4 increased with increasing ammonia concentration outside the cell, it was highest at 66mM

Enzyme Activities

  • levels of NADPH-glutamate dehydrogenase(GDH), NAD-GDH and Gs activity were meaured to see if ammonia concentrations affected them
  • when input ammonia concentrationincreased, levels of NADPH-GDH decreased
  • Fig 3.
    • the decrease in NADPH-GDH was coupled with GDH1 decrease
    • NAD-GDH activity increased dratically as ammonia concentration increased from 29-61mM
    • decreases in the levels of GS transferase activity were seen as ammonia concentration levels increased
  • overall the data shows that the concentration of ammonia is thekey factor that controls how S. cerevisiae regulates its nitrogen metabolism
  • changes in nitrogen metabolism are controlled by the concentrations of ammonia inside or outside the cell

Ten New Terms

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