Kara M Dismuke Week 2 Journal

Definitions

1. dehydrogenase: enzyme that oxidizes a substrate by transferring hydrogen to an acceptor that is either NAD/NADP or a flavin enzyme. An enzyme that is used to remove hydrogen from its substrate, which is used in the cytochrome (hydrogen carrier) system in respiration to produce a net gain of ATP.
   • http://www.biology-online.org/dictionary/Dehydrogenase
2. ammonia: common Name for NH3, a strongly basic, irritating, colourless gas which is lighter than air and readily soluble in water. It is formed in nature as a by-product of protein metabolism in animals. Industrially, it is used in explosives, fertiliser, refrigerants, household cleaning solutions, etc.
   • http://www.biology-online.org/dictionary/Ammonia
3. flux: total amount of a quantity passing through a given surface per unit time. Typical quantities include (magnetic) field lines, particles, heat, energy, mass of fluid, etc.
   • http://www.biology-online.org/dictionary/Flux
4. glutamate: major fast excitatory neurotransmitter in the mammalian central nervous system
   • http://www.biology-online.org/dictionary/Glutamate
5. glutamine: crystalline amino acid occurring in proteins; important in protein metabolism.One of the 20 amino acids that are commonly found in proteins.
   • http://www.biology-online.org/dictionary/Glutamine
6. proline: one of 20 amino acids directly coded for in proteins. Structure differs from all the others, in that its side chain is bonded to the nitrogen of the _ amino group, as well as the _ carbon. This makes the amino group a secondary amine and so proline is described as an imino acid. Has strong influence on secondary structure of proteins and is much more abundant in collagens than in other proteins, occurring especially in the sequence glycine proline hydroxyproline. A proline rich region seems to characterise the binding site of SH3 domains.
   • http://www.biology-online.org/dictionary/Proline
7. permease: 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.
   • http://www.biology-online.org/dictionary/Permease
8. transferase: suffix to the name of an enzyme indicating that it transfers a specific grouping from one molecule to another, for example acyl transferases transfer acyl groups.
   • http://www.biology-online.org/dictionary/Transferase
9. transcription: first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript. As opposed to DNA replication, transcription results in an RNA complement that includes the nucleotide uracil (U) in all instances where thymine (T) would have occurred in a DNA complement. Also unlike DNA replication where DNA is synthesized, transcription does not involve an RNA primer to initiate RNA synthesis.
   • http://www.biology-online.org/dictionary/Transcription
10. metabolite: any substance produced by metabolism or by a metabolic process.
    • http://www.biology-online.org/dictionary/Metabolite
      • metabolism: The process involving a set of chemical reactions that modifies a molecule into another for storage, or for immediate use in another reaction or as a by product. It includes processes for cell growth, reproduction, response to environment, survival mechanisms, sustenance, and maintenance of cell structure and integrity. It is made up of two categories: catabolism and anabolism.
      • http://www.biology-online.org/dictionary/Metabolism

Outline

1. What is the main result presented in this paper?
   • See how particular type of yeast reacts to being put under different conditions
     • Nitrogen limitation
     • Nitrogen excess
     • Glucose limitation
   • Main point:
     • Ammonia concentrations play a role in regulating nitrogen metabolism of Saccharomyces cerevisiae (particular type of yeast) (as determined by keeping ammonia flux levels constant)
     • Note: changes may be attributed to…
       • extracellular or intracellular concentrations of ammonia
       • changes in levels of intracellular metabolites (e.g. alpha-ketoglutamate, glutamate, or glutamine)
2. What is the importance or significance of this work?
   • Ammonia- great to grow particular type of yeast [preferred nitrogen source]
   • Nitrogen metabolism affects gene expression and enzyme activity
   • Ammonia concentration may regulate S. cerevistae in that in may act as a nitrogen sensor
3. What were the limitations in previous studies that led them to perform this work?
   • Previous studies: ammonia concentration= has notable influence
   • But, in these studies, cultures used have different external ammonia concentrations and in rate of ammonia assimilation
   • Thus conclusion: influence lies with flux as opposed to concentration
   • This study: kept levels of flux constant, thus test influence of solely ammonia concentrations on gene expression & enzyme activities
4. General Methods
   • Fixed glucose concentration (100mM)
   • Fixed dilution rate (.15 h-1)
   • Different ammonia concentrations: 29, 44, 61, 66, 78, 90, 114, 118 mM
5. Briefly state the result shown in each of the figures.
   1. FIGURE 1: PHYSIOLOGICAL PARAMETERS
      • 1A: Ammonia Flux
        • x-axis: ammonia concentration (in mM)
        • y-axis:
          • White squares: residual ammonia concentration (in mM)
          • White triangles: biomass (gl-1)
          • Black circles: ammonia flux (mmolg-1h-1)
        • Measurements: increasing levels of ammonia to see the effects on residual ammonia concentration, size of biomass, and ammonia flux of the yeast cells
        • Results (from graph): as ammonia levels increased (from limitation to excess)…
          • the residual ammonia concentration were constant (at about .022mM) for concentration levels of ammonia less than 61mM, but for ammonia concentration greater than 61mM, residual ammonia concentration levels also increased in a linear fashion
          • the biomass size increased when ammonia levels increased from 4.9 and 61 mM, but for ammonia levels greater than 61mM, the biomass size remained the same, relatively speaking (at 8.2 g liter-1)
          • ammonia flux into biomass stayed about the same (1.1mmol g-1h-1)
          • ammonia flux= (dilution rate x [(input ammonia concentration) – (residual ammonia concentration]/biomass)
          • Note: this factors all three of the things previously studied: ammonia concentration, residual ammonia concentration, and biomass size (i.e. it is a ratio of sorts)
        • Trend Observed: By studying the graph, we can observe that there is a relationship between ammonia concentration and residual ammonia concentration (namely as the former increases, after reaching a certain point, the latter increases) and there is a relationship between ammonia concentration and biomass (namely as the former increases, the latter tends to increase).
      • 1B: Carbon Metabolism
        • x-axis: ammonia concentration (in mM)
        • y-axis:
          • Black squares: CO2 produced (mmolg-1h-1)
          • Black triangles: O2 consumed (mmolg-1h-1)
          • White circles: Respiratory Quotient
        • Measurements: increasing levels of ammonia to see the effects on oxygen consumption and carbon dioxide production of the yeast cells
        • Results (from graph): as ammonia levels increased (from limitation to excess)…
          • Under ammonia limitation (29, 44, and 61 mM of ammonia), CO2 production levels decreased but for ammonia concentration levels >61mM, CO2 production levels remained relatively constant
          • Under ammonia limitation (29 and 44mM of ammonia), O2 consumption levels increased but for ammonia concentration levels >44mM, O2 consumption levels remained relatively constant
          • Thus it is no surprise that the respiratory quotient (when ammonia concentration > 44mM), remained relatively constant; granted, upon the increase in ammonia concentration from 29 to 44mM, the respiratory quotient decreases sharply.
            • Note: respiratory quotient= (CO2 produced/ O2 consumed)
        • Trend Observed: at low ammonia concentrations, the behavior O2 and CO2 differs (O2 consumption levels increase and CO2 production levels decrease), but at higher ammonia concentrations, there is no significant difference in their behavior
      • 1C: Alpha-ketoglutarate, Intracellular Glutamate, Intracellular Glutamine
        • x-axis ammonia concentration (in mM)
        • y-axis
          • levels of alpha-ketoglutarate (mumolg-1) [LEFT PANEL OF Fig. 1C]
          • levels of intracellular glutamate (mumolg-1) [CENTER PNEL OF Fig. 1C]
          • levels of intracelullar glutamine (mumolg-1) [RIGHT PANEL OF Fig. 1C]
        • Measurements: increasing levels of ammonia to observe behavior of alpha-ketoglutarate, intracellular glutamate, and intracelullar glutamine in the yeast cells
        • Results (from graph): as ammonia concentration increased (from limitation to excess)…
          • alpha-ketoglutarate concentration decreased
            • with the greatest decrease coming from the increase in ammonia concentrations from 44mM to 61mM
          • intracellular glutamate concentration increased
            • with the greatest increase occurring during the increase in ammonia concentrations from 44mM to 61mM
          • intracellular glutamine concentration increased
            • increased in a linear fashion until ammonia concentration level reached 114mM, at which point it started to decline
        • Trend Observed: with a constant ammonia flux and increasing ammonia concentrations, intracellular concentrations of glutamate and glutamine increase while the concentration of alpha-ketoglutarate decreases
          • A further experiment could be done to try to determine how these three come together in collectively effectively the yeast cells (e.g. is there one that dominates, what is the relationship between each pair should they be paired up, etc.)
   2. FIGURE 2: Northern RNA Analyses
      • Affects of increasing ammonia concentration on RNA levels of nitrogen-regulated genes
      • Left Panel of Figure 2
        • x-axis: ammonia concentration (mM)
        • y-axis: percent expression of
          • GDH1 [white triangles]
          • GDH2 [black triangles]
      • Center Panel of Figure 2
        • x-axis: ammonia concentration (mM)
        • y-axis: percent expression of
          • GAP1 [black squares]
          • PUT4 [white squares]
      • Right Panel of Figure 2
        • x-axis: ammonia concentration (mM)
        • y-axis: percent expression of
          • GLN1 [black diamonds]
          • HIS4 [white circles]
          • ILV5 [black circles]
      • Measurements: increasing levels of ammonia to observe behavior of nitrogen-regulated genes (in terms of RNA expression)
        • Quantified via X-ray films at different exposure times
      • Results (from graph): as ammonia concentration increased (from limitation to excess)…
        • Left Panel (of Figure 2)
          • GDH1 RNA levels remained relatively constant at ammonia concentrations < 78mM, but GDH1 RNA levels decreased at ammonia concentrations >78 mM
          • GDH2 RNA levels couldn’t be observed at ammonia concentrations <61 mM, but when the ammonia concentration levels increased from 61mM to 90mM, GDH2 RNA levels increase sharply (only to then decline once ammonia levels increased past 90mM)
        • Middle Panel (of Figure 2)
          • At 29 and 44 mM ammonia concentrations, changes in GAP1 and PUT4 RNA levels were minimal
            • However, at ammonia levels >44mM, GAP1 and PUT4 RNA levels decreased
          • And, at 118mM ammonia, the GAP1 RNA had all but disappeared (granted, PUT4 RNA was still present)
        • Right Panel (of Figure 2)
          • Greater levels of ammonia concentrations → increased RNA amount of nitrogen-regulated genes GLN1, ILV5 and HIS4 (the first two maxed out at ~66mM ammonia and the latter maxed out at ~78mM ammonia)
          • After maxing out, all three decreased in a somewhat similar pattern
      • Trend Observed (from graph)
        • Left Panel (of Figure 2)
          • Ammonia concentration repressed RNA expression of nitrogen-regulated genes (in the case of the decrease)
          • However, in the case of the observed increase, concentration induced RNA expression of nitrogen-regulated genes
        • Middle Panel (of Figure 2)
          • With ammonia flux kept constant, we observed that as ammonia concentrations increased, levels of GAP1 and PUT4 increased
        • Right Panel (of Figure 3)
          • As ammonia concentrations increase, the levels of GLN1, ILV5, and HIS4 increase until a certain point and then begin decreasing
          • Note: Note: similar behavior between GLN1, ILV5, and HIS4…
   3. FIGURE 3: Enzyme Activities
      • x-axis: ammonia concentration (mM)
      • y-axis:
        • NADPH-GDH (mumolmin-1mg-1) [black circles]
        • NAD-GDH (mumolmin-1mg-1) [black diamonds]
        • GS transferase (mumolmin-1mg-1) [black squares]
        • GS (mumolmin-1mg-1) [black triangles]
      • Measurements: increasing levels of ammonia to observe effects on enzyme activity levels
        • values are averages of three independent samples +/- the standard error of the mean
        • measured under Vmax conditions
      • Results from Graph:
        • Top Graph (of Figure 3)
          • As ammonia levels increase, NADPH-GDH levels decrease steadily
          • Although slight increase from 61mM to 66mM ammonia
        • Middle Graph (of Figure 3)
          • From 29 to 61 mM ammonia: sharp increase in NAD-GDH levels
          • > 61 mM ammonia→ no more large increase in NAD-GDH levels (until 96mM ammonia)
        • Bottom Graph (of Figure 3)
          • Increase ammonia concentration to 61mM sees a slight decrease in GS transferase activity level and slight decrease in GS activity
          • But >61mM ammonia → no further changes
      • Trend
        • Top Graph [of Figure 3]
          • As ammonia levels increase, levels of NADPH-GDH decrease
          • Also decrease in GDH1 so can’t be attributed completely to changing ammonia concentrations
        • Middle Graph [of Figure 3]
          • As ammonia levels increase, levels of NAD-GDH increase steadily (only decrease from 66mM to 78 mM ammonia)
        • Bottom Graph [of Figure 4]
          • Ammonia limitation sees a decrease in GDH2 and GS levels, but once ammonia levels increase (approaching and then reaching excess), GDH2 and GS levels level off (remain relatively constant)
          • Note: GDH2 and NAD-GDH responses to changing levels of ammonia → can’t really be attributed to changing ammonia concentrations (regulation mainly occurs at transcription levels)
6. What is the overall conclusion of the study and what are some future directions for research?
   • Basic conclusion (repeated from above): Changing levels of ammonia concentration effects nitrogen metabolism in S. cerevistae in different ways (held ammonia flux levels constant)
   • Future directions for research:
     • repeat experiment to see if get same results (validity)
     • change physiological parameters, test other amino acids, test other enzymes
     • hold ammonia levels and ammonia flux levels constant and test for other factors that may influence nitrogen metabolism

User: Kara M Dismuke

BIOL398-04/S15:Week 2