William A. C. Gendron Week 2
Biology Terms from the paper
- Glutamate dehydrogenase: "An important enzyme involved in the deamination of amino acids." Deamination is the removal of the amino group (-NH2) from an amino acid. Creates ammonia which eventually becomes urea.
- Proline: "A heterocyclic, non-polar imino acid that is present in all proteins studied to date." Heterocyclic molecules are ring molecules with more than one element.(Carbon and something else).
- Acetate: AKA ethonoate. "the anion, CH3COO−"
- Acetaldehyde: AKA ethanal. "Direct oxidation product of ethanol." "CH3CHO The traditional name for ethanal."
- Alpha-ketoglutarate: "−OOC–[CH2]2–CO–COO−" In Krebs cycle, key part of glutaric acid(helps form aa glutamine, and acts as a nitrogen transporter.
- Glutamate: "Anion of the amino acid glutamic acid." It also has neural functions.
- Glutamine: "Amino acid commonly found in proteins" and related to glutamate.
- Permease: "A membrane-bound protein in bacteria that is responsible for transport of a specific substance in or out of the cell."
- Glutamine synthetase: Catalyzes the interaction between glutamine, ATP and xanthosine 5′‐phosphate.
- NADPH-glutamate dehydrogenase: This enzyme helps with urea synthesis and it converts glutamate to α-ketoglutarate.
What is the main result presented in this paper?
- Changes in the concentration of ammonia alters the nitrogen metabolism of S. cerevisiae. The focus of this paper was to use a continuous culture also know as a chemostat to verify that it is actually the concentration that determines the metabolism rather than changes in the flux. This is a problem in previous papers where they do not maintain as rigid of a control over the flux.
What is the importance or significance of this work?
- This paper is important for several reasons: First, this is an important metabolic pathway. Understanding the mechanisms and how the organism reacts is important to biological study. Secondly, it is valuable to be able to narrow down the actual variables in a field of study. Otherwise, it would not be possible to figure out anything concrete. Finally, it helps look at regulatory features of eukaryotes and therefore this study can have biomedical applications. By looking at the homology of this pathway between the organisms, we can get a better understanding of our own metabolism.
What were the limitations in previous studies that led them to perform this work?
- I stated this previously but a key one was that they did not standardize flux between the groups. I would imagine that this would at least be partly responsible for difference in biomass if one system had a higher flux rate.
What were the methods used in the study?
- The yeast S. cerevisiae were grown in chemostats with fixed flow rates, a fixed concentration of 100mM glucose and varying ammonia concentrations.
- Concentrations: 29, 44, 61, 66, 78, 90, 96, 114 and 118mM of ammonia.(Independent variable for all other measurements).
- They have more details in another paper which they cite.
- They measured various dependent variables.
- Biomass and ammonia were measured by previously described methods.
- Also measured oxygen, carbon dioxide and then extrapolated the respiratory quotient from those.
- Alpha-ketoglutarate, glutamate and glutamine concentrations were all measured. (micromoles per gram).
- Northern Blot was used to analyze expression levels of GDH1, GDH2, GAP1, PUT4, GLN1, HIS4 and ILV5 levels.
- Enzyme activity was measured for NADPH-GDH, NAD-GDH and GS transferase.
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?
- Every graph used ammonia concentration as its x-axis.
- Figure 1
- Graph A shows residual ammonia concentration(mM), biomass(g/l) and ammonia flux. Residual ammonia only become visible once biomass reaches its peak for the container. Flux remains relatively stable as is desired. This was measured by weighing the dried samples. The graph shows that ammonia can only increase growth to a certain extent and then it accumulates residual. Other aspects are the limiting factors.
- Graph B shows the oxygen consumption and carbon dioxide excretion. These were measured by Uras3G CO, analyser and a Magnos4G 0, analyser and were then used to extrapolate the quotient which is also displayed on this graph. This shows that with the lowest amounts of ammonia that O2 consumption is low while the CO2 production is high and therefore the respiration quotient is high. With increased ammonia, the O2 increases and becomes stable while CO2 decreases and becomes stable. Quotient decreases and stabilizes as a result. Ammonia can push O2 consumption and CO2 production to a certain extent, after which other limiting factors prevent further growth.
- Graph Section C displays the metabolite, alpha-ketoglutarate, glutamate and glutamine, concentrations. This was measured by using HPLC analysis. Alpha-ketoglutarate shows high stable levels at 29 and 44mM but decreases and becomes stable at 61-120mM. Glutamate and glutamine show low levels at low ammonia and increase to peaks at 114mM of ammonia. Ammonia accelerates reactions that create glutamate and glutamine while decreasing alpha-ketoglutarate levels.
- Figure 2
- These graphs show the expression levels of nitrogen regulated genes: GDH1, GDH2, GAP1, PUT4, GLN1, HIS4 and ILV5. GDH2 spikes at 78 and 118. GDH1 steadily decreases after 78 mM. Both GAP1 and PUT4 show spikes at 44mM but then decrease and become steady at a low level rapidly. GLN1, HIS4 and ILV5 show something resembling a parabolic arc with peaks between 44 mM and 78mM. These were determined by Northern Blotting. Ammonia levels seem to have ideal amounts when it comes to causing these gene expressions. Too little or too much can alter the expression.
- Figure 3
- I could not see the paper which describes the methods of how they analyzed the enzymes. NADPH-GDH was negatively correlated with increased ammonia. NAD-GDH was positively correlated. GS transferase and GS synthase were both slightly negatively correlated with ammonia concentration.
- This yeast species reacts to ammonia concentration changes by adjusting their metabolism in multiple ways. The controls to this are linked to the concentration gradient between the cell and its surroundings or the changes to the metabolites. Further research could look at specifying which of these is accurate and by looking for the machinery driving these changes.