Cycle 5: Integrated Metabolism Flashcards
Basics of measuring respiration in isolated mitochondria: what do you need?
Measure respiration via oxygen concentration
- Start with litres of Chlamy cells
- Grind up cells
- Purify intact mitochondria
- Wash mitochondria
- Incubate them in a buffer solution
Change in respiration rate (oxygen consumption) in isolated mitochondria by addition of NADH, ADP & Pi, uncoupler
Respiration rate is proportional to oxygen consumption and inversely proportional to oxygen concentration.
1) Isolated mitochondria (ADP washed) - no cellular respiration occurs, constant oxygen concentration
2) Addition of NADH - increase in respiration rate, decrease in oxygen concentration
3) Addition of ADP and Pi - further increase in respiration rate, further decrease in oxygen concentration
4) Addition of uncoupler - even greater increase in respiration rate, even greater decrease in oxygen concentration
Can you explain why the rate changes with each addition?
1) Isolated mitochondria - no substrate, nothing to drive electron transport!!, so no cellular respiration
2) Addition of NADH - drives electron transport, increases rate of cellular respiration
3) Addition of ADP and Pi - makes ATP and dissipates the proton gradient in the intermembrane compartment, proton pumpting becomes easier, which increase the rate of electron transport
4) Addition of 2,4 DNP - uncoupled rate of electron transport is the highest rate, no energy required to dissipate proton gradient, so as long as there is substrate, the pumps are working as fast as they can!
Respiratory control: define it, what is the mechanism.
Respiratory control refers to the phenomenon where the rate of electron transport, or O2 consumption, adjusts based on the availability of ADP and Pi.
Without ADP and Pi, it takes a lot of energy to pump protons into high proton concentration intermembrane compartment, keeping levels of electron transport low (and thus, cellular respiration), because it’s harder for the pumps to work. As ADP and Pi levels rise, protons are pumped via ATP synthase, evening out the proton gradient and making it easier for the Complexes to pump protons, increasing the rate of cellular respiration.
Autotrophic metabolism: Link(s) between chloroplast and cellular respiration.
LINK - G3P from photosynthesis can be respired to make ATP for the rest of the cell.
Chlamy has both chloroplasts (fridge, way to source the food) and mitochondria (food making). ATP is made by the light reactions in the stroma and consumed by the Calvin cycle, where it becomes ADP. G3P is transported outside of the chloroplast via a G3P transporter. G3P is a carbon intermediate of glycolysis and is converted to pyruvate, which goes through the citric acid cycle and oxidative phosphorylation.
Two distinct roles served by G3P that is exported form the chloroplast into the cytosol: energy source and carbon source.
G3P is an energy source for the rest of the cell that can be respired to obtain ATP. G3P can also be converted to glucose or siphoned off to biosynthetic reactions to act as the carbon backbones of organic molecules, effectively acting as a carbon source (and increases the cell’s weight!)
Can Chlamy grow in the dark? Why acetate and not glucose?
Chlamy can grow in the dark, because they are capable of heterotrophic metabolism. For Chlamy to grow without a light source, it needs a way to source carbon compounds without photosynthesis. It has no method to uptake glucose from the environment, since it has no glucose transporter (no evolutionary advantage). However, it can grow on acetate since it has an acetate transporter. Acetate is respired as Acetyl CoA, which goes through the citric acid cycle and oxidative phosphorylation to get ATP. It’s also a source of carbon that can be used to drive biosynthetic reactions.
The concept of Mixotrophic metabolism.
Mixotrophic metabolism combines autotrophic and heterotrophic metabolisms for optimal growth. Chlamy grows fastest via mixotrophic growth (i.e. growing under ideal light conditions, for autotrophic growth, in TAP, for heterotrophic growth).
Gas exchange in respiration and photosynthesis involve both CO2 and O2…you should know which produces / consumes which one of these two molecules.
Photosynthesis - consumes CO2, generates O2
Respiration - consumes O2, generates CO2
This idea that in whole Chlamy cells your measurements of either O2 production or CO2 fixation will underestimate the actual rate…because of respiration.
Measuring [O2] production during photosynthesis underestimates the actual rate, since some O2 is consumed during cellular respiration. Measuring [CO2] fixation during photosynthesis underestimates the actual rate, since CO2 is produced during cellular respiration.
How to measure photosynthesis using gas exchange….and understand the units of carbon fixation.
Photosynthesis can be measured via the rate of CO2 fixation. The units of carbon fixation are µmol of CO2 consumed per min per cell.
Basics of a light response curve…understand the shape of the curve: Linear portion (light-limited region) and Plateau (light-saturated region) in relation to actual photosynthetic processes.
The light response curve has a:
- y-intercept, which represents the rate of respiration, assumed to be constant
- linear portion at low light intensities, where rate of CO2 fixation (or photosynthesis) increases linearly with light intensity, as more CO2 can be used in the Calvin cycle
- plateau at high light intensities, where other factors limit the rate of photosynthesis beyond just increasing light
How the light-saturated rate could be increased (two potential ways..Rubisco content or CO2).
Light-saturated rate can be increased by increasing the concentration of CO2 or enzyme catalyst (rubisco) in the environment.
Even when a chloroplast is present…how does one determine the rate of cellular respiration?
The y-intercept of the light response curve gives the rate of cellular respiration.
Definition of Light Compensation Point…link it to concepts of Carbon Gain and Carbon loss (i.e the dorm room plant your parents got you!)
The light compensation point refers to the x-intercept in a light intensity vs. rate of CO2 fixation graph. It is the point at which light intensity is sufficient for the rate of photosynthesis to equal the rate of respiration.
In other words, the level of carbon gain and carbon loss are the same. Plants cannot grow below or at LCP, because they must have positive rate of CO2 fixation for growth (net carbon gain).
