Lab 9: Isolation of Mushroom Mitochondria and Assay for Succinate Dehydrogenase Flashcards
which membrane of the mitochondria is enriched with enzymes necessary for the reactions of cell resp?
the inner mito membrane. contains ETC
where is the ETC located in the mito?
in the inner membrane and cristae
T/F: Mitochondria can synthesize their own proteins
true. they have minimal DNA that allows for some protein synthesis
what theory explains why mitochondria has DNA and other organelles do not?
the endosymbiont theory
the citric acid cycle takes place in the ____ of the mitochondria, generating reduced electron carriers _____ and _____, which go to the ___ to produce ATP through the process of ____ _____.
the citric acid cycle takes place in the MATRIX of the mitochondria, generating reduced electron carriers NADH and FADH2, which go to the ETC to produce ATP through the process of OXIDATIVE PHOSPHORYLATION.
succinate dehydrogenase utilizes ______ (FAD) to oxidize succinate into _______
succinate dehydrogenase utilizes FLAVIN ADENINE DINUCLEOTIDE (FAD) to oxidize succinate into FUMARATE
where is succinate dehydrogenase located? What is the alternative name for this enzyme?
unlike other citric acid cycle enzymes, succ deHyd. is located IN the inner membrane, and is also known as COMPLEX II of the ETC
How many protons and electrons does FADH2 carry?
carries 2 e- and 2 protons
usually, what electron carrier does FADH2 donate its electrons to?
FADH2 gives the electrons to ubiquinone in the ETC.
3 Main stages of Cell respiration
1) mobilization of acetyl coA from glucose, FA’s or protein
2) citric acid cycle
3) ETC ad oxidative phosphorylation
How many ATP’s does NADH yield? FADH2?
NADH yields 3 ATPs, FADH2 yields 2 ATPs
How did we quantify the activity of succinate dehydrogenase in this experiment? Mechanism?
we used an artificial electron acceptor DCIP instead of FADH2 givings its electrons to ubiquinone. When DCIP is reduced with succinates electrons, it transforms from BLUE to CLEAR, resulting in a quantifiable color change via spectrophotometry.
How do we make sure that all of the electrons from FADH2 are being given to DCIP and not Ubiquinone?
disable ubiquinone and the entire electron transport chain by adding Sodium Azide, which prevents the reduction of Oxygen to water. With ubiquinone out of the picture, DCIP is now the ONLY available electron acceptor, allowing us to fully quantify the activity of succinate dehydrogenase.
DCIP is ____ when oxidized, and _____ when reduced
DCIP is BLUE when oxidized, and CLEAR when reduced.
If you were to add a competitive inhibitor of succinate dehydrogenase, what would you expect to happen? What would happen to the color of the solution if you added DCIP electron acceptor?
there will be a decrease in rate of succinate oxidation because the inhibitor is competing for the active site of succ. de Hyd. Because less succinate is being oxidized, less DCIP is being reduced, and there for the solution will stay Blue.
a standard curve (micromolar vs absorbance) of DCIP activity was determined to be y=0.019x. When measuring a cuvette filled with succinate dehydrogenase, a notable change in absorbance from 0.295 to 0.033 over a period of 25 minutes. What is the succinate de hydrogenase activity in microM/minute?
0.551 microM/min
What happens to SDH activity when you add more mitochondrial pellet to the cuvette with DCIP?
increased mito pellet= increased SDH activity= DCIP gets reduced= cuvette gets more clear = decrease in abs reading
What happens to SDH activity if you add malonate?
malonate is a competitive inhibitor that competes for the active site of SDH with succinate. Malonate cannot be oxidizd however, and thus there will be less DCIP reduction/SDH activity in the presence of malonate.
What happens to SDH activity if you did not add sodium azide?
you would not be able to quantify ALL of SDH’s activity, making it APPEAR as though there is a decrease in activity.
-normally, DCIP competes with ubiquinone and an e- acceptor of FADH2’s electrons. Sodium azide INCREASES the chances that e- from succinate gets transferred to DCIP rather than Ub. by blocking the ETC.
- without the presence of Azide, the ETC will be functional and ubiquinone can accept electrons from FADH2, which IS NOT QUANTIFIABLE.
- SDH is not inhibited, the reaction is still ongoing, however, we cannot see all of it because some e- are being passed to Ub.
What happens to SDH activity if you DID NOT ADD SUCCINATE?
would probably not see any activity because there is no reactant present.
- no reactant= no oxidation= no DCIP reduction = no quantifyable color change, indicating decreased enzymatic activity.
What happens if you place BOILED mitochondria to cuvette with DCIP?
would not see activity because SDH is denatured due to heat. denaturation may change conformation of SDH and render SDH inactive, making it incapable of oxidizing succinate.
to obtain the mitochondria of the mushroom to conduct the experiment, what should you do?
1) homogenize the mushroom
2) centrifuge
3) discard nuclear pellet
4) centrifuge the nuclear supernatant
5) discard the post-mito supernatant, and KEEP THE MITO PELLET
