Lab 9: Isolation of Mushroom Mitochondria and Assay for Succinate Dehydrogenase

Question 1

which membrane of the mitochondria is enriched with enzymes necessary for the reactions of cell resp?

Answer 1

the inner mito membrane. contains ETC

Question 2

where is the ETC located in the mito?

Answer 2

in the inner membrane and cristae

Question 3

T/F: Mitochondria can synthesize their own proteins

Answer 3

true. they have minimal DNA that allows for some protein synthesis

Question 4

what theory explains why mitochondria has DNA and other organelles do not?

Answer 4

the endosymbiont theory

Question 5

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 ____ _____.

Answer 5

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.

Question 6

succinate dehydrogenase utilizes ______ (FAD) to oxidize succinate into _______

Answer 6

succinate dehydrogenase utilizes FLAVIN ADENINE DINUCLEOTIDE (FAD) to oxidize succinate into FUMARATE

Question 7

where is succinate dehydrogenase located? What is the alternative name for this enzyme?

Answer 7

unlike other citric acid cycle enzymes, succ deHyd. is located IN the inner membrane, and is also known as COMPLEX II of the ETC

Question 8

How many protons and electrons does FADH2 carry?

Answer 8

carries 2 e- and 2 protons

Question 9

usually, what electron carrier does FADH2 donate its electrons to?

Answer 9

FADH2 gives the electrons to ubiquinone in the ETC.

Question 10

3 Main stages of Cell respiration

Answer 10

1) mobilization of acetyl coA from glucose, FA’s or protein
2) citric acid cycle
3) ETC ad oxidative phosphorylation

Question 11

How many ATP’s does NADH yield? FADH2?

Answer 11

NADH yields 3 ATPs, FADH2 yields 2 ATPs

Question 12

How did we quantify the activity of succinate dehydrogenase in this experiment? Mechanism?

Answer 12

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.

Question 13

How do we make sure that all of the electrons from FADH2 are being given to DCIP and not Ubiquinone?

Answer 13

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.

Question 14

DCIP is ____ when oxidized, and _____ when reduced

Answer 14

DCIP is BLUE when oxidized, and CLEAR when reduced.

Question 15

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?

Answer 15

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.

Question 16

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?

Answer 16

0.551 microM/min

Question 17

What happens to SDH activity when you add more mitochondrial pellet to the cuvette with DCIP?

Answer 17

increased mito pellet= increased SDH activity= DCIP gets reduced= cuvette gets more clear = decrease in abs reading

Question 18

What happens to SDH activity if you add malonate?

Answer 18

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.

Question 19

What happens to SDH activity if you did not add sodium azide?

Answer 19

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.

Question 20

What happens to SDH activity if you DID NOT ADD SUCCINATE?

Answer 20

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.

Question 21

What happens if you place BOILED mitochondria to cuvette with DCIP?

Answer 21

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.

Question 22

to obtain the mitochondria of the mushroom to conduct the experiment, what should you do?

Answer 22

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