Look at Figure 1 and think about why SDH may be easier to assay than malonate de
ID: 166942 • Letter: L
Question
Look at Figure 1 and think about why SDH may be easier to assay than malonate deydrogenase (MDH) or isocitrate dehydrogenase (ICD) by this protocol? HINT: Why choose to assay this particular enzyme and not one of the other eight involved in the TCA cycle? PROVIDE TWO DISTINCT REASONS.
Look at Figure 2 the ETC system
a.) You used sodium azide in your assay. Provide a detailed (in your own words) scientifically based (referenced) explanation as to what specific components of ETC this assay component is impacting.
b.) If sodium azide was NOT added in this enzyme assay, what might be the issue? Again provide scientifically based (referenced) explanation. HINT: think about what normally would be going on in this pathway and what youa re doing in the enzyme assay to "reroute" things on this pathway.
DIFFERENTIAL CENTRIFUGATION Centrifugation separates cell components on the basis of size and density. The larger Repeated centrifugation at progressively and denser components experience the greatest centrifugal force and move most higher speeds will fractionate cell rapidly. They sediment to form a pellet at the bottom of the tube, while smaller, less homogenates into their components. dense components remain in suspension above, a portion called the supernatant. SUPERNATANT 3 SUPERNATANT 1 SUPERNATANT LON-SPEED VERY HIGH-SPEED MEDIUM-SPEED HIGH-SPEED CENTRIFUGATION a CENTRIFUGATION CENTRIFUGATION ENTRIFUGATION Ce homogenate PELLET 1 PELLET 3 PELLET 4 PELLET 2 whole cells mitochondria microsomes ribosomes nuclei lysosomes other small viruses large macromolecules cytoskeletons peroxisomes vesiclesExplanation / Answer
Answer:
1. Why SDH may be easier to assay than malonate deydrogenase (MDH) or isocitrate dehydrogenase (ICD) by this protocol?
a) Succinate dehydrogenase (SDH) is the only enzyme in the TCA cycle that is embedded in the inner mitochondrial membrane (termed as complex II). By using differential centrifugation technique, the mitochondria can be easily separated from the other organelles and the activity of the SDH can be measured.
b) Succinate dehydrogenase is also the only enzyme in the TCA pathway that participates in the ETC (electron transport chain). It is easy to assay this enzyme by monitoring the release of electrons by using an appropriate dye which acts as an artificial electron acceptor.
2) You used sodium azide in your assay. Provide a detailed (in your own words) scientifically based (referenced) explanation as to what specific components of ETC this assay component is impacting.
b.) If sodium azide was NOT added in this enzyme assay, what might be the issue?
Answer:
a) Sodium azide inhibits the Heme groups of cytochromes in Cytochrome oxidase (complex IV). Succinate dehydrogenase is assayed using DCIP dye that act as an artificial electron acceptor. In the oxidized form, the dye is blue in color. However, upon reduction the dye becomes colorless. Addition of sodium azide ensures that the electrons from the oxidation of succinate are being used by dye alone (since azide blocks the transfer of electrons from cytochrome a in complex IV to oxygen). The oxidation of succinate to fumarate can then be measured by monitoring the reduction in the color of the dye.
b) If sodium azide was not added then the normal flow of the electrons through the Electron transport chain to oxygen would resume and it would not be possible to determine the SDH activity.
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.