BIOL 334 – Final Exam Study Sheet - Questions with Verified Answers
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Module
BIOL 334
Institution
BIOL 334
BIOL 334 – Final Exam Study Sheet
Short Answer
1. A biochemist used an oxygen (O2) electrode to study the rate of O2 consumption by purified, functional
mitochondria isolated from skeletal muscle and brown fat tissue of a mammal. Each preparation was
separately placed in an aerated solution a...
1. A biochemist used an oxygen (O2) electrode to study the rate of O2 consumption by purified, functional
mitochondria isolated from skeletal muscle and brown fat tissue of a mammal. Each preparation was
separately placed in an aerated solution and maintained at physiological pH and temperature.
After adding 5 mM pyruvate to each mitochondrial preparation they noted that O2 uptake by mitochondria from
skeletal muscle was completely dependent upon the subsequent addition of 1 mM ADP and 1 mM inorganic
phosphate (Pi), whereas mitochondria from brown fat showed a significant rate of pyruvate-dependent O2
consumption, even in the absence of any added ADP or Pi.
(a) Why was O2 consumption by mitochondria purified from human skeletal muscle totally dependent
upon the combined presence of pyruvate, ADP, and Pi?
• Pyruvate oxidation requires high energy electrons shuttled by NADH and FADH2 to the ETC. The ETC
eventually reduces (↓) O2 into H2O while establishing proton motive force (PMF) across the inner mito.
membrane (IM)
• Without the substrates ADP and Pi, ATP synthase of the IM cannot function to dissipate PMF (the H+
gradient across the IM)
• ETC cannot function unless PMF is continually dissipated by the ATP synthase. H+ ions have to keep
moving down/across their [ ] gradient on the IM even though the ATP synthase ‘rotary molecular motor’ into
the matrix
(b) Why did pyruvate addition to brown fat mitochondria trigger significant O2 consumption in the absence
of any added ADP or Pi?
• Brown fat mitos. contain ‘thermogenin’ a proton ionophore channel of the IM
• Thermogenin: Allows PMF without proton/H+ movement for ATP synthesis. (ADP + Pi not required)
(c) One minute after adding pyruvate, 5mM potassium cyanide (KCN) was added to each mitochondrial
prep. After KCN addition O2 consumption by mammalian muscle and brown fat mitochondria became
completely undetectable, whereas the rate of O2 consumption by snail muscle mitochondria was
reduced by about 60%
Why did O2 consumption by mammalian muscle or brown fat mitochondria stop when KCN was added?
(note: your answer should include name of specific location(s) where KCN binds).
• O2 consumption stopped because in the ETC electrons are transferred from NADH and FADH2 to complex
V (4) which bind to oxygen
• However, CN irreversibly binds to complex V (4), thus blocks electrons transfer to oxygen. Therefore,
oxygen cannot be reduced and thus preventing respiration
(d) Why was O2 consumption by snail (mollusk) mitochondria only partially inhibited when KCN was
added?
• Molluscs have the AOX that bypass complex V so they remain unaffected by cyanide, allowing electron
transfer to oxygen and allowing respiration
• This is called ‘cyanide resistant respiration’, some flux still travels through the traditional pathway which is
why oxygen consumption is partially reduced
, (e) What is thermogenin and how does it promote heat production by brown fat tissue of human babies or
hibernating mammals?
Thermogenin: an uncoupling protein
• Promotes heat production as it is a mammalian ‘ionophore’ that dissipates PMF (without ATP synthesis),
leading to thermogenesis
• Allows PMF to be dissipated without coupling proton movement down it’s [ ] gradient to ATP synthesis
• No dependence on ADP + Pi
(f) Why does addition of 5 mM cyanide block any respiratory O2 uptake by mitochondria isolated from
skeletal muscle or brown fat tissue (when they are also being incubated in the presence of pyruvate,
ADP and Pi)?
CN- (Cyanide) • Potent inhibitor of cytochrome oxidase
• Blocks electron transfer to O2
• Inhibits COX
• Inhibits complex IV (5)
(g) Why do respiring mitochondria isolated from the foot muscle of a gastropod mollusc (i.e. snail)
continue to consume O2 following the addition of 5 mM cyanide?
Mollusc • Their IM also contains AOX which bypasses COX to allow continued electron transfer
• AOX bypasses complexes I, III, and IV instead of transferring electrons direction from
NAD(P)H to ubiquinone, and hence O2
• AOX is not inhibited by CN-
2. Annotation of an animal’s genome sequence predicted that a specific gene (GI:623450) encodes a
putative citrate synthase. Describe an experimental strategy that would confirm that this predicted citrate
synthase gene definitely encodes an active citrate synthase enzyme.
1) Bacterial overexpression: express the specific gene (GI:623450), which will show recombinant ENZ has
citrate synthase activity
2) Purify citrate synthase from the same species to show that a portion of its aa. sequence matches the
corresponding portion of deduced aa. sequence from the annotated species
3. Calcium (Ca2+) ions participates in the ‘fine control’ of respiration in aerobic mammalian muscle.
What is/are the metabolic control mechanism(s) by which an increased Ca2+ concentration rapidly enhances
the rate of pyruvate oxidation to 3CO2 molecules in contracting mammalian muscle cells (myocytes)?
PDC • Converted from inactive phosphorylated form to dephosphorylated form
• Pyruvate rapidly oxidizes to acetyl-CoA + CO2
TCA cycle • Activated
• Acetate group of acetyl-CoA is rapidly oxidized to O2
4. Modification (restructuring) of the chemical composition of membrane lipids is an important biochemical
adaptation of many animals and plants to changes in their external environment.
, (a) What type of modification occurs to their membrane phospholipids when a cold-blooded (i.e.
‘poikilothermic’) marine animal migrates up to warmer, shallow, surface waters following an extended
stay in much colder, deep waters of the ocean’s ‘abyss’ (e.g. >2,000 meters)?
1. Fatty acids of the membrane P-lipids become more saturated
2. Fatty acids become less saturated (less double bonds)
(b) Why does this modification need to occur (i.e. how is it beneficial for optimal membrane function)?
1. Maintain optimal ‘semi-fluidity’ of membranes
2. Needed for membrane (transporter) proteins to properly function
(c) This type of adaptive response is known as “________homoviscous_________ adaptation”.
(d) What type of modification occurs to membrane lipids of plant cells after they have been subjected to an
extended period of nutritional phosphate (Pi) starvation?
Phospholipids are replaced by sulfonyl-lipids
5.
(a) Why do our skeletal muscle cells use carbohydrates (i.e. glucose stored as glycogen) to fuel energy
metabolism during ‘burst’ muscle work (e.g., a 100-meter sprint), but not fatty acids derived from
storage lipids?
• Only glucose is used to produce ATP when there is an absence of O2 via glycolytic pathway
• Fatty acid catabolism cannot produce ATP in absence of O2
(b) Why would ‘burst’ muscle work by humans be impossible if the glycolytic pathway was not rapidly and
massively activated (as occurs in rapidly contracting skeletal muscle cells during a 100-meter sprint)?
Burst muscle work requires too much ATP for aerobic oxidation to keep up, therefore anaerobic metabolism
(glycolysis) is needed
(c) How does the AMP-activated protein kinase contribute to the “Pasteur effect” (i.e. enhanced glycolytic
flux) when heart muscle cells become hypoxic (as would occur during a heart attack)?
AMPK is a primary sensor of cell’s ‘adenylate energy change’ and mediates most effects of decreased [↓] ATP
[ ] due to stresses that inhibit ATP production (like a heart attach) and/or stimulate ATP use
(d) How does the AMP-activated protein kinase (AMPK) contribute to the ‘Pasteur Effect’ (enhanced
glycolytic flux) during burst muscle work?
AMPK actives muscle PFK2, which increases F26P2 [ ], which then activates PFK1 to increase glycolytic flux
(this is the ‘Pasteur Effect’)
(e) What are the two major reasons that humans cannot perform ‘burst’ muscle work (such as an ‘all out’
sprint) for more than about 10 to 20 seconds.
• Fermentable fuel is exhausted quickly
• Lactate buildup is toxic
• Anaerobic metabolism produces less ATP
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