MITOCHONDRIAL LAB

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MITOCHONDRIAL LAB

• We are alive because we make a lot of ATP and ATP
  makes (nonspontaneous) chemical reactions take
  place
• We make about 95 of our ATP in the mitochondria
• We will isolate mitochondria, and study one
  enzymatic step in the pathway to making ATP

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Mitochondria and disease

• CU MED CENTER MITOCHONDRIAL MALFUNCTION LEADS TO
  PARKINSONS DISEASE (SHAKING AS YOU AGE)
• IF YOU RESTRICT YOUR DIET, YOU MAY LIVER LONGER
  HEALTHIER MORE CALORIES TAKEN IN, MORE
  NAD IS USED TO CARRY ELECTRONS (NOTE THAT FAD
  ALSO CARRIES ELECTRONS). HOWEVER, NAD IS ALSO
  NEEDED FOR THE SIR2 PROTEIN THAT PREVENTS AGING
  SYMPTOMS. THUS, LOTS OF SUGAR INTAKE, NAD IS
  NOT AVAILABLE TO SIR2 YOU AGE FASTER.
• Bad mitochondria may be related to Diabetes

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4. Aging (less ATP made by mitochon-dria)
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How we make most of our ATP AEROBIC RESPIRATION
GLUCOSE IS BROKEN DOWN (HIGH ENERGY CHEMICAL
BONDS BROKEN, ATOMS REMOVED) TO STRIP OFF
ENERGETIC ELECTRONS.ENERGY FROM ELECTRONS IS
USED TO MAKE ATP in the MITOCHONDRIAOXYGEN (02)
ACCEPTS THE SPENT (LOW ENERGY) ELECTRONS AEROBIC
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PARTS OF AEROBIC RESPIRATION

• GLYCOLYSIS occurs in the cytoplasm (glucose
  broken in HALF to produce 2 pyruvate molecules)
  (ch. 9)
  (some list other step as intermediate
  step moving pyruvic acid into the
  mitochondrion)
• TCA CYCLE (or Krebs cycle)- where what is left
  of glucose is broken all the way down to C02 and
  all the electrons are stripped off
• Electrons are carried (by NADH or FADH2) to the
  electron transport chain and ATP synthase where
  ATP is made from electron energy (ch. 10)

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PARTS OF AEROBIC RESPIRATION
Part 1.
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Part 3
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CH. 9 GLYCOLYSIS

• GLYCOLYSIS IS A SERIES OF 12 CHEMICAL REACTIONS
  OCCURING IN THE CYTOPLASM
• TAKE GLUCOSE (LIKE JET FUEL) AND STRIPS OFF ITS
  ELECTRONS IN THE HIGH ENERGY COVALENT BONDS
• THIS BREAKS THE COVALENT BONDS AND BREAKS GLUCOSE
  IN HALF PRODUCING TWO 3 CARBON MOLECULES CALLED
  PYRUVATE
• LATER, ENERGY FROM THESE ELECTRONS WILL BE USED
  TO MAKE ATP

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ENERGETIC ELECTRONS TAKEN FROM GLUCOSEGIVEN TO
NADWHICH CARRIES ELECTRONS INTO MITOCHONDRIA
ELECTRONS GIVEN TO NAD is a REDUCTIONUSE
MNEMONIC OILRIG
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Ch. 10 Last 2 Parts of Aerobic Respiration take
place in the Mitochondrion. ATP made at inner
membrane
FOLDING MEMBRANE DOES WHAT? (HINT GUT FOLDS)
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Last 2 Parts involve the Electron Transport
Chain (where electrons are stripped of their
energy, energy used to pump Protons H)Followed
by allowing H to move back, turning ATP Synthase
to make ATP. See SUMMARY ANIMATION FROM OUR
TEXTBOOKD\cell biol 3611\mito
respiration\respiration 1418m.mov
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Electron transport animation from Virtual cell
web site see LINK ON our Cell Lab web site
LAST STEP ATP SYNTHASE IS LIKE A LITTLE
MOLECULAR TURBINE TURBINE IS ROTATED BY MOVEMENT
OF H THEN TURBINE MAKES ATP. Animations

• D\cell biol 3611\mito respiration\ETCAdvanced.wmv

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ANIMATIONS OF CHEMIOSMOSIS

• D\cell biol 3611\mito respiration\chemiosmosis2.s
  wf
• D\cell biol 3611\mito respiration\ATP SYNTHASE
  MBC 14_1.mov
• D\cell biol 3611\mito respiration\17 ELEC TRANS
  CHAIN.MPG
• D\cell biol 3611\mito respiration\ATPGradientAdva
  nced.wmv
• (NOTE THAT SOME ANIMATIONS TALK ONLY OF THE H
  CONCENTRATION GRADIENT, IGNORING THE VERY
  IMPORTANT ELECTRICAL GRADIENT FOR THE H!!)

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We will study one enzyme in the TCA Cycle (see
Ch. 10 esp figures used here) Succinate
Dehydrogenasethis enzyme breaks two chemical
bonds and removes two H atoms from what is left
of glucose.
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Succinate Dehydrog. Is located here
Succin Dehydrog Actually binds Membrane proteins
Of the Electron Transport Chain
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Pyruvate comes in from the cytoplasm into the
mitochondrion, it is broken down in the TCA
cycle to C02 and water. We will study TCA step 6
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In this reaction, once again what is left of
glucose is broken down further by breaking bonds
and removal of 2 H atoms. FADH2 carries the
excited electrons to the electron transport chain
(to make ATP from electron energy)
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Better view of reaction note the two H atoms
that are removed are on different carbons and on
opposite sides (trans, not cis)
In the lab, the electrons are not given to FAD,
but we add a dye that changes its absorbance
when it takes the electrons (change in
absorbance recorded by spectrophotometer)
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Succinate Dehydrogenase is an enzyme substrate
succinate binds in the active site (similar to
enzymes below)
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Characteristics of Succ. Dehyd.

• As it breaks chemical bonds between Carbon and
  Hydrogen (C-H) in succinate, it takes the excited
  electrons and the Hydrogen atoms (actually
  hydride) from the chemical bonds and gives them
  to FAD
• FAD becomes FADH2
• FADH2 transfers the electrons to the electron
  transport chain.
• Energy from excited electrons used to make ATP

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Contd

• Succ. Dehyd. Is an Integral (?) membrane protein
  in the inner mitochondrial membrane (hard to
  remove from membrane, hard to study)
• All other TCA cycle enzymes are soluble (located
  in the matrix)
• If we add a reducible dye, the dye not FAD will
  pick up the electrons
• OILRIG oxidation is loss of electrons, reduction
  is gain of electrons.
• So, dye (or FAD) is reduced, succinate is
  oxidized to fumarate

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Contd

• Succinate dehyd. has a size of 100,000 daltons.
  Average protein is 50,000 daltons how many
  amino acids in succ dehy? (/100)
• Also contains 8 iron atoms,
• Fe (iron) atoms help in the transfer of electrons
  from succinate to FAD.
• Has two subunits (so it has quarternary
  structure)
• Has higher activity than any other TCA cycle
  enzyme

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Succinate Dehydrogenase is turned on or off
through allosteric regulation (page 144-145).

• Allosteric regulation is how the body controls an
  enzyme (competitive inhibition is typically
  artificial or external to the body)
• ATP or reduced coenzyme Q are allosteric
  activators of Succ Dehyd
• Allosteric activators typically bind somewhere
  between the subunits of Succ Dehyd (not the
  active site) to stimulate the enzyme activity
• Allosteric inhibitors act similarly to inhibit

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Competitive Inhibition

• Inhibitor resembles Substrate
• This is not how the body/cell regulates enzymes
  (typically) - some medicines work this way
• So this method is artificial and used in test
  tubes to study an enzyme
• The inhibitor can bind to the active site
  (preventing the normal substrate from binding)
  but the inhibitor cannot form the product
• So, both the inhibitor and Substrate compete for
  the active site of the enzyme
• If the substrate is in excess, the inhibitor will
  not inhibit

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Competitive Inhibitors resemble the normal
substrate (but cannot be turned into productso
they tie up enzymes by binding to their active
site)
Malonate
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Competitive Inhibitors resemble the normal
substrate -but cannot be turned into productso
they tie up enzymes by binding to their active
site. Malonate is a molecule that looks like
succinate, but it cannot be made into fumaric
acid (product) so malonate is a competitive
inhibitor. Malonate is in a COMPETITION for the
active site of the enzyme with succinate-- which
ever is in higher concentration typically
wins!Some medicines are competitive inhibitors
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Other competitive inhibitors

• Other dibasic acids (means that they have two
  carboxylic acid functional groups C00-) can act
  as competitive inhibitors
• The other dibasic acids inhibit because the
  distance between the two C00- is about the same
  as the distance in succinate.
• The active site of succinate dehydrogenase must
  have two charges that are separated by the same
  distance

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Note that as long as the spacing between the two
ends is same as in Succinate, get competitive
inhibition. Even two negative charges of
pyrophosphate can act as a negative inhibitor
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SUCCINATE FITS INTO ACTIVE SITE (SOME OTHER
DIBASIC ACIDS HAVE SAME SPACING BETWEEN
NEGATIVE CHARGES)
0 C - C - C C 0
0-
0-


ACTIVE SITE- WHERE SUBSTRATE OR COMPETITIVE
INHIBITORS BIND. HERE, FIND AMINO ACIDS WHERE
THEIR R GROUP HAS CHARGE
SUCCINATE DEHYDROGENASE
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So, we will isolate mitochondria using
centrifugation, and study Succinate
DehydrogenaseTo Isolate Organelles, you
homogenize the cell and then use centrifugtation
to Isolate the organelle
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A rotor moves round and round, and heavy
particles move to the bottom of the test tube
fasterText p. 322-323, Sixth Ed
Differential Centrifugation
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So, we will 1. isolate mitochondria from Xenopus
liver and 2. follow Succinate Dehydrogenase
activity by adding Succinate 3. add a
competitive inhibitor called Malonate to reduce
Succ Dehyd activity
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Enzyme Kinetics

• If little substrate is around, there will be very
  little enzyme activity and the rate of the
  reaction will be slow
• If there is more substrate around, the enzyme
  will be more active and the reaction will be
  faster
• At a certain point, even if you raise the
  substrate concentration further, the rate of the
  reaction will not increase
• THIS IS SATURATION KINETICS
• (page 136 to 139 in text 6th ed)

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Rate of Reaction (slope of OD600 vs. time)
Saturation at higher substrate
concentrations because all enzyme working as
hard as they can- the enzymes Are saturated!
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Vm maximum velocity or rate of the reactionKm
a measure of enzyme-substrate affinity (low Km
means high affinity)

• Obtain Vm by going over from the Y axis (rate of
  the reaction when it first begins) to where the
  rectangular hyperbola levels off
• Obtain Km by going down the Y axis to one half
  Vm, then going over to the line in the graph,
  then going down to X axis.
• You can also obtain the values by using a Double
  Reciprocal Plot (Fig. 6-12 and 6-13).

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Succinate
Rate is initial slope for each concentration of
succinate (in OD600 Versus time)
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Double Reciprocal Plot