Download Mitochondrial Lab - University of Colorado Denver

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
Mitochondria and disease:
1. CU MED CENTER: MITOCHONDRIAL
MALFUNCTION LEADS TO PARKINSON’S
DISEASE (SHAKING AS YOU AGE)
2. 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.
3. Bad mitochondria may be related to Diabetes
4. Aging
(less ATP
made by
mitochondria)
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 MITOCHONDRIA
OXYGEN (02) ACCEPTS THE SPENT
(LOW ENERGY) ELECTRONS= AEROBIC
PARTS OF AEROBIC RESPIRATION:
1) 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)
2) TCA CYCLE (or Kreb’s cycle)- where what is left
of glucose is broken all the way down to C02 and
all the electrons are stripped off
3) Electrons are carried (by NADH or FADH2) to
the electron transport chain and ATP synthase
where ATP is made from electron energy (ch. 10)
PARTS OF AEROBIC
RESPIRATION
Part 1.
2
Part 3
CH. 9: GLYCOLYSIS
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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
ENERGETIC ELECTRONS
TAKEN FROM GLUCOSE
GIVEN TO NAD+
WHICH CARRIES ELECTRONS
INTO MITOCHONDRIA
ELECTRONS GIVEN TO NAD is
a REDUCTION
USE MNEMONIC: OILRIG
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)
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 TEXTBOOK…
D:\cell biol 3611\mito
respiration\respiration 1418m.mov
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
ANIMATIONS OF CHEMIOSMOSIS

D:\cell biol 3611\mito
respiration\chemiosmosis2.swf

D:\cell biol 3611\mito respiration\ATP SYNTHASE MBC
14_1.mov
D:\cell biol 3611\mito respiration\17 ELEC TRANS
CHAIN.MPG

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D:\cell biol 3611\mito
respiration\ATPGradientAdvanced.wmv
(NOTE THAT SOME ANIMATIONS
TALK ONLY OF THE H+
CONCENTRATION GRADIENT, IGNORING THE VERY
IMPORTANT ELECTRICAL GRADIENT FOR THE H+!!)
We will study one enzyme in the
TCA Cycle
(see Ch. 10; esp figures used here)
Succinate Dehydrogenase
this enzyme breaks two chemical
bonds and removes two H atoms
from what is left of glucose.
Succinate Dehydrog.
Is located here…
Succin Dehydrog
Actually binds
Membrane proteins
Of the Electron Transport
Chain
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…
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)
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)
Succinate Dehydrogenase is an
enzyme; substrate succinate binds in
the active site (similar to enzymes below)
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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Cont’d
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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
Cont’d
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
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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
Competitive Inhibitors resemble the
normal substrate (but cannot be turned
into product—so they tie up enzymes by
binding to their active site)
Malonate
Competitive Inhibitors resemble the normal
substrate -but cannot be turned into
product—so 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
Other competitive inhibitors…
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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
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:
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
So, we will isolate mitochondria
using centrifugation, and study
Succinate Dehydrogenase
To Isolate Organelles, you
homogenize the cell and then use
centrifugtation to Isolate the
organelle
A rotor moves
round and round,
and heavy
particles move to
the bottom of the
test tube faster
Text: p. 322-323,
Sixth Ed
Differential
Centrifugation
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
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)

Rate of
Reaction
(slope of
OD600
vs. time)
Saturation at higher
substrate concentration
because all enzyme
working as hard as
they can- the enzymes
Are saturated!
Vm = maximum velocity or rate of the
reaction
Km = a measure of enzyme-substrate
affinity (low Km means high affinity)
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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).
= [Succinate]
Rate is initial
slope for each
concentration
of succinate (in
OD600
Versus time)
Double
Reciprocal
Plot