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JOHN ESSIGMANN: Let's take a
look at storyboard number 10.

00:00:24.090 --> 00:00:26.040
Back in earlier
sessions, we talked

00:00:26.040 --> 00:00:28.650
about the detail of glycolysis.

00:00:28.650 --> 00:00:30.270
One of the points
that I emphasized

00:00:30.270 --> 00:00:32.520
is the fact that it's
necessary to maintain

00:00:32.520 --> 00:00:37.020
redox neutrality in the
cytoplasm of a mammalian cell.

00:00:37.020 --> 00:00:39.960
It's also necessary to
maintain redox neutrality

00:00:39.960 --> 00:00:44.070
and prokaryotic cells,
not just eukaryotic cells.

00:00:44.070 --> 00:00:47.400
At the glyceraldehyde
3-phosphate dehydrogenase step

00:00:47.400 --> 00:00:52.650
of glycolysis, NAD+ was
consumed and converted to NADH.

00:00:52.650 --> 00:00:57.480
That means we have to find a
way to convert NADH back to NAD+

00:00:57.480 --> 00:01:01.140
in order to make glycolysis
a continuous process.

00:01:01.140 --> 00:01:03.690
Back in lectures three and four,
I said that there were three

00:01:03.690 --> 00:01:08.610
ways to convert the NADH back
to NAD+ These were alcoholic

00:01:08.610 --> 00:01:11.460
fermentation in anaerobic cells.

00:01:11.460 --> 00:01:15.320
Homolactic fermentation, again,
in an anaerobic environment.

00:01:15.320 --> 00:01:17.100
And the third is
respiration, which occurs

00:01:17.100 --> 00:01:19.350
in the presence of oxygen.

00:01:19.350 --> 00:01:22.950
I'm going to loop back now
and revisit this topic.

00:01:22.950 --> 00:01:25.770
I want to spotlight
three general strategies

00:01:25.770 --> 00:01:29.820
that cells use to achieve redox
neutrality in the cytoplasm.

00:01:29.820 --> 00:01:32.250
The first is lactate
dehydrogenase.

00:01:32.250 --> 00:01:36.420
The second is called the
glycerol-3-phosphate shuttle.

00:01:36.420 --> 00:01:39.930
And the third is called the
malate-aspartate shuttle.

00:01:39.930 --> 00:01:44.130
Panel A of this figure shows
the cytoplasm working in concert

00:01:44.130 --> 00:01:45.540
with the mitochondrion.

00:01:45.540 --> 00:01:47.640
You can see depicted
on the left,

00:01:47.640 --> 00:01:49.620
the pathway of glycolysis.

00:01:49.620 --> 00:01:52.590
In the middle,
pyruvate dehydrogenase.

00:01:52.590 --> 00:01:55.630
And to the right, the citric
acid cycle or TCA cycle,

00:01:55.630 --> 00:01:57.870
which we just covered.

00:01:57.870 --> 00:02:00.540
There are two important boundary
conditions for the discussion

00:02:00.540 --> 00:02:01.870
we're about to have.

00:02:01.870 --> 00:02:04.140
The first is that
oxaloacetate is,

00:02:04.140 --> 00:02:07.260
as I mentioned earlier,
present only in very small

00:02:07.260 --> 00:02:09.930
concentrations within
the cell and especially

00:02:09.930 --> 00:02:11.850
in the mitochondrion.

00:02:11.850 --> 00:02:13.990
As a consequence,
the mitochondrion

00:02:13.990 --> 00:02:17.490
does not have a transporter
to allow it to escape.

00:02:17.490 --> 00:02:19.380
In other words,
its concentration

00:02:19.380 --> 00:02:21.870
is preserved at
about one micromolar

00:02:21.870 --> 00:02:24.420
inside the mitochondrion.

00:02:24.420 --> 00:02:27.840
The second boundary condition
concerns the fact that NAD+

00:02:27.840 --> 00:02:34.320
and NADH, as well as NADP+ and
NADPH cannot go directly across

00:02:34.320 --> 00:02:37.120
the mitochondrial membrane.

00:02:37.120 --> 00:02:39.360
So in other words, there
are two separate pools

00:02:39.360 --> 00:02:41.070
of this nucleotide co-factor.

00:02:41.070 --> 00:02:42.480
One in the cytoplasm.

00:02:42.480 --> 00:02:44.310
One in the mitochondrion.

00:02:44.310 --> 00:02:46.350
I'll come back to the
importance of the two

00:02:46.350 --> 00:02:48.870
pools in just a few minutes.

00:02:48.870 --> 00:02:50.910
Let's look first,
here in panel A,

00:02:50.910 --> 00:02:54.270
at the mechanism by which
lactate dehydrogenase achieves

00:02:54.270 --> 00:02:57.120
redox neutrality
in the cytoplasm.

00:02:57.120 --> 00:03:00.720
We've already covered this,
so this is a bit of a review.

00:03:00.720 --> 00:03:03.810
Note that you see NAD+
getting converted to NADH

00:03:03.810 --> 00:03:05.040
in the cytoplasm.

00:03:05.040 --> 00:03:07.470
That's at that GAPDH
or glyceraldehyde

00:03:07.470 --> 00:03:09.920
3-phosphate dehydrogenase step.

00:03:09.920 --> 00:03:11.820
The hatched lines
that you see represent

00:03:11.820 --> 00:03:13.890
the flow of electrons.

00:03:13.890 --> 00:03:16.290
In other words, electrons
flow from glucose

00:03:16.290 --> 00:03:18.900
and they end up in NADH.

00:03:18.900 --> 00:03:21.940
Then the lactate
dehydrogenase enzyme

00:03:21.940 --> 00:03:26.580
transfers the electrons
from NADH into lactate.

00:03:26.580 --> 00:03:29.040
So these electrons
from glucose are

00:03:29.040 --> 00:03:31.890
involved in the reduction
of the ketone functionality

00:03:31.890 --> 00:03:36.210
of pyruvate into the alcohol
functionality of lactate.

00:03:36.210 --> 00:03:39.090
And that's where
the electrons stay.

00:03:39.090 --> 00:03:41.310
The other product of this
reaction, as you'll see,

00:03:41.310 --> 00:03:45.240
NAD+ which is now available
to enable the oxidation

00:03:45.240 --> 00:03:47.610
of the next molecule of glucose.

00:03:47.610 --> 00:03:50.640
What happens to the
lactate that's produced?

00:03:50.640 --> 00:03:53.700
In a working muscle
cell, that lactate

00:03:53.700 --> 00:03:56.190
will escape from the
cell, go into the blood,

00:03:56.190 --> 00:03:58.860
and then go to the liver
or another organ that's

00:03:58.860 --> 00:04:02.550
capable of doing the
pathway of gluconeogenesis.

00:04:02.550 --> 00:04:04.830
As I've mentioned in the
past, gluconeogenesis

00:04:04.830 --> 00:04:07.080
is a pathway by which
non carbohydrate

00:04:07.080 --> 00:04:11.820
precursors, such as lactate,
are built back up into glucose.

00:04:11.820 --> 00:04:14.230
Keep that working
muscle scenario in mind,

00:04:14.230 --> 00:04:16.110
because I'm going to
come back to it later

00:04:16.110 --> 00:04:18.630
when I talk about
physiological responses

00:04:18.630 --> 00:04:21.720
to stress, such as what I'll
call the fight and flight

00:04:21.720 --> 00:04:23.550
scenario.

00:04:23.550 --> 00:04:26.490
That's all I'm going to say
for now about the LDH shuttle.

00:04:26.490 --> 00:04:28.350
That is, lactate
dehydrogenase shuttle

00:04:28.350 --> 00:04:31.410
in panel A, which is the
first of the three pathways

00:04:31.410 --> 00:04:35.340
by which redox neutrality is
maintained in the cytoplasm.

00:04:35.340 --> 00:04:38.040
The second pathway
to retain redox

00:04:38.040 --> 00:04:41.040
neutrality is the
glycerol-3-phosphate shuttle.

00:04:41.040 --> 00:04:43.590
This pathway is particularly
active in the brain

00:04:43.590 --> 00:04:45.570
and in skeletal muscle.

00:04:45.570 --> 00:04:47.790
Once again, follow
the hatched lines

00:04:47.790 --> 00:04:51.199
to follow the path of electrons
as they go from glucose.

00:04:51.199 --> 00:04:52.740
And ultimately, in
this case, they're

00:04:52.740 --> 00:04:57.000
going to end up being deposited
into oxygen to form water.

00:04:57.000 --> 00:05:01.520
Starting at the top, you see
in NAD+ being reduced to NADH

00:05:01.520 --> 00:05:04.920
at the glyceraldehyde
3-phosphate dehydrogenase step,

00:05:04.920 --> 00:05:06.410
GAPDH.

00:05:06.410 --> 00:05:08.870
Next, we're going to
temporarily borrow

00:05:08.870 --> 00:05:13.130
a molecule of dihydroxyacetone
phosphate, DHAP.

00:05:13.130 --> 00:05:15.140
DHAP is a ketone.

00:05:15.140 --> 00:05:19.430
And what we're going to do is
deposit the electrons from NADH

00:05:19.430 --> 00:05:22.610
into the ketone functionality
to make the alcohol,

00:05:22.610 --> 00:05:24.710
glycerol-3-phosphate.

00:05:24.710 --> 00:05:27.350
The source of the
electrons was NADH.

00:05:27.350 --> 00:05:29.730
And now you've accomplished
your chemical goal,

00:05:29.730 --> 00:05:33.290
which was to restore the
NAD+ pool -- the cytoplasm,

00:05:33.290 --> 00:05:37.880
but we borrowed a molecule of
dihydroxyacetone phosphate.

00:05:37.880 --> 00:05:40.640
And we've somehow
got to get that back.

00:05:40.640 --> 00:05:45.020
Let me point out, at this point,
that the reduction of DHAP

00:05:45.020 --> 00:05:47.180
to glycerol-3-phosphate
was accomplished

00:05:47.180 --> 00:05:51.410
by the cytoplasmic form of the
enzyme glycerol-3-phosphate

00:05:51.410 --> 00:05:56.120
dehydrogenase, which catalyzed
step 2 on the storyboarded.

00:05:56.120 --> 00:05:59.400
We're going to deal more with
coenzyme q in the next lecture.

00:05:59.400 --> 00:06:01.505
But for now, it's a
molecule, specifically

00:06:01.505 --> 00:06:05.270
a quinone, that's easily reduced
to its hydroquinone form,

00:06:05.270 --> 00:06:07.130
called QH2.

00:06:07.130 --> 00:06:10.850
The structures of q, in
QH2, are shown in the box.

00:06:10.850 --> 00:06:13.790
QH2 is in the
mitochondrial membrane.

00:06:13.790 --> 00:06:16.130
In glycerol-3-phosphate
dehydrogenase

00:06:16.130 --> 00:06:18.200
the mitochondrial
version of it is

00:06:18.200 --> 00:06:22.640
present in the outer part of the
mitochondrial inner membrane.

00:06:22.640 --> 00:06:27.230
Like NADH and FADH2,
QH2, the hydroquinone,

00:06:27.230 --> 00:06:30.670
is what I've called a
mobile electron carrier.

00:06:30.670 --> 00:06:33.750
QH2 is going to allow the
electrons that started out

00:06:33.750 --> 00:06:37.280
in glucose or in any
LDH of the gap DH step,

00:06:37.280 --> 00:06:40.070
to flow through the electron
transport chain, which we'll

00:06:40.070 --> 00:06:41.570
come to in the next lecture.

00:06:41.570 --> 00:06:45.470
And flow into oxygen, which
is reduced to form water.

00:06:45.470 --> 00:06:49.040
This terminal reduction
is shown in step five.

00:06:49.040 --> 00:06:52.010
Effectively, in the
glycerol-3-phosphate shuttle

00:06:52.010 --> 00:06:57.890
we're using oxygen in order to
oxidize NADH back to NAD+ And

00:06:57.890 --> 00:07:01.130
once again, maintaining a
constant supply of NAD+ is

00:07:01.130 --> 00:07:04.220
necessary in order to make
glycolysis a continuous

00:07:04.220 --> 00:07:05.580
process.

00:07:05.580 --> 00:07:09.890
Panel C shows a third
strategy for maintaining redox

00:07:09.890 --> 00:07:12.060
neutrality in the cytoplasm.

00:07:12.060 --> 00:07:14.990
This is called the
malate-aspartate shuttle.

00:07:14.990 --> 00:07:19.310
and this pathway is operative
in heart, liver, and kidney.

00:07:19.310 --> 00:07:21.950
To the left we see the
production of NADH,

00:07:21.950 --> 00:07:25.730
just as we did in the
previous two small pathways.

00:07:25.730 --> 00:07:27.740
At step one, let's
assume that there's

00:07:27.740 --> 00:07:31.100
a molecule of oxaloacetate
present as part

00:07:31.100 --> 00:07:35.120
of the cytoplasmic
pool of organic acids.

00:07:35.120 --> 00:07:38.180
Oxaloacetate or OA is ketone.

00:07:38.180 --> 00:07:41.630
And the cytoplasmic form of the
enzyme malate dehydrogenase,

00:07:41.630 --> 00:07:44.090
working in the reverse
direction from the one

00:07:44.090 --> 00:07:46.550
that we see operative
in the TCA cycle

00:07:46.550 --> 00:07:49.910
is able to reduce the
oxaloacetate to malate.

00:07:49.910 --> 00:07:54.500
We just reduce to ketone
OA to an alcohol malate.

00:07:54.500 --> 00:07:57.020
In step three, the
accumulating malate

00:07:57.020 --> 00:07:59.120
is transported by a
malate transporter

00:07:59.120 --> 00:08:02.130
into the mitochondrial
matrix, which is, of course,

00:08:02.130 --> 00:08:04.700
the location of the TCA cycle.

00:08:04.700 --> 00:08:06.530
At this point, we're
going to be using one

00:08:06.530 --> 00:08:09.320
of the steps of the TCA cycle.

00:08:09.320 --> 00:08:12.560
Specifically, we're going
to use malate dehydrogenase,

00:08:12.560 --> 00:08:14.600
the mitochondrial
version of the enzyme

00:08:14.600 --> 00:08:17.930
this time, to convert
malate to oxaloacetate.

00:08:17.930 --> 00:08:20.120
That reaction is an oxidation.

00:08:20.120 --> 00:08:23.540
We use the mitochondrial pool
of NAD+ to carry out that

00:08:23.540 --> 00:08:24.740
oxidation.

00:08:24.740 --> 00:08:27.440
In effect, we're using the
electrons that came in from

00:08:27.440 --> 00:08:32.309
malate to reduce NAD+ to end
NADH in the mitochondrion.

00:08:32.309 --> 00:08:35.240
Now, take a careful
look at step four.

00:08:35.240 --> 00:08:38.390
Looking to the left you see the
hatched lines go all the way

00:08:38.390 --> 00:08:42.140
back to glucose, which was
the source of the electrons.

00:08:42.140 --> 00:08:46.280
To the right, the hatched
lines by step five,

00:08:46.280 --> 00:08:50.060
go to the electron transport
chain all the way to oxygen.

00:08:50.060 --> 00:08:53.390
We haven't done the electron
transport chain as yet.

00:08:53.390 --> 00:08:56.660
So you're just going to have
to trust me for a little while.

00:08:56.660 --> 00:09:01.160
There's an enzyme,
NADH dehydrogenase,

00:09:01.160 --> 00:09:03.620
in the mitochondrial inner
membrane that will take

00:09:03.620 --> 00:09:08.120
the electrons from NADH and
eventually regenerate the NAD+

00:09:08.120 --> 00:09:10.840
in the mitochondrial matrix.

00:09:10.840 --> 00:09:13.110
In step five, we're
taking the electrons

00:09:13.110 --> 00:09:16.690
from the NADH produced
by malate dehydrogenase

00:09:16.690 --> 00:09:19.770
and entering those electrons
into the electron transport

00:09:19.770 --> 00:09:20.670
chain.

00:09:20.670 --> 00:09:22.800
Then, in a manner that's
quite similar to what

00:09:22.800 --> 00:09:25.650
we did in the previous shuttle,
the glycerol-3-phosphate

00:09:25.650 --> 00:09:27.690
shuttle, those
electrons are going

00:09:27.690 --> 00:09:30.870
to be transferred to
oxygen to make water.

00:09:30.870 --> 00:09:33.450
Before I go on, let's
review a little bit.

00:09:33.450 --> 00:09:36.580
Between step one and step
two in the cytoplasm,

00:09:36.580 --> 00:09:40.870
we deposited electrons into
oxaloacetate to make malate.

00:09:40.870 --> 00:09:44.830
That step restored in NAD+
levels in the cytoplasm,

00:09:44.830 --> 00:09:46.830
which is what we
wanted to accomplish.

00:09:46.830 --> 00:09:50.670
However, we've consumed a
molecule of oxaloacetate.

00:09:50.670 --> 00:09:52.650
And as I've mentioned
before, the cell

00:09:52.650 --> 00:09:55.470
has to try to preserve the
concentration of this very

00:09:55.470 --> 00:09:57.010
precious molecule.

00:09:57.010 --> 00:10:00.630
We now have to find a way
to restore oxaloacetate

00:10:00.630 --> 00:10:03.550
that we borrowed in step one.

00:10:03.550 --> 00:10:06.300
Now let's look
back at step four,

00:10:06.300 --> 00:10:08.770
where malate was
converted to oxaloacetate

00:10:08.770 --> 00:10:11.080
in the mitochondrial matrix.

00:10:11.080 --> 00:10:14.410
Because the molecule of malate
came from the cytoplasm,

00:10:14.410 --> 00:10:17.140
this is a net increase in
the mitochondrial matrix

00:10:17.140 --> 00:10:19.590
of one unit of malate
and, ultimately,

00:10:19.590 --> 00:10:22.060
one unit of oxaloacetate.

00:10:22.060 --> 00:10:26.050
We need to find a way to get
that molecule of oxaloacetate

00:10:26.050 --> 00:10:28.660
back out into the
cytoplasm, in order to make

00:10:28.660 --> 00:10:31.290
the shuttle a continuous one.

00:10:31.290 --> 00:10:33.960
In the co-factor
section of 5.07,

00:10:33.960 --> 00:10:35.760
JoAnne taught us
about the ways that

00:10:35.760 --> 00:10:39.450
pyridoxal phosphate and
pyridoxamine work, in order

00:10:39.450 --> 00:10:43.920
to put amino groups into organic
acids, such as oxaloacetate.

00:10:43.920 --> 00:10:47.170
And that's what's going
to happen in this case.

00:10:47.170 --> 00:10:51.230
Oxaloacetate is converted
into its amino acid homolog,

00:10:51.230 --> 00:10:52.970
aspartic acid.

00:10:52.970 --> 00:10:56.480
Why did we do this
emanation reaction?

00:10:56.480 --> 00:10:59.960
Well, there's no way to get
oxaloacetate directly out

00:10:59.960 --> 00:11:03.540
of the mitochondria because
there's no transporter for it.

00:11:03.540 --> 00:11:06.710
But there is a good transporter,
the apartheid transporter,

00:11:06.710 --> 00:11:10.460
that will take aspartic
acid out into the cytoplasm.

00:11:10.460 --> 00:11:13.880
So the oxaloacetate is
converted, temporarily,

00:11:13.880 --> 00:11:16.850
into aspartic acid
in the mitochondrion.

00:11:16.850 --> 00:11:19.310
And that aspartic
acid then slips out

00:11:19.310 --> 00:11:23.500
through its transporter
to the cytoplasm.

00:11:23.500 --> 00:11:26.830
Once in the cytoplasm, there's
a similar pyridoxal mediated

00:11:26.830 --> 00:11:29.860
mechanism to deaminate
the aspartate

00:11:29.860 --> 00:11:33.430
to regenerate the cytoplasmic
molecule of acetate

00:11:33.430 --> 00:11:36.660
that we borrowed at step
one a few minutes ago.

00:11:36.660 --> 00:11:39.070
In panel D I summarize.

00:11:39.070 --> 00:11:42.580
That we've looked at three
different small pathways that

00:11:42.580 --> 00:11:46.180
enable the cytoplasm of the
cell to always have enough NAD+

00:11:46.180 --> 00:11:49.320
to oxidize glucose to pyruvate.

00:11:49.320 --> 00:11:52.720
These pathways are first, the
lactate dehydrogenase system.

00:11:52.720 --> 00:11:55.260
Second, the
glycerol-3-phosphate shuttle.

00:11:55.260 --> 00:11:57.970
And third, the malate
departed shuttle.