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

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00:00:24,090 --> 00:00:26,040
Back in earlier
sessions, we talked

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00:00:26,040 --> 00:00:28,650
about the detail of glycolysis.

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00:00:28,650 --> 00:00:30,270
One of the points
that I emphasized

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00:00:30,270 --> 00:00:32,520
is the fact that it's
necessary to maintain

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00:00:32,520 --> 00:00:37,020
redox neutrality in the
cytoplasm of a mammalian cell.

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00:00:37,020 --> 00:00:39,960
It's also necessary to
maintain redox neutrality

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00:00:39,960 --> 00:00:44,070
and prokaryotic cells,
not just eukaryotic cells.

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00:00:44,070 --> 00:00:47,400
At the glyceraldehyde
3-phosphate dehydrogenase step

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00:00:47,400 --> 00:00:52,650
of glycolysis, NAD+ was
consumed and converted to NADH.

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00:00:52,650 --> 00:00:57,480
That means we have to find a
way to convert NADH back to NAD+

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00:00:57,480 --> 00:01:01,140
in order to make glycolysis
a continuous process.

20
00:01:01,140 --> 00:01:03,690
Back in lectures three and four,
I said that there were three

21
00:01:03,690 --> 00:01:08,610
ways to convert the NADH back
to NAD+ These were alcoholic

22
00:01:08,610 --> 00:01:11,460
fermentation in anaerobic cells.

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00:01:11,460 --> 00:01:15,320
Homolactic fermentation, again,
in an anaerobic environment.

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00:01:15,320 --> 00:01:17,100
And the third is
respiration, which occurs

25
00:01:17,100 --> 00:01:19,350
in the presence of oxygen.

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00:01:19,350 --> 00:01:22,950
I'm going to loop back now
and revisit this topic.

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00:01:22,950 --> 00:01:25,770
I want to spotlight
three general strategies

28
00:01:25,770 --> 00:01:29,820
that cells use to achieve redox
neutrality in the cytoplasm.

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00:01:29,820 --> 00:01:32,250
The first is lactate
dehydrogenase.

30
00:01:32,250 --> 00:01:36,420
The second is called the
glycerol-3-phosphate shuttle.

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00:01:36,420 --> 00:01:39,930
And the third is called the
malate-aspartate shuttle.

32
00:01:39,930 --> 00:01:44,130
Panel A of this figure shows
the cytoplasm working in concert

33
00:01:44,130 --> 00:01:45,540
with the mitochondrion.

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00:01:45,540 --> 00:01:47,640
You can see depicted
on the left,

35
00:01:47,640 --> 00:01:49,620
the pathway of glycolysis.

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00:01:49,620 --> 00:01:52,590
In the middle,
pyruvate dehydrogenase.

37
00:01:52,590 --> 00:01:55,630
And to the right, the citric
acid cycle or TCA cycle,

38
00:01:55,630 --> 00:01:57,870
which we just covered.

39
00:01:57,870 --> 00:02:00,540
There are two important boundary
conditions for the discussion

40
00:02:00,540 --> 00:02:01,870
we're about to have.

41
00:02:01,870 --> 00:02:04,140
The first is that
oxaloacetate is,

42
00:02:04,140 --> 00:02:07,260
as I mentioned earlier,
present only in very small

43
00:02:07,260 --> 00:02:09,930
concentrations within
the cell and especially

44
00:02:09,930 --> 00:02:11,850
in the mitochondrion.

45
00:02:11,850 --> 00:02:13,990
As a consequence,
the mitochondrion

46
00:02:13,990 --> 00:02:17,490
does not have a transporter
to allow it to escape.

47
00:02:17,490 --> 00:02:19,380
In other words,
its concentration

48
00:02:19,380 --> 00:02:21,870
is preserved at
about one micromolar

49
00:02:21,870 --> 00:02:24,420
inside the mitochondrion.

50
00:02:24,420 --> 00:02:27,840
The second boundary condition
concerns the fact that NAD+

51
00:02:27,840 --> 00:02:34,320
and NADH, as well as NADP+ and
NADPH cannot go directly across

52
00:02:34,320 --> 00:02:37,120
the mitochondrial membrane.

53
00:02:37,120 --> 00:02:39,360
So in other words, there
are two separate pools

54
00:02:39,360 --> 00:02:41,070
of this nucleotide co-factor.

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00:02:41,070 --> 00:02:42,480
One in the cytoplasm.

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00:02:42,480 --> 00:02:44,310
One in the mitochondrion.

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00:02:44,310 --> 00:02:46,350
I'll come back to the
importance of the two

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00:02:46,350 --> 00:02:48,870
pools in just a few minutes.

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00:02:48,870 --> 00:02:50,910
Let's look first,
here in panel A,

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00:02:50,910 --> 00:02:54,270
at the mechanism by which
lactate dehydrogenase achieves

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00:02:54,270 --> 00:02:57,120
redox neutrality
in the cytoplasm.

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00:02:57,120 --> 00:03:00,720
We've already covered this,
so this is a bit of a review.

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00:03:00,720 --> 00:03:03,810
Note that you see NAD+
getting converted to NADH

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00:03:03,810 --> 00:03:05,040
in the cytoplasm.

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00:03:05,040 --> 00:03:07,470
That's at that GAPDH
or glyceraldehyde

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00:03:07,470 --> 00:03:09,920
3-phosphate dehydrogenase step.

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00:03:09,920 --> 00:03:11,820
The hatched lines
that you see represent

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00:03:11,820 --> 00:03:13,890
the flow of electrons.

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00:03:13,890 --> 00:03:16,290
In other words, electrons
flow from glucose

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00:03:16,290 --> 00:03:18,900
and they end up in NADH.

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00:03:18,900 --> 00:03:21,940
Then the lactate
dehydrogenase enzyme

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00:03:21,940 --> 00:03:26,580
transfers the electrons
from NADH into lactate.

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00:03:26,580 --> 00:03:29,040
So these electrons
from glucose are

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00:03:29,040 --> 00:03:31,890
involved in the reduction
of the ketone functionality

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00:03:31,890 --> 00:03:36,210
of pyruvate into the alcohol
functionality of lactate.

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00:03:36,210 --> 00:03:39,090
And that's where
the electrons stay.

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00:03:39,090 --> 00:03:41,310
The other product of this
reaction, as you'll see,

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00:03:41,310 --> 00:03:45,240
NAD+ which is now available
to enable the oxidation

79
00:03:45,240 --> 00:03:47,610
of the next molecule of glucose.

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00:03:47,610 --> 00:03:50,640
What happens to the
lactate that's produced?

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00:03:50,640 --> 00:03:53,700
In a working muscle
cell, that lactate

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00:03:53,700 --> 00:03:56,190
will escape from the
cell, go into the blood,

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00:03:56,190 --> 00:03:58,860
and then go to the liver
or another organ that's

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00:03:58,860 --> 00:04:02,550
capable of doing the
pathway of gluconeogenesis.

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00:04:02,550 --> 00:04:04,830
As I've mentioned in the
past, gluconeogenesis

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00:04:04,830 --> 00:04:07,080
is a pathway by which
non carbohydrate

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00:04:07,080 --> 00:04:11,820
precursors, such as lactate,
are built back up into glucose.

88
00:04:11,820 --> 00:04:14,230
Keep that working
muscle scenario in mind,

89
00:04:14,230 --> 00:04:16,110
because I'm going to
come back to it later

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00:04:16,110 --> 00:04:18,630
when I talk about
physiological responses

91
00:04:18,630 --> 00:04:21,720
to stress, such as what I'll
call the fight and flight

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00:04:21,720 --> 00:04:23,550
scenario.

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00:04:23,550 --> 00:04:26,490
That's all I'm going to say
for now about the LDH shuttle.

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00:04:26,490 --> 00:04:28,350
That is, lactate
dehydrogenase shuttle

95
00:04:28,350 --> 00:04:31,410
in panel A, which is the
first of the three pathways

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00:04:31,410 --> 00:04:35,340
by which redox neutrality is
maintained in the cytoplasm.

97
00:04:35,340 --> 00:04:38,040
The second pathway
to retain redox

98
00:04:38,040 --> 00:04:41,040
neutrality is the
glycerol-3-phosphate shuttle.

99
00:04:41,040 --> 00:04:43,590
This pathway is particularly
active in the brain

100
00:04:43,590 --> 00:04:45,570
and in skeletal muscle.

101
00:04:45,570 --> 00:04:47,790
Once again, follow
the hatched lines

102
00:04:47,790 --> 00:04:51,199
to follow the path of electrons
as they go from glucose.

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00:04:51,199 --> 00:04:52,740
And ultimately, in
this case, they're

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00:04:52,740 --> 00:04:57,000
going to end up being deposited
into oxygen to form water.

105
00:04:57,000 --> 00:05:01,520
Starting at the top, you see
in NAD+ being reduced to NADH

106
00:05:01,520 --> 00:05:04,920
at the glyceraldehyde
3-phosphate dehydrogenase step,

107
00:05:04,920 --> 00:05:06,410
GAPDH.

108
00:05:06,410 --> 00:05:08,870
Next, we're going to
temporarily borrow

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00:05:08,870 --> 00:05:13,130
a molecule of dihydroxyacetone
phosphate, DHAP.

110
00:05:13,130 --> 00:05:15,140
DHAP is a ketone.

111
00:05:15,140 --> 00:05:19,430
And what we're going to do is
deposit the electrons from NADH

112
00:05:19,430 --> 00:05:22,610
into the ketone functionality
to make the alcohol,

113
00:05:22,610 --> 00:05:24,710
glycerol-3-phosphate.

114
00:05:24,710 --> 00:05:27,350
The source of the
electrons was NADH.

115
00:05:27,350 --> 00:05:29,730
And now you've accomplished
your chemical goal,

116
00:05:29,730 --> 00:05:33,290
which was to restore the
NAD+ pool -- the cytoplasm,

117
00:05:33,290 --> 00:05:37,880
but we borrowed a molecule of
dihydroxyacetone phosphate.

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00:05:37,880 --> 00:05:40,640
And we've somehow
got to get that back.

119
00:05:40,640 --> 00:05:45,020
Let me point out, at this point,
that the reduction of DHAP

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00:05:45,020 --> 00:05:47,180
to glycerol-3-phosphate
was accomplished

121
00:05:47,180 --> 00:05:51,410
by the cytoplasmic form of the
enzyme glycerol-3-phosphate

122
00:05:51,410 --> 00:05:56,120
dehydrogenase, which catalyzed
step 2 on the storyboarded.

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00:05:56,120 --> 00:05:59,400
We're going to deal more with
coenzyme q in the next lecture.

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00:05:59,400 --> 00:06:01,505
But for now, it's a
molecule, specifically

125
00:06:01,505 --> 00:06:05,270
a quinone, that's easily reduced
to its hydroquinone form,

126
00:06:05,270 --> 00:06:07,130
called QH2.

127
00:06:07,130 --> 00:06:10,850
The structures of q, in
QH2, are shown in the box.

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00:06:10,850 --> 00:06:13,790
QH2 is in the
mitochondrial membrane.

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00:06:13,790 --> 00:06:16,130
In glycerol-3-phosphate
dehydrogenase

130
00:06:16,130 --> 00:06:18,200
the mitochondrial
version of it is

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00:06:18,200 --> 00:06:22,640
present in the outer part of the
mitochondrial inner membrane.

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00:06:22,640 --> 00:06:27,230
Like NADH and FADH2,
QH2, the hydroquinone,

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00:06:27,230 --> 00:06:30,670
is what I've called a
mobile electron carrier.

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00:06:30,670 --> 00:06:33,750
QH2 is going to allow the
electrons that started out

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00:06:33,750 --> 00:06:37,280
in glucose or in any
LDH of the gap DH step,

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00:06:37,280 --> 00:06:40,070
to flow through the electron
transport chain, which we'll

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00:06:40,070 --> 00:06:41,570
come to in the next lecture.

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00:06:41,570 --> 00:06:45,470
And flow into oxygen, which
is reduced to form water.

139
00:06:45,470 --> 00:06:49,040
This terminal reduction
is shown in step five.

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00:06:49,040 --> 00:06:52,010
Effectively, in the
glycerol-3-phosphate shuttle

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00:06:52,010 --> 00:06:57,890
we're using oxygen in order to
oxidize NADH back to NAD+ And

142
00:06:57,890 --> 00:07:01,130
once again, maintaining a
constant supply of NAD+ is

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00:07:01,130 --> 00:07:04,220
necessary in order to make
glycolysis a continuous

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00:07:04,220 --> 00:07:05,580
process.

145
00:07:05,580 --> 00:07:09,890
Panel C shows a third
strategy for maintaining redox

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00:07:09,890 --> 00:07:12,060
neutrality in the cytoplasm.

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00:07:12,060 --> 00:07:14,990
This is called the
malate-aspartate shuttle.

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00:07:14,990 --> 00:07:19,310
and this pathway is operative
in heart, liver, and kidney.

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00:07:19,310 --> 00:07:21,950
To the left we see the
production of NADH,

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00:07:21,950 --> 00:07:25,730
just as we did in the
previous two small pathways.

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00:07:25,730 --> 00:07:27,740
At step one, let's
assume that there's

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00:07:27,740 --> 00:07:31,100
a molecule of oxaloacetate
present as part

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00:07:31,100 --> 00:07:35,120
of the cytoplasmic
pool of organic acids.

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00:07:35,120 --> 00:07:38,180
Oxaloacetate or OA is ketone.

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00:07:38,180 --> 00:07:41,630
And the cytoplasmic form of the
enzyme malate dehydrogenase,

156
00:07:41,630 --> 00:07:44,090
working in the reverse
direction from the one

157
00:07:44,090 --> 00:07:46,550
that we see operative
in the TCA cycle

158
00:07:46,550 --> 00:07:49,910
is able to reduce the
oxaloacetate to malate.

159
00:07:49,910 --> 00:07:54,500
We just reduce to ketone
OA to an alcohol malate.

160
00:07:54,500 --> 00:07:57,020
In step three, the
accumulating malate

161
00:07:57,020 --> 00:07:59,120
is transported by a
malate transporter

162
00:07:59,120 --> 00:08:02,130
into the mitochondrial
matrix, which is, of course,

163
00:08:02,130 --> 00:08:04,700
the location of the TCA cycle.

164
00:08:04,700 --> 00:08:06,530
At this point, we're
going to be using one

165
00:08:06,530 --> 00:08:09,320
of the steps of the TCA cycle.

166
00:08:09,320 --> 00:08:12,560
Specifically, we're going
to use malate dehydrogenase,

167
00:08:12,560 --> 00:08:14,600
the mitochondrial
version of the enzyme

168
00:08:14,600 --> 00:08:17,930
this time, to convert
malate to oxaloacetate.

169
00:08:17,930 --> 00:08:20,120
That reaction is an oxidation.

170
00:08:20,120 --> 00:08:23,540
We use the mitochondrial pool
of NAD+ to carry out that

171
00:08:23,540 --> 00:08:24,740
oxidation.

172
00:08:24,740 --> 00:08:27,440
In effect, we're using the
electrons that came in from

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00:08:27,440 --> 00:08:32,309
malate to reduce NAD+ to end
NADH in the mitochondrion.

174
00:08:32,309 --> 00:08:35,240
Now, take a careful
look at step four.

175
00:08:35,240 --> 00:08:38,390
Looking to the left you see the
hatched lines go all the way

176
00:08:38,390 --> 00:08:42,140
back to glucose, which was
the source of the electrons.

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00:08:42,140 --> 00:08:46,280
To the right, the hatched
lines by step five,

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00:08:46,280 --> 00:08:50,060
go to the electron transport
chain all the way to oxygen.

179
00:08:50,060 --> 00:08:53,390
We haven't done the electron
transport chain as yet.

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00:08:53,390 --> 00:08:56,660
So you're just going to have
to trust me for a little while.

181
00:08:56,660 --> 00:09:01,160
There's an enzyme,
NADH dehydrogenase,

182
00:09:01,160 --> 00:09:03,620
in the mitochondrial inner
membrane that will take

183
00:09:03,620 --> 00:09:08,120
the electrons from NADH and
eventually regenerate the NAD+

184
00:09:08,120 --> 00:09:10,840
in the mitochondrial matrix.

185
00:09:10,840 --> 00:09:13,110
In step five, we're
taking the electrons

186
00:09:13,110 --> 00:09:16,690
from the NADH produced
by malate dehydrogenase

187
00:09:16,690 --> 00:09:19,770
and entering those electrons
into the electron transport

188
00:09:19,770 --> 00:09:20,670
chain.

189
00:09:20,670 --> 00:09:22,800
Then, in a manner that's
quite similar to what

190
00:09:22,800 --> 00:09:25,650
we did in the previous shuttle,
the glycerol-3-phosphate

191
00:09:25,650 --> 00:09:27,690
shuttle, those
electrons are going

192
00:09:27,690 --> 00:09:30,870
to be transferred to
oxygen to make water.

193
00:09:30,870 --> 00:09:33,450
Before I go on, let's
review a little bit.

194
00:09:33,450 --> 00:09:36,580
Between step one and step
two in the cytoplasm,

195
00:09:36,580 --> 00:09:40,870
we deposited electrons into
oxaloacetate to make malate.

196
00:09:40,870 --> 00:09:44,830
That step restored in NAD+
levels in the cytoplasm,

197
00:09:44,830 --> 00:09:46,830
which is what we
wanted to accomplish.

198
00:09:46,830 --> 00:09:50,670
However, we've consumed a
molecule of oxaloacetate.

199
00:09:50,670 --> 00:09:52,650
And as I've mentioned
before, the cell

200
00:09:52,650 --> 00:09:55,470
has to try to preserve the
concentration of this very

201
00:09:55,470 --> 00:09:57,010
precious molecule.

202
00:09:57,010 --> 00:10:00,630
We now have to find a way
to restore oxaloacetate

203
00:10:00,630 --> 00:10:03,550
that we borrowed in step one.

204
00:10:03,550 --> 00:10:06,300
Now let's look
back at step four,

205
00:10:06,300 --> 00:10:08,770
where malate was
converted to oxaloacetate

206
00:10:08,770 --> 00:10:11,080
in the mitochondrial matrix.

207
00:10:11,080 --> 00:10:14,410
Because the molecule of malate
came from the cytoplasm,

208
00:10:14,410 --> 00:10:17,140
this is a net increase in
the mitochondrial matrix

209
00:10:17,140 --> 00:10:19,590
of one unit of malate
and, ultimately,

210
00:10:19,590 --> 00:10:22,060
one unit of oxaloacetate.

211
00:10:22,060 --> 00:10:26,050
We need to find a way to get
that molecule of oxaloacetate

212
00:10:26,050 --> 00:10:28,660
back out into the
cytoplasm, in order to make

213
00:10:28,660 --> 00:10:31,290
the shuttle a continuous one.

214
00:10:31,290 --> 00:10:33,960
In the co-factor
section of 5.07,

215
00:10:33,960 --> 00:10:35,760
JoAnne taught us
about the ways that

216
00:10:35,760 --> 00:10:39,450
pyridoxal phosphate and
pyridoxamine work, in order

217
00:10:39,450 --> 00:10:43,920
to put amino groups into organic
acids, such as oxaloacetate.

218
00:10:43,920 --> 00:10:47,170
And that's what's going
to happen in this case.

219
00:10:47,170 --> 00:10:51,230
Oxaloacetate is converted
into its amino acid homolog,

220
00:10:51,230 --> 00:10:52,970
aspartic acid.

221
00:10:52,970 --> 00:10:56,480
Why did we do this
emanation reaction?

222
00:10:56,480 --> 00:10:59,960
Well, there's no way to get
oxaloacetate directly out

223
00:10:59,960 --> 00:11:03,540
of the mitochondria because
there's no transporter for it.

224
00:11:03,540 --> 00:11:06,710
But there is a good transporter,
the apartheid transporter,

225
00:11:06,710 --> 00:11:10,460
that will take aspartic
acid out into the cytoplasm.

226
00:11:10,460 --> 00:11:13,880
So the oxaloacetate is
converted, temporarily,

227
00:11:13,880 --> 00:11:16,850
into aspartic acid
in the mitochondrion.

228
00:11:16,850 --> 00:11:19,310
And that aspartic
acid then slips out

229
00:11:19,310 --> 00:11:23,500
through its transporter
to the cytoplasm.

230
00:11:23,500 --> 00:11:26,830
Once in the cytoplasm, there's
a similar pyridoxal mediated

231
00:11:26,830 --> 00:11:29,860
mechanism to deaminate
the aspartate

232
00:11:29,860 --> 00:11:33,430
to regenerate the cytoplasmic
molecule of acetate

233
00:11:33,430 --> 00:11:36,660
that we borrowed at step
one a few minutes ago.

234
00:11:36,660 --> 00:11:39,070
In panel D I summarize.

235
00:11:39,070 --> 00:11:42,580
That we've looked at three
different small pathways that

236
00:11:42,580 --> 00:11:46,180
enable the cytoplasm of the
cell to always have enough NAD+

237
00:11:46,180 --> 00:11:49,320
to oxidize glucose to pyruvate.

238
00:11:49,320 --> 00:11:52,720
These pathways are first, the
lactate dehydrogenase system.

239
00:11:52,720 --> 00:11:55,260
Second, the
glycerol-3-phosphate shuttle.

240
00:11:55,260 --> 00:11:57,970
And third, the malate
departed shuttle.