Rehab Cell

Physical Medicine and Rehabilitation

#34 Biochemistry Oxidative Phosphorylation/Respiratory Control Lecture for Kevin Ahern’s BB 451/551

Kevin Ahern: Excuse me. Let’s get started. How’s everybody doing? Ready to get this over
with, get out of here, go have a weekend, study for the exam? A couple announcements. An exam in here on Wednesday and material will definitely
cover through today. I haven’t decided exactly
where that cutoff will be. I may announce that today. I may not announce it until Monday. But I will find a
logical place to cut it so don’t sweat that too much. I have scheduled a review session. There will be a review
session on Monday evening at 7:30pm in ALS 4001. And I will videotape
it as I’ve done before and get it posted as
quickly as I possibly can. Alright, we have a fair
amount of material to cover. Yes, Anesia? Student: [inaudible] Kevin Ahern: So the
question is will material on Monday be on the exam,
and I will know better after I see where I get to today. So I will give you an
indication of that by the end of the lecture today. We have some really
cool stuff to talk about and I find that it’s this
component of metabolism that really gets students
understanding that big picture about their bodies, about
energy, and so forth. And so I want to spend some
time getting through this. I don’t want to go too
fast but I also recognize I’ve been talking a little
bit so I’m a little bit behind where I need to be. Last time I spent
some time talking about oxidative phosphorylation
and I think it’s quite straightforward what is happening. We don’t have to worry
about identifying and naming all the individual proteins
in the complex and so forth. That’s not the most important thing. The most important thing is
understanding what’s happening with respect to the
limitations on the process. And that’s a very interesting phenomenon known as respiratory control. I’m going to spend some
time talking about that today and hopefully lead you through that. Before I do that I need
to make sure that I cover a couple of things because
they are considerations for us. So what I’m going to do now
is talk about some membrane shuttles that are relevant for us, so we can see how they play
into this overall picture. Then I’m going to talk
about respiratory control and some uncoupling proteins,
the various things in here, and last I’m going to
come back up and talk about the superoxide dismutase and so forth because I think that’s
actually a little bit out of place for where it needs to be. why do I want to talk about shuttles? The reason I want to talk
about shuttles is that they’re important because
NAD and NADH do not cross the mitochondrial inner membrane. The NAD that’s in the
matrix has to be synthesized there by enzymes in there. It’s not something that can
move across the membrane. Well that poses a
little bit of a problem because we’ve got
that NADH that we made. That’s a little loud isn’t it? That NADH that we made in glycolysis. How we do get the NADH from
glycolysis into the electron transport system if
it has to get inside? Well we have to use things
that carry electrons in, and those are known as shuttles. I’m going to describe
two shuttles to you. I don’t want you to get too,
freaking on the details of these. The first one is actually
quite straightforward. It’s the one you see on the screen. And it’s one that’s
common in insect muscle. Insect muscle very commonly uses this. And you’re going to see a
difference between what insect muscle does and what your cells do. So here we are at the very top. We are in the cytoplasm
and we are trying to get electrons down here into the matrix, or at least into the inner
mitochondrial membrane. How do insects do that? Well the way they do it
is they use this shuttle that you see on the
screen in which they take dihydroxyacetone phosphate, which is an intermediate in glycolysis, and they transfer
electrons to it from NADH thereby creating glycerol 3-phosphate. So glycerol 3-phosphate has acquired those two electrons from NADH. It’s also acquired a couple of protons and that’s what we see here. Glycerol 3-phosphate can
interact with this enzyme here to donate electrons
to FAD and make FADH2. So what we have done, in
essence, is we have converted, we have transferred electrons
to NADH to FAD to make FADH2, and then from FADH2 the
electrons go through the Q cycle, they go into coenzyme Q,
they go into the Q cycle and they go the rest
of the way on through. Now if you remember what
we talked about with respect to the pumping of protons
from Wednesday’s lecture, what we see is that there
were, this is essentially coming in through Complex 2
meaning we’re bypassing the proton pumping of Complex 1, and it means that every
time we shuttle electrons in like this, not we, insects do this, every time they do that they’re in essence only
pumping enough protons to make a couple of ATPs instead of
making enough for three ATPs. This is not a very efficient process. The advantage of this
process is it’s very quick. Very quick. So what they lose in
efficiency they gain in speed. Well that’s one way of getting electrons into the system from the cytoplasm. We use a somewhat different
mechanism that I’m going to show you called the
malate-aspartate shuttle. And it’s shown here. And it looks more complicated
but in reality it’s not. And I’ll tell you why it’s not. We have the same basic
problem that insects have. That is we have to get electrons from the cytoplasm into the matrix. We start with electrons
up here and we’ve got to get them down into here. If you focus your attention,
this is a bit of a dumb figure the way it’s set
up, so you’ve got to focus your attention starting
right here with oxaloacetate. Oxaloacetate out in
the cytoplasm is reduced by electrons from NADH to make malate. That’s a backwards reaction
from the citric acid cycle. So we’re reducing
oxaloacetate to malate. Those electrons now are in malate. Protons there, and
malate gets transferred into the mitochondrial matrix. When it gets in there,
the reverse happens. Malate donates electrons
to NAD to make NADH, and it goes back to oxaloacetate. What have we done? Well in essence we had an NADH out here. Now we have an NADH in here. We have no net loss of energy. Everybody’s happy. What’s all the rest of
that crap on the screen? Well the rest of that crap on the screen is just balancing the equation. We’re not going to worry
about balancing equations. What matters? What matters is how we
get the electrons in. How do they get in? They get in on the back of malate. Malate carries them into the matrix. Malate gets converted
back into oxaloacetate and ultimately oxaloacetate
pops back out over here. That’s all there is to it. Kevin, are you going to ask us to redraw this thing that you see on the screen? Well whenever I use that
voice, what’s the answer always? Class: No. Kevin Ahern: The answer is no! So all this is showing
how we balance this to get back to oxaloacetate. But that’s not the story. The story is right here
where we see malate coming in. Well what we see here, look at this. This is, these are antiports. Malate in, what’s going out? Alpha-ketoglutarate. Here’s an antiport. So what? It doesn’t change the overall story. The overall story is
we’re getting electrons in and this is much more
efficient than insect muscle is because we’re not giving
up any pumping of protons. We start with NADH out,
we end up with NADH in. Yes sir? Student: Is it proton
motive force that will allow oxaloacetate to reduce
to malate and then on the inside just directly
reverse that malate to… Kevin Ahern: Yeah his question
is basically is the difference in the oxidation/reduction
state of these two outsides and insides the driving force for this, and the answer is yes it is. Okay, so that’s the malate-aspartate shuttle that comes in there. Another consideration
that we have to have is that I mentioned last
time that ADP is a limiting thing for oxidative phosphorylation. If we have limiting amounts of ADP. We don’t have enough
ADP-when would that happen? When would we not have enough ADP? When we’re high energy
and we’re sitting around doing nothing eating a lot of food. If we’re not burning ATP to ADP we’re not going to have much ADP
and Complex 5 is going to stop. And when Complex 5
stops, so are the incoming protons going to stop. So it’s pretty important
that whenever ADP becomes available in the
cytoplasm which is where it’s used, it’s very important that
that ADP be transported into the mitochondrion
so that it can be used. Well there’s actually
a shuttle that does that and it’s a shuttle that’s very cool. It swaps ADP for ATP. So ATP that gets made
in the mitochondrion gets kicked out and ADP that gets used in the cytoplasm gets kicked in. It’s a very neat antiport. And it’s an important
antiport because if it required energy we wouldn’t be able
to get anything, right? If we had to make an ATP
to use an ATP to get an ATP out into the cytoplasm
we wouldn’t have any ATP! So all this is showing
is this is an antiport that swaps ADP for ATP. A very important consideration. There are a lot of
important transporters and they look pretty. [laughing] That’s it. That’s all I’m going to say. Was that a test question? “Two seconds Ahern and it’s up there.” I want you guys to get
this stuff down, right? ATP counts. Now ATP counts depend upon
how you count these things. Your book counts it at 30
and that’s because they make certain assumptions
about numbers of ATPs per pair of electrons. I put the number a little higher. It doesn’t really matter. The important point is
there’s approximately 30 to 38 ATPs made per
molecule of glucose oxidized. Why is it uncertain? Well it’s uncertain because
remember we’re kicking protons out of the mitochondrion. We don’t necessarily have
a one-to-one relationship of protons coming in. Once we kick them out they
can go do other things, they can go other places,
so it’s not an even number that’s there. We will for this class assume
three ATPs per pair of electrons, two ATPs, or no I’m
sorry, three ATPs per pair of electrons from NADH
and two ATPs per pair of electrons from FADH2. Well in any event that’s a lot of energy that comes out of a
glucose molecule and that’s why glucose is a very
important energy source for us. Now the energy is only
part of the picture. We have to understand how our
body controls these things. And there’s a lot of control. It’s called respiratory
control and I want to dig into that just a little bit. Well what does respiratory control mean? Well, oxygen consumption
is basically related to the production of ATP. You know that. And how do you know that? Well oxygen is needed
for electron transport and electron transport is needed
for oxidative phosphorylation. So it starts to make sense. If I start exercising, I start running, I need oxygen to make
ATP which I’m burning up when I am exercising. That’s why I start
breathing heavily, alright? Well if we think about
it at the actual molecular scale it’s kind of cool. Let’s think about this for a second. I take off. I start running. What’s the first thing that happens when I take off and I start running? Well my ATP gets converted into ADP because ATPs needed for
muscular contractions. So what happens to
my ADP concentrations? They start increasing, right? As my ADP concentrations
start increasing, what’s going to happen to
oxidative phosphorylation? It’s going to spin. The ATPase is going to
spin, and when it spins what’s going to happen
to proton concentration? Well proton gradient’s
going to start falling because protons are
going to be coming in. Everybody understand that? The proton gradient’s going
to get less as I’m making ATP because protons are
what’s causing the spinning. Protons are coming
into the mitochondrion. When proton concentrations
start falling outside the mitochondrion, what
happens to electron transport? It’s going to speed up. Why? Because there’s nothing
stopping those protons from being pushed out. Before I started exercising
I had a high proton gradient. Now I’ve started reducing
that proton gradient and all the sudden the complexes wake up and start kicking protons
out, and when they kick protons out what happens to electrons? Electrons flow. And as electrons flow,
where do they have to go to? They have to go to oxygen and
that’s why breathing heavily. Really cool. What happens if I were to put my head into a paper bag and take off for a jog? Student: Run into stuff. Kevin Ahern: Run into stuff. [laughing] It was a trick question. He got the answer. I put my head in a paper bag,
I don’t have enough oxygen. What’s going to happen? Let’s think about what would
happen in that scenario. I don’t have enough
oxygen, so what’s the first thing that’s going to stop? Electron transport’s
going to stop, right? Electron transport stops, what
happens to proton gradient? Student: It goes down. Kevin Ahern: It’s going to go down. And why is it going to go down? I’m making ATP. I’m pulling protons, or
I’m letting protons flow in to make ATP but there’s nothing putting more protons out there. All the sudden instead of
having a proton gradient I have nothing. What’s going to happen
to my ATP synthesis? Well it’s going to “vrerrrr,”
and it’s going to stop. It’s the reason I suffocate. I suffocate because I
can’t make enough ATP to support what I’m trying to do. How about I take cyanide and
I try to do the same thing? What would happen? Student: You’d die faster. Kevin Ahern: What would happen? Exactly the same thing. Cyanide’s going to
stop electron transport. If I stop electron transport, okay, if I stop electron
transport there’s not going to be any proton pumping. No proton pumping going on,
same thing’s going to happen. I’m hosed. Let’s think about I’m sitting around, eating pizza, drinking
beer, watching the tube, and not thinking about BB 450, which always causes stress and
a certain amount of energy burn. So we’re not doing anything. We’re sitting there
and our energy levels are very high because
we’re not burning ATP. So our ATP concentrations are high. When our ATP concentrations are high that means our ADP
concentration are low. And if our ADP concentrations are low, what’s happening with Complex 5? Nothing. It’s not spinning. It stops spinning because ADP is needed for the spinning just like protons
are needed for the spinning. You’ve got a ton of protons
up here but there’s nothing down here to allow
the spinning to occur. There’s no ADP. So what’s going to happen in that case? Well my ATP concentrations are high. My proton gradient is going to do what? It’s going to get higher,
and higher, and higher, until I can’t get any higher. And the complexes can’t
push any more protons out because the gradient’s so
high they can’t beat it. So electron transport is going to stop. Electron transport is going to stop. What happens when
electron transport stops? Well when electron transport
stops NADH concentrations go up because we’re not converting
it back into NAD anymore. When NADH concentrations go up, what happens to the citric acid cycle? Student: [inaudible] Kevin Ahern: Right? It gets better. Or it gets worse. [laughing] Citric acid cycle stops. What happens to the
concentration of citrate? Hmm, trick question. Well it turns out, do we
need NADH-or I’m sorry, do we need NAD to make citrate? No we don’t. We need NAD to convert
isocitrate to alpha-ketoglutarate but we can make citrate just fine. Why do I mention citrate? Well citrate concentrations we would agree would go up, right? Why is that important? Because citrate is the way
that cells take acetyl CoA out into the cytoplasm. So citrate gets moved out into
the cytoplasm, it’s a shuttle, and it gets cleaved into
oxaloacetate and acetyl CoA. Why is that important? Because as we start dumping acetyl CoA out into the cytoplasm that’s
how we make fatty acids. When we’re not exercising,
we’re not burning our ATP, we’re going to make fatty acids. If we eat more than we burn, a
very basic principle of dieting, if we eat more than we burn
we’re going to make fatty acids. It’s very simple respiratory control. Very very simple respiratory control. Let’s think about that magic diet drug I talked about the other day. I go to bed, I take my magic diet drug. What’s going to happen? Magic diet drug pokes a hole in my inner mitochondrial membrane. What happens to the proton gradient? It goes down, right? It’s going to go down. What happens to my Complex
5 production of ATP? What happens? No ATP made. My body needs ATP. So as ADP concentrations go
up, my body is starting to go, “Whoa, better get something going.” Proton gradient is falling. As proton gradient is
falling what happens to the citric acid cycle? Let’s back up. What happens to electron transport? Up or down? Up, right? It’s up because there’s no
proton gradient to stop it. It’s going like crazy. What happens to oxygen consumption? Up, because electron
transport is going like crazy. I am going to sleep and I am going, [panting] I’m panting heavily as I am sleep. What happens to my body temperature? Up because I’m doing
all this metabolism. What happens to my use of glucose? Up. What happens, and we’ll see this later, what happens to my
burning of fatty acids? Up. So all these things are doing
the magic diet drug thing. They are in fact burning
all that stuff off, okay? I just hope I don’t kill myself. Yes? Student: So how would you die from it? Would you die from, like, starvation? Kevin Ahern: Would you
die from like starvation. [laughing] Well that’s a good question. I’m not sure you’d last that long. Because if we think about it,
let’s put my head in the bag. One of the things that’s
killing me is I’m not making enough ATP and I don’t last
very long if I don’t do that. So, I suspect if you had enough of that, you probably wouldn’t last very long. You wouldn’t have a chance to starve. That’s probably what
would happen to you. Nasty stuff. But fun to think about. Okay, yes? Student: So lethality
was just a dosing issue? Kevin Ahern: Okay here we go. “Lethality is just a dosing issue.” [laughing] Just like arsenic poisoning is
just a dosing issue too, right? Same principle. Questions about that because
I’m almost about ready to tell you about a photosynthetic fish. But I’ll stay quiet, any questions? Does that make sense? Could you guys take it
through those steps and I said, “Hey, here’s what we’re doing,” and you could predict what
would happen in those scenarios? [class murmuring] There’s a question. Karen. Student: So how is
using a paper bag help when you’re hyperventilating? Kevin Ahern: How does
using a paper bag help when you’re hyperventilating? Well A, hopefully you’re
not doing this for too long. Let’s see, what would happen? When you’re hyperventilating
what you’re doing is you’re producing, well
anytime you’re breathing you’re producing carbon dioxide. If you’re hyperventilating,
the more carbon dioxide that you produce and the
less you get rid of probably the lower the pH of
your blood is falling and I’m guessing it’s related to that, but I don’t its the
answer to the question. You can change the pH of the blood pretty readily with carbon dioxide. That’s one of the
reasons it’s poisonous, so yeah, I don’t know. If somebody finds the answer
to that that’d be kind of cool. Probably because your brain is racing with a little bit of oxygen so you put carbon dioxide in there
you might also be changing some chemistry there as well. Okay, let’s think about
photosynthetic fish. This is a really cool
thing for us to consider. And I can guarantee you
this principle is solid. I think you’ll see it’s solid
once I explain it to you. You’ve learned how proton
gradients are important. Well there’s a protein
I talked about earlier in the term that I said,
“I’m going to remind you “about this when I go to talk
about a photosynthetic fish.” Anybody remember what the protein was? Student: Bacteriorhodopsin Kevin Ahern: Bacteriorhodopsin. So bacteriorhodopsin I’ll
remind you is a protein that is found in some
photosynthetic bacteria. And it’s a membrane
protein in the bacterium. And what does it do? Well it’s in the membrane, so
it’s got a little channel there. And that little channel allows
protons to pass through it. However, there’s a barrier. There’s a guard that stops protons from just passing through it. You kind of want to have
that because, otherwise, you’d have no proton gradient. The guard that’s there is a really
interesting and cool molecule. It’s vitamin A. Vitamin A is in the
middle of this little chamber of bacteriorhodopsin. Well why is that important? Well vitamin A as we will
learn later in the term is a molecule that is light sensitive. You know vitamin A is
needed for your vision. And what you will
learn is that vitamin A, being light sensitive,
changes its chemical structure as a result of exposure to light. There’s a bond in vitamin A
that’s very light sensitive and when light hits it, it
changes from cis to trans and trans to cis and back and forth, and back and forth, and back and forth. Doing this. Well in the middle of this
chamber is this vitamin A, and vitamin A has got this
little ring structure here that we can think of like a hand. So when light hits it, it does this… swish, swish, swish. Each time grabbing a proton, kicking protons out of
the bacterial cell wall. When you turn the light
off it just lays there. When you turn the light on it does this. We’ve got the beginnings. We have something that will pump protons under the control of light. That’s really useful if you
want make a photosynthetic fish. How do you make a photosynthetic fish? You take bacteriorhodopsin,
you put the gene in… You guys, anybody here with aquariums? You like those little clear
fish you can see through? Oh these are the ones you want to have. [laughing] Because light goes right through ’em! And you put it in their mitochondrion, so that bacteriorhodopsin
is in their inner membrane of their mitochondrion and
guess what’s going to happen? You turn the light on the
fish, you’re going to be pumping protons but it doesn’t
cost you any glucose. It’ll make ATP only under
the control of light. That’s a photosynthetic fish. Cool stuff. Now, it’s not the same as a plant because what plants do
is they also assimilate carbon dioxide out of the atmosphere. This won’t assimilate carbon
dioxide from the atmosphere because it’s just
simply pumping protons, but what it will do is make ATP. Yes? Student: Will the fish get fat? Kevin Ahern: Will the fish get fat? Well that’s a good question. I would wager this fish,
this theoretical fish, and I’ve talked to some experts in this, and they claim that
it’s an interesting idea. I don’t know anybody who’s done it. You guys, it’s out there if you
want to go do it, like I said. Well, will it get fat? I claim this fish will need less food than virtually any fish on earth. It will need some food. Why? Because it has to have a carbon source. But if we want to think about
neat ways to make protein that don’t take much energy, a photosynthetic fish might
be a real cool way to do it. All you’ve got to do
is give it a reasonable carbon source and they can
make stuff more efficiently than any other fish that’s out there. You think of fish farming and so forth. Kind of a cool thing to do. Yeah? Student: Would this
fish die if it was dark? Kevin Ahern: Would the
fish die if it was dark? Well we can speculate
on a couple things. The people I talked to
said they suspect the fish might die if it’s light. [laughing] I’ll tell you why. Why might it die if it’s light? Well with a pump that, first of all, hasn’t evolved with the fish, you can probably create a
pretty intense proton gradient that just might fry the bacteria. So my idea is if you make this
fish, you grow it in the dark. It’s going to be like a regular fish. It’s going to eat like a regular fish. It’s going to be hungry
like a regular fish. And it’s not going to be any different than a regular fish as
long as it’s in the dark. But if you put the fish in the light and you might start
seeing things happen. [class laughing] I mean it might be kind of a cool thing. There’s a YouTube video for you, right? There’s a fish in the dark, you know. You turn on the light. Vree, vree, vree, you
know, it’s going like crazy. I don’t know. So it might get fat. It might die in the light. I don’t know. I’d like somebody to make that fish and we could do the experiment. It’d be kind of a fun thing to do. Student: Self-tenderizing fish. Kevin Ahern: A self-tenderizing fish. So anyway that’s my
photosynthetic fish idea. Yes sir? Student: You know
[inaudible] where you said about [inaudible] proton gradients. Once it runs out of ATP [inaudible], wouldn’t it be one of the reasons it would be so acidic
outside the cell… Kevin Ahern: I’m not sure
I understand your question. Say again? Student: Well, it has such
a high proton concentration on the outside, and it runs on ATP, [inaudible] ATP. So, that high concentration of protons and high acidity would damage the cell. Kevin Ahern: Okay, so his
question really relates to the nature of the proton gradient as a result of this action. That’s why I said it hasn’t
evolved under the conditions that, you know, we’ve all evolved under. So while a bacterium
might be able to tolerate a certain level of gradient,
a mitochondrion might not. So a couple things might happen. One, you might fry it. When I say fry it, that
voltage gradient it could create would be greater than would be normal for an electron transport
system for example. So that voltage
difference might literally just burn the membrane. The other possibility is you might pump enough protons out of the mitochondrion that you’d acidify the cytoplasm. And if you do that then you’d
have some real problems too. So it’s hard to say. Yes sir? Student: Could you use
it to solve world hunger? [laughing] Kevin Ahern: Well, hey, don’t laugh. I think it’s actually a very interesting way to make animal protein. Yeah I do. Making animal protein
isn’t necessarily the most efficient way to solve world hunger, but you could produce in
my opinion with something like this a lot more
animal protein for much less cost in terms of food
energy that would be needed. But it’s got to work. I mean if we fry the fish it’s
not going to do us any good. Fried fish before, you fry
it right there in the thing. “Honey I’m hungry,”
and you turn the light on and the fish goes “tsssst.” [laughing] Student: Are there very many
isoforms of bacteriorhodopsin or is it pretty dialed
in to one particular… Kevin Ahern: Are there many
isoforms of bacteriorhodopsin. That’s a good question. I think, I suspect there
are a variety of forms. And I suspect you could also
tweak it so that you might find wavelengths of light that you could have it be sensitive to and
not other ones where it’s not, where you could grow
it one light and then maybe make it pumping
in another form of light, another wavelength of light? Okay, so you like my idea. Well you start to see what
we can do with gradients. I mean proton gradients
and ion gradients are really interesting
and really cool things that we can do things with. I want to tell you about a couple, or at least one, of
biological relevance. And this is one that
you have in your body, and in fact a variety of
organisms have in their body. Let’s think about that
situation of the magic diet drug. The magic diet drug causes
problems because it’s letting protons come in and it’s not making ATP. And what was one of the
byproducts they said of that? Heat, right? Is this a way to generate
heat, and the answer is it is. It turns out our body has a collection of cells known as brown fat. And brown fat has
a very interesting protein in it called uncoupling protein. Uncoupling protein, okay? What does uncoupling protein do? Basically it does the
same thing as a diet drug. It pokes a hole in the
inner mitochondrial membrane and allows protons to come in. And because it allows
protons to come in, what happens to electron transport? Electron transport goes crazy. As electron transport goes
crazy, what’s the byproduct? Citric acid cycle goes crazy. Citric acid cycle goes
crazy we have heat. Brown fat in humans is
located near the spinal cord. And this is my own personal pet theory but my explanation for
this is that it’s important that we keep our nerve system
at a reasonably constant temperature even if our
extremities get colder. The reason being that
even when we’re cold we need to be able to respond
to our environment quickly. If we’re out there and that grizzly bear is chasing us we don’t
want to have to go slower, or recognize the grizzly bear’s slower because of the fact
that it’s cold out there. So when we’re cold
we’re going to protect that nervous system
and brown fat kicks in. So when that kicks in, and it does kick in when the brown fat gets very cold, and allows that to happen. Well why doesn’t that
kill our brown fat cells? It doesn’t kill our brown fat cells because uncoupling
protein gets regulated. We can think of this as being a chamber that allows protons to come through, but that chamber can get plugged up. And it does get plugged up. It’s plugged up by palmitic acid. When the cell is at the
point where it doesn’t need to generate any more heat
it plugs this uncoupling protein up and the proton flow stops and everything goes back to normal. There’s your diet drug. There’s your diet drug. Okay, cook stuff. There’s imaging for
your brown fat active and inactive depending on temperature and you can see again
sort of back here around the spinal cord you see
where this stuff is laid in. Cool. Yes sir? Student: So, when you say the diet drug, is the magic diet secret
to sleep in the freezer? Kevin Ahern: The magic trick
is to sleep in the freezer? [laughing] No I didn’t say that. I said the magic diet drug
is to get uncoupling protein to work the way you want
it to when you want it to. That’s the diet drug. Yes sir? Student: So doesn’t that decrease
over time naturally anyway? It’s mostly infants and
hibernal animals that have that? Kevin Ahern: His question
is does it change over time. And it’s true. Infants do have more brown fat
than we do and it does change. Student: Could you repeat what regulates it again? Kevin Ahern: What
regulates it is palmitic acid will plug it up. So cells just simply plug
it up with palmitic acid. Student: Based on temperature? Kevin Ahern: I’m sorry? Student: Based on temperature? Kevin Ahern: Based
on temperature, right. And it’s only found, as
far as I know, in brown fat. Okay let’s see what
else did I have here. I had, this is a nice schematic. It reminds us of all the
players in this process. It reminds us what happens
if we stop various things. So let’s stop something here. Let’s stop ADP going to ATP. That stops this. That stops this. That stops this. That stops this. We stop it here, we’ve got it. Now what I’ve just described
to you is a phenomenon that’s there that we almost
always have in our body. It’s called tightly
coupled mitochondria. And tightly coupled
mitochondria means that there are no holes in the membrane. And when there are no
holes in the membrane oxidative phosphorylation
depends on electron transport, and electron transport requires
oxidative phosphorylation. Because if I stop this the
gradient starts getting high. If I stop this, no gradient to
make oxidative phosphorylation. So tight coupling occurs
when the mitochondrial inner membrane is intact. And that’s why that protein
is called uncoupling protein. Because it’s allowing
protons to flow back in. It’s no longer intact. The diet drug is uncoupling
oxidative phosphorylation from electron transport. When they’re tightly coupled, no holes. When we put holes in, they’re uncoupled. And let’s see, there’s that
magic diet drug right there. Very simple compound. And this illustrates a variety of things that are used as energy
sources of a proton gradient. Obviously you’ve seen ATP. Flagella in bacteria can
use a proton gradient. Active transport you already saw that with the lactose permease. You saw how that worked. Electron potential, I haven’t
really talked about that. Heat production, you’ve seen
how brown fat can do that. And we won’t talk about it
here but NADPH synthesis, that’s photosynthesis. Proton gradients are
used as energy sources in chloroplasts to make
NADPH and also to make ATP. Proton gradients are pretty useful. The last thing I said
I was going to talk about here and then
I’ll actually start some new material is a
reactive oxidation species. And they’re interesting
and they’re important. Whenever we don’t complete
that cycle of four electrons going through the
electron transport system to completely reduce an oxygen we create a reactive oxygen species. And reactive oxygen
species get their name from the fact that they’re
extraordinarily reactive. They cause damage. One of the things that
they’ll do we’ll talk about later in the term is that
if you have reactive oxygen floating around in your cell
and it’s not taken care of, it’ll oxidize guanine
residues in your DNA. It’ll put an oxygen on a guanine
where there wasn’t one before. It creates a molecule
called 8-oxoguanine. 8-oxoguanine is a very,
very potent mutagen. The reason it’s a potent mutagen is because 8-oxoguanine will
form base pairs with adenine. G paired with A is
not a good career move. So protecting your DNA,
protecting against reactive oxygen species is very important. Part of that protection is
what you see on the screen. We have enzymes that
do their best to reduce the concentration of
reactive oxygen species. They have to do that. If they don’t, we have problems. The enzyme you see
on the screen is known as superoxide dismutase. A mouthful of a name. And what it’s job is to do
is to reduce the concentration of this reactive oxygen
molecule you’ll see here. O2 with an extra electron. That’s known as a superoxide. Somewhere along the line
electrons didn’t match right and this guy’s
got an extra electron. That guy’s extraordinarily reactive. It will in fact create 8-oxoguanine. Bang! Without even thinking about it. What you see depicted in blue and red is the enzyme, superoxide dismutase. And look what it’s doing. Here’s the enzyme. The enzyme exists in two states, an oxidized state and a reduced state. What mechanism is it using? We talked about different
that enzymes work. Anyone remember what
mechanism we’re choosing? Nobody? We talked about order displacement. We talked about random displacement. What’s that? Anybody remember Ping-Pong? The enzyme’s in one state
and then it flipped to another and then it flipped back and
then it flipped back, right? That’s why it was Ping-Pong, Ping-Pong, also known
as double displacement? Look at what the enzyme’s doing. The enzyme in the oxidized state is able to accept an electron. It takes an electron
from this superoxide and becomes in the reduced state. It’s now got the
electron that the oxygen had and look what it does. It releases oxygen. This guy’s fine, no problems. Well we’ve got to get the enzyme back to its original state. To get the enzyme back
to its original state we get another one of these. We’re getting double
duty out of this enzyme. And we add the electron
from here to superoxide, add a couple of protons and
we create hydrogen peroxide. And now the enzyme’s back where it was and we have hydrogen peroxide
which is also fairly reactive. However it’s not as
reactive as superoxide. That’s one thing. And number two, we have
an enzyme known as catalase that’ll break this guy down. So we’ve effectively taken
something that’s very poisonous, very detrimental to our longevity, and we have in fact wiped it out. Through a pingpong mechanism. Now one of the things about this enzyme that’s really interesting
is that this enzyme is known to be defective
in certain people who have Lou Gehrig’s disease. Amyotrophic lateral sclerosis, a small percentage of
the people who have that, in fact, that was the
way that the significance of the enzyme was originally discovered, a small percentage of the
people who have that disease have a defective enzyme
for superoxide dismutase. And the thinking is that
one of the reasons that ALS, Lou Gehrig’s disease, is a
neurodegenerative disease, the thinking is that over time, these neurons that lack
this enzyme accumulate reactive oxygen species and
they basically get destroyed by the superoxides that are in there. Now the disease itself
is very complicated and it’s quite clear,
I shouldn’t say quite clear but it’s not clear why this is not found in all of the patients. But for people who
have a genetic tendency to get it this enzyme has
in fact been implicated. Yes sir? Student: When these superoxides
are initially formed, do they preferentially attack
the mitochondrial genome just because of proximity? Kevin Ahern: They don’t. Well, okay-so his question
is will superoxides preferentially attack
the mitochondrial genome. They will preferentially attack whatever the first thing it is that they hit. But yes since they’re
in the mitochondrion that’s why we see it,
that’s one of the reasons we see damage to the mitochondria. I see that older mitochondria
look older than new ones do. That’s one reason. And you’re right, the
mitochondrial membrane, the mitochondrial DNA being
there is much more likely to be oxidized than anything
else is, and in fact we see more mutations in the
mitochondrial genome. We see them at a faster rate than we see in the nuclear genome. So again, probably because of all these reactive oxygen species. Anesia? Student: Does enzyme
activity decrease as we age? Kevin Ahern: Does enzyme
activity decrease as we age? Good question. I don’t know that that’s the case. The question of activity
though is one of how active an individual enzyme is and also how many enzymes we’re making. And I can’t tell you
definitively the answer to that. I will point out that there’s not just one superoxide dismutase. We have several. And so their separate
functions aren’t completely understood about why we have,
you know, which ones doing what. Yes sir? Student: Is this the cost of doing business? Kevin Ahern: Say again? Student: Is this the
cost of doing business? Kevin Ahern: Ah, very good question. Is this the cost of doing business? You are exactly right. The is the cost of doing business, yep. Student: Is there any
link between dietary uptake of antioxidants and superoxides? Kevin Ahern: Oh boy, very good question. Is there any link between the intake, that is the eating of antioxidants, and the level of superoxides
that are present in the body? There are at least some
suggestions that yes, there are differences between
not taking versus taking. It’s one of the reasons, in
fact many of you probably know the Linus Pauling Institute here at OSU, one of their major focuses
is understanding the role of antioxidants in human health. And there’s some phenomenal work that’s coming out of what has happened, what they’re finding out
about the role of antioxidants. Antioxidants of course,
that was Linus Pauling’s, maybe one of the
reasons that Linus Pauling took those many grams of
acetic, of ascorbic acid that he did was because of
its antioxidant properties. And so there’s some
really interesting things with respect to that. In fact there’s a couple
clinical trials going on that look very interesting
with respect to vitamin C, from what I’ve heard. And so there are some cool
things that are happening there. As we will see when we talk later, when I talk later about the movement of cholesterol in the body, we will see how levels of antioxidants in the bloodstream may play
a role in helping to reduce the levels of atherosclerosis
because reactive oxygen species that damage LDLs probably help atherosclerotic plaques to form. So yeah there’s some really
good reasons to take antioxidants and be careful of some
of the crap that you eat. So… Let’s see where am I at? Well I’ve got a couple minutes. Let me just finish up here. We are a little behind so
otherwise I would let you go. So that is what I wanted to say. Well actually I will just
show you this since I was here. Again, Medical links. Free radicals are not just important for Lou Gehrig’s disease. We see free radicals
implicated in a variety of diseases that you see on the screen and there are many, many others. The list is growing
and growing and growing. There are a lot of people who
believe, and with good reason, that superoxides play
a role-or I’m sorry, that reactive oxygen species
play a role in the aging process. And we think about a
protective mechanism that we might have for reducing those and reducing the incidence, or not the incidence
but the rate of aging. And there are some good
reasons to think of that. Now I’m not going to
start a whole new chapter. I will point out that
when we come back on Monday I will start talking about
synthesis of membrane lipids. Let’s call the exam through today. So we’ll start new material
for the new exam on Monday. See you guys on Monday. Student: I had a question I wasn’t sure I wanted to ask in class. Kevin Ahern: Okay. Student: When you were
talking about brown fat around the spinal column
and saying that [inaudible], why is it that they’re
now, when it comes to, spinal injuries in sports,
injecting people with IV cold material at that location? Kevin Ahern: Well cold
material’s going to reduce the level of damage. So damage is one thing. Escaping from a grizzly
bear is another, right? Student: Yeah. Kevin Ahern: So very different
kinds of things but yeah, that’s the reason. Student: I was like, ‘why would you [inaudible]?” Kevin Ahern: Yep. Make sense? Student: If you want to use
it, you want to keep it warm. Kevin Ahern: Right. You’re not going to be
escaping from a grizzly bear when you’ve just had your
spinal cord half severed, right? [END]

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