 Let's get going. Again, I'll have office hours from nine to ten today and probably we will try to move them into the lab if a lot of people come again. It's pretty crowded in 2084 but if other rooms are not available we have to stick in 2084. So today I'm going to finish talking about biological membranes and I wanted to start off. Can I have the overhead on showing you something I should have shown you on Wednesday? I was talking on Wednesday a lot about membrane proteins and I mentioned that one way people study membrane proteins is through this technique of sodium dodecyl sulfate, polyacrylamide gel electrophoresis and this is what one gets. This is an actual gel which has been stained with a dye. Every one of these blue bands represents a different protein. The protein, the material under study here is actually a photosynthetic membrane complex. In some regards it's similar to the photosynthetic membrane complex. I showed you the structure of it the last lecture but this is a complex from higher plants. What one can do is isolate this complex out of the membrane using detergents, something I talked about on Wednesday. Fortunately in this case the complex is active. It does what it's supposed to do so one can then study the activity of the complex and try to understand what you can see there is one very high molecular weight to low molecular weight. One very high molecular weight protein and then a bunch of low molecular weight proteins and try to understand what each of these proteins does in the overall activity of this complex, of this biological system. The first thing to do is to try to get a clear picture of what proteins are in this material and then to try to go further and try to elucidate their specific role in the activity of that complex. For example, this is a complex that contains chlorophyll. If you don't stain this gel with a protein stain, some of these protein bands are actually green because they still have bound chlorophyll so you immediately get some information about which proteins might bind chlorophyll and which don't. Anyway this is an example of an SDS page gel that you probably will, most of you might encounter sometime in your future life. Okay so what I was talking about last time at the end of the hour was transport, biological transport. Oh great, I was going to say can you see the board. I firstly described simple diffusion which is the movement of a molecule across a membrane with the concentration gradient and that movement is driven strictly by the hydrophobicity or hydrophilicity of the compound because as you now know the biological membrane is a phospholipid bilayer with hydrophobic protein stuck in it. So simple diffusion there is no protein involvement. In facilitated diffusion, again you are still moving molecules from a high concentration gradient to a low concentration gradient but now there are proteins involved. It is protein mediated and what was shown in figure 73 are the two kinds of proteins that are involved in facilitated diffusion. One is a carrier protein, a protein that actually binds the molecule being moved on one side of the membrane, undergoes some change in shape presumably which allows the molecule to move across the membrane and be deposited on the other side of the membrane. So that is a carrier, however there are also channel proteins which essentially form a pore in the biological membrane through which certain molecules can move and the pore, the size of the pore is going to dictate to some degree what specificity there is in moving a molecule across the membrane. The third type of transport which we will talk about in a little more detail is active transport which is also protein mediated with carriers. So again there are carriers picking up so-called picking up molecules on one side of the membrane and somehow moving them across the membrane but active transport can move molecules against the concentration gradient. So if I have a membrane like this and I have a lot of molecules of solute on this side and very few molecules on this side it is going to be difficult to move molecules against the thermodynamic concentration gradient. The molecules will naturally want to move in this direction, however cells have to do this, they have to move molecules against the gradient period and to do this they require an energy source because this is a thermodynamically unfavorable process. I want to briefly mention going from this to the different types of carrier proteins that exist that we can characterize. This is also shown in figure seventy-four, summarized it on the board. There are three types of carriers, uniport, simport and antiport and you have to remember what each of these are. Uniport is a carrier that transports one molecule across the membrane. It can be in either direction but it's one molecule at a time. A simport is transporting two molecules. It's a coupled transport. Two molecules are being transported not necessarily simultaneously but they are being transported in the same process in the same direction. So it says at the legend of this figure, which I was just reading, it says simultaneously or sequential transfer but the point is there are now two molecules being transported and they both have to go together. You can't put one in and then wait an hour and then the other one will come in. They are essentially moving together and finally an antiport transports two molecules but in this case one is going in one direction and one is going in the other direction. These types of carrier proteins are involved in both facilitated diffusion and in active transport. The channel proteins are only involved in facilitated diffusion. I just want to say a word or two about active transport and then move on. Active transport as I said involves two molecules being transported but transported in the opposite direction to each other. The classic protein which is viewed as an antiport molecule, an active transport molecule is a protein known as, it goes by various names. It is called the sodium potassium pump. It's called the sodium potassium transporter. It's called the sodium potassium ATPase. You will find all of these names used for this one protein and what this protein does is it moves sodium ions out of the cell and potassium ions into the cell and it is actually a very very important protein in maintaining the ionic balance of both of these ions within the cell. As I said it requires an energy source so if you look at figure 75 you can see that there is this compound which we will talk about in much more detail in about well probably a week, the end of next week. ATP which is sort of the biological energy source. ATP and you don't have to worry about this yet because you don't have all the information about the ATP and how it works. ATP is hydrolyzed. It's chopped up. One phosphate is removed. Energy is released through this process and then energy is somehow coupled to the movement of these ions across the membrane. Each of the ions, sodium and potassium are being moved against their concentration gradients. Now you might think the simplest thing to think visualize of this enzyme is that one sodium comes out and then there is a binding site or something on the protein. It picks up a potassium and one potassium goes in and this happens sequentially. Next time the same thing happens. The problem with that model is the stoichiometry is not one to one. Three sodiums are moved out and two sodiums are moved in. So it's a more complicated model that has to be put forth and as I said this is a very important enzyme in the membrane. I don't remember maybe Mike you remember. How much of the ATP of a cell is used by this enzyme? It's like 50% or something. An enormous amount of the ATP that a cell makes during respiration and or photosynthesis is consumed by this one enzyme. So this is really an important enzyme. You'll hear more about this enzyme later on in terms of how it functions in various physiological systems. Okay. What I briefly want to mention now is the traffic of larger molecules across membranes, the processes of exocytosis and endocytosis. We've talked a little bit about endocytosis already which is molecules being moving out of cells. If you look at figure 76 it very simply shows you that these processes which involve larger molecule transport like proteins or polysaccharides involve vesicles. Endocytosis and exocytosis involve vesicles and they involve vesicles that fuse with the membrane to take molecules in and that's what's shown in the top figure of figure 76 and endocytosis. You've got a whole bacterial cell and the membrane encloses that cell. Some of you may have seen something like this in the lab last week when you were looking at paramecium or amoeba or the classic example. The whole bacteria is surrounded by the membrane and that is taken into the cell and that new vesicle with a bacterium in it will generally fuse with the lysosome and all its contents will be degraded. Exocytosis, oh and there are three types of endocytosis. There is a phagocytosis which refers to large materials, particles, cells, there's penocytosis which refers to liquids which are encapsulated by the membrane and then there is a receptor mediated endocytosis where there are regions of the membrane that have receptors on it for specific molecules such as cholesterol. The exocytosis process is what occurs when proteins are secreted from cells such as we talked about in terms of secretory vesicles being formed from the Golgi complex moving to the plasma membrane. Again, there's a fusion of these vesicles with the membrane and the membrane is essentially opened and the material inside the vesicles is deposited outside the cell. That's all I'm going to talk about. In terms of membranes, we talked a lot about the structure of membranes, the structure of hydrophobic proteins in membranes and transport processes. I think you can see that transport is a very important problem for a cell based on the fact that these membranes have to be permeable to things coming and going so it really is important. Okay, we're going to switch gears now and at this point I always bring in, I need some volunteers. Come up here. I need at least two volunteers for this and I need two more volunteers for this. Here we go. Don't fall. I'm sorry. You didn't sign a release when you came in here that if you get injured we're not liable. Okay, so what we're going to start to talk about now is metabolism. Metabolism is basically the series of reactions that occurs in the cell that produces all the compounds the cell needs, amino acids, nucleotides, carbohydrates, lipids, and also breaks down these molecules. And these are the metabolic pathways that exist within cells. And as you can see this is, I don't want to say it's overwhelming, but it's overwhelming. Some of these are very specific pathways, for example I mentioned heme, somewhere in here it shows you how a heme is synthesized, somewhere else it shows you how vitamins are made. Fortunately for us and you guys we will primarily, if I can find it, oh this is two, we will talk about one small subset of these reactions and I can't even find them now. This looks familiar and I don't know where it is. This looks familiar. Anyway, we will talk about in some detail the process of respiration and in plants the process of photosynthesis. But we will not talk about probably 95% of the material on these two graphs, on these two schemes, but I think I can guarantee that you will have to talk about this when you take biochemistry. At least when I took biochemistry because the professor teaching in those days was a world expert on amino acids and how they were synthesized, we learned how every amino acid in the cell was synthesized and what happened to it. So you're going to go into much more detail, but we're going to limit ourselves to a couple of the key pathways in the metabolic pathways and those are related to energy production. So thank you guys for assisting. There's another one of these graph, these schemes in the GSI office on the board behind. I think it's a smaller version. Now again one of the things I just mentioned is that much of the metabolism in a cell occurs in the cytosol. Remember that. We will be talking about mitochondria and chloroplasts in particular, but a lot of the processes that I don't talk about like I don't talk about the synthesis of amino acids or nucleotides or carbohydrate polymers, those will occur in the cytosol. So the cytosol is very, very important in the overall metabolic processes of a cell. I didn't put this up, but I will. What is happening in a cell is there are macromolecules, you know what these are, which are being converted and this is a heterotrophic cell, not an autotrophic cell. You should know what that means, which are being converted to carbon dioxide and water. This is a process which produces energy. Energy is released and that energy is used for the synthesis of macromolecules, proteins, from, what do I want to call them, building elements, simple, I'll just say simpler, simple compounds, which are in this case amino acids, carbohydrates, monomeric, monosaccharides, et cetera, fats, to lipids, things like that. So we have in the cell a constant breakdown of macromolecules and a constant synthesis of macromolecules back and forth and presumably there is some reasonable balance between these two processes until maybe you get old and things start to break down. The key point here that I want to make is that there is energy involved. Energy is generated in the degradation reactions and energy is utilized in the synthetic reactions. So I spend a few minutes talking about something we call free energy, free energy changes in biological systems. I used to talk in some detail about the first and second laws of thermodynamics, but I don't think that's terribly important for the points that we want to make in terms of biology. So what I talk a bit now about is free energy and the free energy changes that occur in biological systems because we will spend some time trying to understand how much energy is available in certain reactions, what is that energy converted to, what does it mean when we say energy? What does it mean here? What is this energy that is being produced and used? So I'm going to give you a bit of the layman's presentation of free energy changes and what they mean. Here is a definition of free energy. I'm sure most of you have seen this. Free energy is a measure of useful work. The reason I don't talk about the first and second law of thermodynamics is they deal with energy changes of the world essentially, the entire system. I don't think we're interested in that. We're interested in energy changes that are occurring in cells and are occurring as specific biological reactions occur. So the free energy function, which is a measure of useful work, is much more useful to us in this context. It is a measure, free energy is equal to the basic heat content of molecules minus something that you've probably heard about, this entropy factor which is related to the disorder of the system. This is not something you have to memorize. You'll notice that I've put on these little zeros, delta G0, delta H0, delta S0. These are measurements that are made under what physical chemists call standard conditions. Standard conditions I know include pH 1 and high concentrations of the reactants. They are not physiological conditions, but the reason people use standard conditions versus non-standard conditions is everybody can then come back to the same benchmark. We would be much more interested in the free energy change under non-standard cellular conditions, but of course they're going to change. A cellular condition might be pH 6, it might be pH 8. Reactants may be 10 millimolar, 1 millimolar, 0.1 millimolar, so it's difficult to really compare those numbers. But what we have is, you'll find tables, they're large tables. Biochemists were very taken with this equation in the sort of 50s and 60s and people were measuring the free energy change for various reactions in biological systems and you will see tables where you can find delta G0 for various reactions. The free energy function is giving us a measure of the useful work out of a particular process, out of a particular reaction. When I say free energy change, I'm always talking about a specific reaction. In this case I've put up, hope you can see this, two reactions, one, both of which are very important to what we will talk about in the future. This is glucose, C6H1206, reacting with molecular oxygen to produce CO2 in water and this is one of the rare, almost somewhat balanced equations I will put up. I don't usually put up balanced equations. This is not a chemistry question. This is a reaction that releases energy. It's called exergonic for that reason. It's called catabolic degradation. Degradative reactions are called catabolic reactions and the free energy change when glucose is oxidized completely to carbon dioxide in water is minus 686 kilocalories per mole. Minus means the energy is being released. Now that number has absolutely no meaning to you. I could say it was 10 million, I could say it was 13. You wouldn't have any basis for understanding what that number means. The number will become more important as we talk about the processes of respiration, which is what this is, and photosynthesis. If I write this reaction in the opposite direction where 6 CO2s and 6 waters are being converted into glucose plus oxygen, the free energy change for that reaction is plus 686 kilocalories per mole. The directionality of the reaction does not affect the free energy change. What affects the free energy change is the nature of the reactants and the nature of the products. Now if I carry this reaction out, I'll just talk about glucose oxidation, if I carry this reaction out in a cell, a liver cell or a bacterial cell, the free energy change for the reaction will be minus 686 kilocalories per mole because I have not changed the products and the reactants. What one often sees, two often sees, when you have a free energy change which is negative, is that this is a spontaneous reaction and this is really a bad term. You'll see it in many books, spontaneous. So a negative free energy change for your reaction is a spontaneous reaction. What does that mean? Well, it implies something that doesn't occur. The reaction is not going to occur spontaneously. What it means is energy is released when it's negative. When the free energy change for a reaction is negative, there's a release of energy. That's all it means. When free energy is positive for a reaction, that means the reaction requires energy. So respiration here is releasing that much energy. Photosynthesis requires energy. Where does that energy come from? It comes from sunlight. I think everybody realizes these two reactions are probably the most basic reactions in our biological sphere, our biosphere, whereby photosynthesis is driving the synthesis of carbohydrates and producing molecular oxygen and we, the heterotrophs, are using those compounds to produce energy for our biosynthetic processes. So the free energy change for a reaction is independent of where it occurs. So I've given you the example of a cell. If I measure glucose oxidation in a cell, it will be minus 686 kilocalories. If I measure that reaction in a yeast cell, it's going to be minus 686 kilocalories. If I take my glucose and I put it in a calorimeter, a pressure bomb, essentially, and I pump that thing full of oxygen and the glucose gets burned, I'm going to release 686 kilocalories of energy. So it is independent of the particular pathway that these systems follow because E. coli, a yeast cell, a liver cell, and a calorimeter may not be carrying out the reaction, the oxidation of glucose in the same manner, but if the products are CO2 and water, you are going to release 686 kilocalories of energy. Okay. Now I come to my most favorite part of this class because I do this wonderful demonstration for you. And this demonstration, Mike, has seen this 50 times by now. This demonstration always works. It's great. You do science and it doesn't always work. So here's an experiment. I have in this envelope about 10 grams I'd say of a white powder, not dangerous. It's glucose. And I pour that glucose out on the counter here. And everybody watched the glucose because glucose is going to react with molecular oxygen and 686 kilocalories of energy is going to be produced. Right? That's what we've just learned. Let's do that away. I'm not aware that anything is happening. Okay. Unfortunately, I always hope that they'll leave the glucose on the counter till Monday. I usually end up giving this lecture on Friday and that we could come in and the glucose would still be there. But they're very efficient in the back room there. They clean the counter. They clean the boards. The glucose is, it doesn't disappear. It is scraped off the table. What's wrong here? We've got glucose. We've got oxygen. I presume there's oxygen in this room, right? And nothing is happening, I would say. And if I were to wait a very long time, nothing would happen. And so what's the problem here? The problem is that this is a thermodynamic determination. When glucose is oxidized, 686 kilocalories is released, but it doesn't give us any information on how long that process is going to take. It doesn't give us any kinetic information, kinetics. Kinetics deals with the speed of reactions. Clearly, if we were to wait eons and eons and eons, yes, glucose would be oxidized by molecular oxygen. But cells don't have eons and eons and eons of time. Cells cannot function simply by thermodynamics. Cells have to function on a kinetic basis. That is, we need a system that is going to make reactions occur rapidly. If I were to take this glucose and put it in with an E. coli cell, it would be gone in minutes or seconds. If I were to put it in a liver cell, it would be gone in minutes or seconds. So there's something that goes on in a cell that allows reactions to occur at a reasonable rate. Biological cells have things that are known as catalysts. And we would not be alive today as we are without biological catalysts. Biological catalysts. What are catalysts? Catalysts are compounds that increase the rate of a reaction without being consumed. So these, how do you spell consumed? I don't know if there's one M or two M's. So there are molecules in cells which are allowing these cells to oxidize glucose and everything else in very, very short periods of time. And they are being used over and over and over. And they're not being consumed during this process. Well, when I took biochemistry, which was way back when, I won't tell you, I won't embarrass myself by telling you when. We learned that biological catalysts are proteins and these are known as enzymes. Enzymes were the only biological catalyst known. So it was nice and simple because then we could just study proteins and we would be studying these biological catalysts. And one of the wonders of all science, particularly, I know, biology, is that you have these, I wouldn't say they're rules, you have these dogmas that are established, such as all biological catalysts are proteins. And it just takes one crummy, sneaky little experiment to destroy that concept. And this was one of the biggies because in the 1970s or so, there were people who were working with nucleic acids, in particular RNA, and they isolated or were working with an RNA that appeared to catalyze certain reactions and you'll hear more about these reactions later when Bob talks to you. And when they discovered what they thought was an activity associated with the RNA, of course nobody believed them because all biological catalysts are proteins. So, you know, one thing you do is you continue to do experiments. So there are various ways you can study this problem. You can take your preparation and, for example, I mentioned that if you heat up chloroplast, heat up proteins, they denature, they aggregate, so you take your preparation and you boil it and all the proteins will be destroyed and you find that this preparation still had activity. You treat the preparation with proteolytic enzymes, these are enzymes that chop up proteins. You work as hard as you can to convince yourself that there are no proteins in the preparations and ultimately that's what they did. And they discovered what are now known as ribosomes, ribosomes, which are catalytic RNA molecules. And you'll hear something about these when you talk about ribosomes in the next part of the class. But we can no longer say that all biological catalysts are proteins. We now say yes there are biological catalysts, some of them are proteins, the enzymes, some of them are ribosomes. This work was done by Tom Cech who was a postdoc here and now I think is still at the University of Colorado and Sidney Altman who I think is still at Yale and guess what? They got a Nobel Prize for this work because it really revolutionized their biology. So there's a lesson here. If you ever go into a research lab and somebody tells you, well we know this is this, maybe it is and maybe it isn't. And the beauty of science is you can do experiments. If you can't do experiments, you're not doing science. Okay, so what are enzymes and how do they work? That's what we're going to be talking about for the next 10 or 15 minutes and we will be talking about mostly next week because enzymes are, I think they're the most important components in cells because what they do is they make reactions occur at reasonable rates, reasonable for the overall life of the organism. So they are pretty important compounds. Fortunately, these fortunately for people who work in this field, we have an enormous amount of information on enzymes structure, enzyme activity and enzyme function. I think this is one of the modern major advances of modern biochemistry since the 1950s to 2000, the last 50 years, that we really understand how enzymes work in some detail. We have fortunately an enormous amount of structural information. Our advancing knowledge has benefited from the ability to crystallize enzymes, study their structure and understand what they're doing. Okay, so we have a very classic reaction that we'll talk about, AB plus CD. These are two reactants which are converted to AC plus BD. If we look at this reaction in a funny kind of way, what we find if we plot here the progress of the reaction versus the free energy change for the reaction. This is a classic picture which is in your book and is all over. So we start off at some free energy level where we have AB and CD and the reaction will proceed and you have to put some energy into the system. Every reaction requires an input of energy to get the molecule, the bonds stretched, twisted or whatever, so there's an activation energy that is required and once that activation energy is attained then you can convert these compounds into products. So we've got up here, the way I have done it is I've got A, B, C, D. In other words, we have altered these bonds by the absorption of energy from the environment and then these bonds are converted so that at the end of this reaction we have AC and BD. I don't want to make any mistakes. This energy here is the activation energy that had to be initially put into the system. The difference between this starting point and this ending point, this represents the free energy change for this reaction, the delta G zero. This reaction has a negative free energy change, that is energy is being released as these two compounds are being formed. I could draw this in a different way, I could draw it like this, actually you should think about it. Think about what this profile would look like if the reaction had a positive free energy change. This is the uncatalyzed reaction. If I add an enzyme or a catalyst to this reaction, what happens is it does not affect the starting material or the products. So I still have A, B, C, D and at the end I'm still going to have AC, B, D. So we start here but what one finds is that, I'm probably exaggerating this but that's okay to make the point, that the activation energy for the process drops and I think you should be able to see that if I have two processes and they both have, are going to end up with the same products that the yellow line is going to occur faster than the white line because it's a shorter route essentially. So what a catalyst does, what an enzyme does is it lowers the activation energy for the reaction. It does not affect the starting material, it does not affect the energetics of the overall reaction. In the words delta G is still the same. If this represents minus 10 kilocalories, I start off here, I end up there, I have minus 10 kilocalories released in both processes so the catalyst is not affecting the free energy change for the reaction but it is making the reaction faster by lowering the activation energy of the process. And again the catalyst, this is plus catalyst, cannot be consumed in the process, that does not mean it's not altered, this is a point that people often are confused with. When I talk about the mechanism of this process you'll see the catalyst which now I mean as we'll say enzyme, the enzyme of course is going to interact with these compounds and is going to undergo changes that allow this reaction to occur but at the end the enzyme is going to go back to a starting point from which it came so that it can do it over and over and over. So what we have to talk about and we're going to talk about in some detail is how do enzymes work. Well first thing I'd like to mention is this thing about how we name enzymes and unfortunately this is a little awkward because enzymes are named in a very unsystematic manner. You will generally see the term aces, something ace, that's an enzyme. And for example I've got a big table here, I'm not going to put it all up. You have enzymes that are hydrolysis such as the enzymes in lysosomes, they hydrolyze bonds, they add water across a bond, okay? Nucleases, these are enzymes involved in the breakdown of nucleic acids. Proteases, these are enzymes involved in the breakdown of proteins. Synthases are synthesizing things, isomerases are isomerizing things. One group of enzymes that we will talk about are known as kinases and these are enzymes that are involved in putting a phosphate group on a substrate or on a protein. Phosphatases we'll talk about, these are enzymes that are involved in taking off phosphates from proteins. So you know there's no sort of grand order to order all of this. These are just the names of enzymes and they are trivial names but they are historically still used. When we talk about glycolysis for example, there are ten steps in glycolysis. Each enzyme has a very specific name and function and they're the old names. Hexokinase is the first enzyme in glycolysis. It means it's involved with a hexose and it's involved in some way with a phosphate group, putting it maybe on a sugar. So you've sort of got to, you just have to come to grips with this. This is the way enzymes are named. What enzymes do is they react with a compound which is known as the substrate. So you have a substrate and you have an enzyme. That's beautiful. And the enzyme will convert the substrate into products. That's pretty simple. It's not quite that simple in life. For example, there is table sugar, sucrose, our old disaccharide friend which in the presence of an enzyme known as sucrase produces fructose plus glucose. Actually this reaction has a free energy change just to come back to this point that I saw here before. Delta G zero of minus seven kilocalories per mole. You know that if you take sucrose and you put it in water it doesn't break down to fructose and glucose. It's sucrose. And that's again, that's the issue that I was mentioning that the kinetic component is very important and in many cases overrides the importance of the free energy change for this reaction. Here's a reaction that you're going to do next week in lab. Starch with an enzyme known as amylase producing maltose. So you're going to all become enzymologists next week because you're going to study this particular enzyme, its properties, but what it's doing is taking starch which is a carbohydrate polymer or polysaccharide and breaking it down. Not all the way to glucose. Maltose is a disaccharide. That's as far as it goes. Enzymes are fairly specific. The reason we have so many different classes of enzymes is they have a specificity to them. An enzyme that reacts with a carbohydrate will not react with an amino acid. An enzyme that reacts with a phosphate will not react with a sulfate. So there is an enormous specificity that exists which means there are lots and lots of enzymes. The enzyme that breaks down sucrose to fructose and glucose will probably not break down lactose which is another disaccharide or will not break down maltose. And that's because, and I'll say this and then I'll stop, there are specific interactions between the substrate, sucrose, and the enzyme sucrose binding which allows that interaction to occur and other compounds which may look a little bit like sucrose are sufficiently different so sucrose will not react with those compounds. I think it's a good place to stop. On Monday I'm going to talk about the details of how enzymes work and how their activity is regulated.