Outline for Lecture #5 C2006/F2402 '14 OUTLINE FOR LECTURE #5 Last updated 02/03/14 05:49 PM (c) 2014 Dr. Deborah Mowshowitz , Columbia University, New York, NY Handouts: 5A-- Measurement & Classification of Transport* 5B -- Models for Active Transport *Handout 5A is in the CW handout folder -- accessible to registered students via the left menu on the Courseworks (CW) web site. All handouts with copyrighted material are in this folder. (Extra paper copies available after lecture in Boxes on 7th floor of Mudd.) I. Review of Classification of types of transport. See handout 4C, Sadava table 6.1, or Becker table 8-1. Note: The terms used for the various types of transport are variable, and you will see different terms used in different sources. The features of each type (1-5 on handout 4C) are more important than the names. II. How is Transport of Small Molecules Measured? A. Need a suitable experimental set up. A common method: using RBC ghosts. How is it done? Put resealed ghosts in solution with some concentration of X on the outside and none on the inside. At appropriate time intervals, you take a sample, centrifuge out the ghosts, and measure the amount of X in the ghosts. You repeat with different starting concentrations of [X]out B. What do you learn from doing this? You can tell active transport from passive. You can tell if a carrier or pump protein is required for transport (as vs. none or a channel). You can measure the properties of the transport protein (if any) -- equivalents of Km and Vmax.. C. Details of experimental set up -- See handout 5A. Put ghosts in solution with some concentration of X inside and outside: Co = concentration outside = [X]out ( [X]out = some fixed value to start) Ci = concentration inside = [X]in ( usually [X]in = 0 to start) You measure Ci as a function of time. This generates 'curve #1' -- the top set of three curves on 5A. You repeat with different starting values of Co. This generates the data plotted in 'curve #2' -- the second set of three curves on 5A. file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect5.14.html[2/3/2014 6:56:06 PM] Outline for Lecture #5 D. How to check your understanding? Be sure you can answer the following: In terms of types of transport, which cases on Handout 4C does this procedure allow you to tell apart? Which set of curves on handout 5A will give you the information you need? How do you get the 'Km and Vmax'? (Details of each case & meaning of each curve are discussed in detail below.) III. What do Results of Measurement Look Like? What do they mean? A. Curve # 1 -- Uptake of X vs time: Measure [X]in at increasing times at some starting, outside concentration of X; plot conc. of X inside vs. time. Curve always levels off -- but at what value? This allows you to distinguish active and passive transport. 1. For active transport of neutral molecules, at equilibrium, [X]in will exceed [X]out. 2. For passive transport of neutral molecules, at equilibrium, [X]in will equal [X]out. Notes: (1) If X is charged, the situation is more complicated, as explained below. (2) Concentration of X outside is essentially fixed and is the same as the starting concentration outside (Co). This is because the amount taken up is relatively small. Why? Because the volume inside the cells is much smaller than the volume outside the cells. Questions: If you measure carrier-mediated uptake a second time, using a higher starting concentration of X, what will happen? (1) Will the slope of curve #1 (flux) be the same? (2) Will the curve level off at the same value? B. Curve #2 -- Uptake of X vs concentration: Measure initial rate of uptake of X (from curve #1) at varying concentrations of added (outside) X; plot rate of uptake (flux) vs. initial concentration of [X]out. See handout or Becker fig. 8-5 (8-6). This allows you to find out what sort of protein (if any) is involved in transport. 1. If an enzyme-like protein (carrier or pump) is involved in transport, curve will be hyperbolic -- carrier or pump protein will saturate at high [X] just as an enzyme does. Why? If [X] is high enough, all protein molecules will be "busy" or engaged, and transport reaches a max. value. Adding more X won't increase the rate of transport. (Same as reaching Vmax with a V vs [S] curve for an enzyme.) 2. If no protein, or a channel-like protein, is involved in transport, curve will be linear (at physiological, that is reasonable, concentrations of X.). There is no time consuming event such as the binding of X or a major conformational change in the protein that limits the rate of the reaction at high [X]. Note: for a channel the curve will saturate at extremely high levels of X. These saturating levels are not usually reached in practice. C. Summary Comparison of Curve #1 vs Curve #2. For both curves, you are considering the reaction Xout ↔ Xin. So what's the big difference? (Most of this should be discussed as we go along, but is summarized here for reference.) 1. In Curve #1, you are looking at how the concentration of Xin varies with time (starting with a fixed concentration of Xout, and no X inside.). a. Curve shows uptake as a function of time. b. (Initial) Slope of the curve = rate of uptake (with time as the variable) | c. Plateau value = yield = final value of [X]in when curve #1 levels off (when rate in = rate file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect5.14.html[2/3/2014 6:56:06 PM] Outline for Lecture #5 out). d. Note that curve #1 ALWAYS levels off. e. Same idea as plotting P formed (or S used up) vs time for an enzyme catalyzed reaction. 2. In Curve #2, you are looking at how the rate of uptake (flux) -- initial slope of curve #1 -- varies for different starting concentrations of [X]out. a. Curve shows uptake as a function of concentration of X added (outside). b. (Initial) Slope of the curve = rate of change of uptake (with [X]out as the variable) = flux c. This curve levels off * only if a protein must bind to X and/or change conformation significantly in order to move X. d. Same idea as a plotting V vs S for an enzyme-catalyzed reaction. Gives you the properties of the transport protein. *at physiological values of [X]out IV. Kinetics and Properties of each type of Transport -- How you tell the cases apart. All the cases below refer to the reaction [X]in ↔ [X]out. All the important features are summarized in the table on handout 5A. A. Simple Diffusion (Case 1) 1. Curve #1 (concentration of substance X inside plotted vs. time) plateaus at [X]in = [X]out. 2. Curve #2 (rate of uptake of X plotted vs concentration of X added outside) does not saturate. 3. Energy: a. Reversibility: Rxn ( X in ↔ X out) is strictly reversible. o b. Keq = 1; Standard free energy change (ΔG ) = 0; at equil. [X]in = [X]out c. ΔG. Actual free energy change (ΔG) and direction of transport depends on concentration of X. If [X] is higher outside, X will go in and vice versa. 4. Importance. Diffusion across a membrane: Used by steroid hormones, some small molecules, gases. Only things that are very small or nonpolar can use this mechanism to cross membranes. Diffusion through liquid (no membrane involved): Materials -- usually small molecules -- can diffuse in or out of capillaries by diffusing through the liquid in the spaces between the cells. (The cells surrounding capillaries do not have tight junctions, except in the brain.) More on this next time. B. Carrier mediated Transport = Facilitated Diffusion using a carrier protein (Case 3). Note we are deferring case 2. file:///C|/Users/dbm2/Documents/COURSES/C2006/current-lectures14/lect5.14.html[2/3/2014 6:56:06 PM] Outline for Lecture #5 1. Curve #1 same as above (case 1) 2. Curve #2 saturates. See Becker fig. 8-5 (8-6), or Sadava fig. 6.12 (6.14) 3. Mechanism: Carrier acts like enzyme or permease, with Vmax, Km etc. Carrier can be considered an enzyme (permease) that catalyzes: Xout ↔ Xin Carrier is specific, just like an enzyme. Will only catalyze movement of X and closely related compounds. 4. Energy as above (case 1) -- substance flows down its gradient, so transport is reversible, depending on relative concentrations in and out. 5. Example -- GLUT family of proteins. Transport of glucose across a membrane (down its gradient) requires a GLUT (glucose transporter/carrier) protein. For mech. of action, see Becker fig. 8-7 (8-8). a. Role of GLUTs: Glucose enters and/or exits most cell using a GLUT protein. b. Different cell types make different GLUT proteins. Proteins are called GLUT1, GLUT2, GLUT4 etc. (1). RBC contain GLUT1 (See Becker fig. 8-2) (2). Liver cells contain GLUT2 (3). Muscle and adipose tissue contain GLUT4 c. Direction: Which way the glucose goes, in or out of the cell, depends on the relative concentrations of glucose on the two sides of the membrane, not on the GLUT protein present. An analogy: a revolving door. Some molecular examples: (1). GLUT1 & GLUT4 transport glucose into their respective cells (in vivo). (2). GLUT2 in liver cells can transport glucose in or out (in vivo), depending on the level of glucose in the blood.