Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

● A portion of the structure of heparin, a carbohydrate having anticoagulant properties. It is used by blood banks to prevent the clotting of blood during donation and storage and also by physicians to prevent the formation of life-threatening blood clots in patients recovering from serious injury or surgery. This sulfated pentasaccharide sequence in heparin binds with high affinity to antithrombin III, accounting for this anticoagulant activity. The 3-O-sulfate marked by an asterisk is essential for high-affinity binding of heparin to antithrombin III.

Many of the functions of proteoglycans involve the binding of specific proteins to the glycosaminoglycan groups of the proteoglycan. The glycosaminoglycan binding sites on these specific proteins contain multiple basic amino acid residues. The amino acid sequences BBXB and BBBXXB (where B is a basic amino acid and X is any amino acid) recur repeatedly in these binding domains. Basic amino acids such as lysine and arginine provide charge neutralization for the negative charges of glycosaminoglycan residues, and in many cases, the binding of extracellular matrix proteins to glycosaminoglycans is primarily charge-dependent. For example, more highly sulfated glycosaminoglycans bind more tightly to fibronectin. Certain protein–glycosaminoglycan interactions, however, require a specific carbohydrate sequence. A particular pentasaccharide sequence in heparin, for example, binds tightly to antithrombin III (Figure 9.33), accounting for the anticoagulant properties of heparin. Other glycosaminoglycans interact much more weakly.

Proteoglycans May Modulate Cell Growth Processes

Several lines of evidence raise the possibility of modulation or regulation of cell growth processes by proteoglycans. First, heparin and heparan sulfate are known to inhibit cell proliferation in a process involving internalization of the glycosaminoglycan moiety and its migration to the cell nucleus. Second, fibroblast growth factor binds tightly to heparin and other glycosaminoglycans, and the heparin–growth factor complex protects the growth factor from degradative enzymes, thus enhancing its activity. There is evidence that binding of fibroblast growth factors by proteoglycans and glycosaminoglycans in the extracellular matrix creates a reservoir of growth factors for cells to use. Third, transforming growth factor has been shown to stimulate the synthesis and secretion of proteoglycans in certain cells. Fourth, several proteoglycan core proteins, including versican and lymphocyte homing receptor, have domains similar in sequence to epidermal growth factor and complement regulatory factor. These growth factor domains may interact specifically with growth factor receptors in the cell membrane in processes that are not yet understood.

Proteoglycans Make Cartilage Flexible and Resilient

Cartilage matrix proteoglycan is responsible for the flexibility and resilience of cartilage tissue in the body. In cartilage, long filaments of hyaluronic acid are studded or coated with proteoglycan molecules, as shown in Figure 9.34. The hyaluronate chains can be as long as 4 m and can coordinate 100 or more proteoglycan units. Cartilage proteoglycan possesses a hyaluronic acid binding domain on the NH2-terminal portion of the polypeptide, which binds to hyaluronate with the assistance of a link protein. The proteoglycan–hyaluronate aggregates can have molecular weights of 2 million or more.

The proteoglycan–hyaluronate aggregates are highly hydrated by virtue of strong interactions between water molecules and the polyanionic complex.

Proteoglycan

O-linked oligosaccharides

SerSer

Carboxylate

group

Core protein

Link protein

Hyaluronic acid

Core protein

● Hyaluronate (see Figure 7.33) forms the backbone of proteoglycan structures, such as those found in cartilage. The proteoglycan subunits consist of a core protein containing numerous O-linked and N- linked glycosaminoglycans. In cartilage, these highly hydrated proteoglycan structures are enmeshed in a network of collagen fibers. Release (and subsequent reabsorption) of water by these structures during compression accounts for the shock-absorbing qualities of cartilaginous tissue.

294 Chapter 9 ● Membranes and Cell Surfaces

PROBLEMS

1.In Problem 1(b) in chapter 8 (page 257) you were asked to draw all the phosphatidylserine isomers that can be formed from palmitic and linolenic acids. Which of the PS isomers are not likely to be found in biological membranes?

2.The purple patches of the Halobacterium halobium membrane, which contain the protein bacteriorhodopsin, are approximately 75% protein and 25% lipid. If the protein molecular weight is 26,000 and an average phospholipid has a molecular weight of 800, calculate the phospholipid to protein mole ratio.

3.Sucrose gradients for separation of membrane proteins must be able to separate proteins and protein–lipid complexes having a wide range of densities, typically 1.00 to 1.35 g/mL.

a. Consult reference books (such as the CRC Handbook of Biochemistry) and plot the density of sucrose solutions versus percent sucrose by weight (g sucrose per 100 g solution), and versus percent by volume (g sucrose per 100 mL solution). Why is one plot linear and the other plot curved?

b. What would be a suitable range of sucrose concentrations for separation of three membrane-derived protein–lipid complexes with densities of 1.03, 1.07, and 1.08 g/mL?

4.Phospholipid lateral motion in membranes is characterized by

FURTHER READING

Aspinall, G. O., 1982. The Polysaccharides, Vols. 1 and 2. New York: Academic Press.

Bennett, V., 1985. The membrane skeleton of human erythrocytes and its implications for more complex cells. Annual Review of Biochemistry 54:273–304.

Bretscher, M., 1985. The molecules of the cell membrane. Scientific American 253:100–108.

Collins, P. M., 1987. Carbohydrates. London: Chapman and Hall.

Davison, E. A., 1967. Carbohydrate Chemistry. New York: Holt, Rinehart and Winston.

Dawidowicz, E. A., 1987. Dynamics of membrane lipid metabolism and turnover. Annual Review of Biochemistry 56:43–61.

Doering, T. L., Masterson, W. J., Hart, G. W., and Englund, P. T., 1990. Biosynthesis of glycosyl phosphatidylinositol membrane anchors. Journal of Biological Chemistry 265:611–614.

Fasman, G. D., and Gilbert, W. A., 1990. The prediction of transmembrane protein sequences and their conformation: An evaluation. Trends in Biochemical Sciences 15:89–92.

Feeney, R. E., Burcham, T. S., and Yeh, Y., 1986. Antifreeze glycoproteins from polar fish blood. Annual Review of Biophysical Chemistry 15:59–78.

Frye, C. D., and Edidin, M., 1970. The rapid intermixing of cell surface antigens after formation of mouse–human heterokaryons. Journal of Cell Science 7:319–335.

Gelb, M. H., 1997. Protein prenylation, et cetera: Signal transduction in two dimensions. Science 275:1750–1751.

Glomset, J. A., Gelb, M. H., and Farnsworth, C. C., 1990. Prenyl proteins in eukaryotic cells: A new type of membrane anchor. Trends in Biochemical Sciences 15:139–142.

a diffusion coefficient of about 1 10 8 cm2/sec. The distance traveled in two dimensions (across the membrane) in a given time is r (4Dt)1/2, where r is the distance traveled in centimeters, D is the diffusion coefficient, and t is the time during which diffusion occurs. Calculate the distance traveled by a phospholipid across a bilayer in 10 msec (milliseconds).

5.Protein lateral motion is much slower than that of lipids because proteins are larger than lipids. Also, some membrane proteins can diffuse freely through the membrane, whereas others are bound or anchored to other protein structures in the membrane. The dif-

fusion constant for the membrane protein fibronectin is approximately 0.7 10 12 cm2/sec, whereas that for rhodopsin is about

310 9 cm2/sec.

a. Calculate the distance traversed by each of these proteins in 10 msec.

b. What could you surmise about the interactions of these proteins with other membrane components?

6.Discuss the effects on the lipid phase transition of pure dimyristoyl phosphatidylcholine vesicles of added (a) divalent cations, (b) cholesterol, (c) distearoyl phosphatidylserine, (d) dioleoyl phosphatidylcholine, and (e) integral membrane proteins.

Gordon, J. I., Duronio, R. J., Rudnick, D. A., Adams, S. P., and Gokel, G. W., 1991. Protein N-myristoylation. Journal of Biological Chemistry 266: 8647–8650.

Jain, M. K., 1988. Introduction to Biological Membranes, 2nd ed. New York: John Wiley & Sons.

Jennings, M. L., 1989. Topography of membrane proteins. Annual Review of Biochemistry 58:999–1027.

Jentoft, N., 1990. Why are proteins O-glycosylated? Trends in Biochemical Sciences 15:291–294.

Kjellen, L., and Lindahl, U., 1991. Proteoglycans: Structures and interactions. Annual Review of Biochemistry 60:443–475.

Knowles, B. H., Blatt, M. R., Tester, M., et al., 1989. A cytosolic -endo- toxin from Bacillus thurigiensis var. israelensis forms cation-selective channels in planar lipid bilayers. FEBS Letters 244:259–262.

Koblan, K. S., Kohl, N. E., Omer, C. A., et al., 1996. Farnesyltransferase inhibitors: A new class of cancer chemotherapeutics. Biochemical Society Transactions 24:688–692.

Lasky, L. A., 1995. Selectin–carbohydrate interactions and the initiation of the inflammatory response. Annual Review of Biochemistry 64:113–139.

Lennarz, W. J., 1980. The Biochemistry of Glycoproteins and Proteoglycans. New York: Plenum Press.

Lodish, H. F., 1991. Recognition of complex oligosaccharides by the multisubunit asialoglycoprotein receptor. Trends in Biochemical Sciences 16:374 –377.

Marchesi, V. T., 1984. Structure and function of the erythrocyte membrane skeleton. Progress in Clinical Biology Research 159:1–12.

Marchesi, V. T., 1985. Stabilizing infrastructure of cell membranes.

Annual Review of Cell Biology 1:531–561.

McNeil, M., Darvill, A. G., Fry, S. C., and Albersheim, P., 1984. Structure and function of the primary cell walls of plants. Annual Review of Biochemistry 53:625–664.

Op den Kamp, J. A. F., 1979. Lipid asymmetry in membranes. Annual Review of Biochemistry 48:47–71.

Park, H-W., Boduluri, S. R., Moomaw, J. F., et al., 1997. Crystal structure of protein farnesyltransferase at 2.25 Angstrom resolution. Science 275: 1800–1804.

Pigman, W., and Horton, D., 1972. The Carbohydrates. New York: Academic Press.

Rademacher, T. W., Parekh, R. B., and Dwek, R. A., 1988. Glycobiology.

Annual Review of Biochemistry 57:785–838.

Robertson, R. N., 1983. The Lively Membranes. Cambridge: Cambridge University Press.

Ruoslahti, E., 1989. Proteoglycans in cell regulation. Journal of Biological Chemistry 264:13369–13372.

Seelig, J., and Seelig, A., 1981. Lipid conformation in model membranes and biological membranes. Quarterly Review of Biophysics 13:19–61.

Sefton, B., and Buss, J. E., 1987. The covalent modification of eukaryotic proteins with lipid. Journal of Cell Biology 104:1449–1453.

Sharon, N., 1980. Carbohydrates. Scientific American 243:90–102.

Sharon, N., 1984. Glycoproteins. Trends in Biochemical Sciences 9:198–202.

Singer, S. J., and Nicolson, G. L., 1972. The fluid mosaic model of the structure of cell membranes. Science 175:720–731.

Singer, S. J., and Yaffe, M. P., 1990. Embedded or not? Hydrophobic sequences and membranes. Trends in Biochemical Sciences 15:369–373.

Tanford, C., 1980. The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed. New York: Wiley-Interscience.

Towler, D. A., Gordon, J. I., Adams, S. P., and Glaser, L., 1988. The biology and enzymology of eukaryotic protein acylation. Annual Review of Biochemistry 57:69–99.

Unwin, N., and Henderson, R., 1984. The structure of proteins in biological membranes. Scientific American 250:78–94.

Wirtz, K. W. A., 1991. Phospholipid transfer proteins. Annual Review of Biochemistry 60:73–99.

It takes a membrane to make sense out of disorder in biology. You have to be able to catch energy and hold it, storing precisely the needed amount and releasing it in measured shares. A cell does this, and so do the organelles inside. . . . To stay alive, you have to be able to hold out against equilibrium, maintain imbalance, bank against entropy, and you can only transact this business with membranes in our kind of world.

LEWIS THOMAS, “The World’s Biggest

Membrane,” The Lives of a Cell (1974)

OUTLINE

296

Chapter 10

Membrane Transport

“Drawbridge at Arles with a Group of Washerwomen” (1888) by Vincent van Gogh

(Rikjsmuseum Kroller-Muller; photo by Erich Lessing/Art Resource)

Transport processes are vitally important to all life forms because all cells must exchange materials with their environment. Cells must obviously have ways to bring nutrient molecules into the cell and ways to send waste products and toxic substances out. Also, inorganic electrolytes must be able to pass in and out of cells and across organelle membranes. All cells maintain concentration gradients of various metabolites across their plasma membranes and also across the membranes of intracellular organelles. By their very nature, cells maintain a very large amount of potential energy in the form of such concentration gradients. Sodium and potassium ion gradients across the plasma membrane mediate the transmission of nerve impulses and the normal functions of the brain, heart, kidneys, and liver, among other organs. Storage and release of calcium from cellular compartments controls muscle contraction, and also the response of many cells to hormonal signals. High acid concentrations in the stomach are required for the digestion of food. Extremely high hydrogen ion gradients

are maintained across the plasma membranes of the mucosal cells lining the stomach in order to maintain high acid levels in the stomach yet protect the cells that constitute the stomach walls from the deleterious effects of such acid.

In this chapter, we shall consider the molecules and mechanisms that mediate these transport activities. In nearly every case, the molecule or ion transported is water-soluble, yet moves across the hydrophobic, impermeable lipid membrane at a rate high enough to serve the metabolic and physiologic needs of the cell. This perplexing problem is solved in each case by a specific transport protein. The transported species either diffuses through a channel-form- ing protein or is carried by a carrier protein. Transport proteins are all classed as integral membrane proteins (Chapter 9), ranging in size from small peptides to large, multisubunit protein complexes.

Some transport proteins merely provide a path for the transported species, whereas others couple an enzymatic reaction with the transport event. In all cases, transport behavior depends on the interactions of the transport protein not only with solvent water but with the lipid milieu of the membrane as well. The dynamic and asymmetric nature of the membrane and its components (Chapter 9) plays an important part in the function of these transport systems.

From a thermodynamic and kinetic perspective, there are only three types of membrane transport processes: passive diffusion, facilitated diffusion, and active transport. To be thoroughly appreciated, membrane transport phenomena must be considered in terms of thermodynamics. Some of the important kinetic considerations also will be discussed.

10.1 ● Passive Diffusion

Passive diffusion is the simplest transport process. In passive diffusion, the transported species moves across the membrane in the thermodynamically favored direction without the help of any specific transport system/molecule. For an uncharged molecule, passive diffusion is an entropic process, in which movement of molecules across the membrane proceeds until the concentration of the substance on both sides of the membrane is the same. For an uncharged molecule, the free energy difference between side 1 and side 2 of a membrane (Figure 10.1) is given by

The difference in concentrations, [C2] [C1], is termed the concentration gradient, and G here is the chemical potential difference.

Passive Diffusion of a Charged Species

For a charged species, the situation is slightly more complicated. In this case, the movement of a molecule across a membrane depends on its electrochemical potential. This is given by

where Z is the charge on the transported species, is Faraday’s constant (the charge on 1 mole of electrons 96,485 coulombs/mol 96,485 joules/volt • mol, because 1 volt 1 joule/coulomb), and is the electric potential difference (that is, voltage difference) across the membrane. The second term in the expression thus accounts for the movement of a charge across a potential

Concentration C1 Concentration C2

∆ G = RT ln [C2] [C1]

FIGURE 10.1 ● Passive diffusion of an uncharged species across a membrane depends only on the concentrations (C1 and C2) on the two sides of the membrane.

298 Chapter 10 ● Membrane Transport

Z ∆Ψ < 0

FIGURE 10.2 ● The passive diffusion of a charged species across a membrane depends upon the concentration and also on the charge of the particle, Z, and the electrical potential difference across the membrane, .

Facilitated diffusion

υ

Passive diffusion

S

FIGURE 10.3 ● Passive diffusion and facilitated diffusion may be distinguished graphically. The plots for facilitated diffusion are similar to plots of enzyme-catalyzed processes (Chapter 14) and they display saturation behavior.

difference. Note that the effect of this second term on G depends on the magnitude and the sign of both Z and . For example, as shown in Figure 10.2, if side 2 has a higher potential than side 1 (so that is positive), for a negatively charged ion the term Z makes a negative contribution to G.

In other words, the negative charge is spontaneously attracted to the more positive potential—and G is negative. In any case, if the sum of the two terms on the right side of Equation 10.2 is a negative number, transport of the ion in question from side 1 to side 2 would occur spontaneously. The driving force for passive transport is the G term for the transported species itself.

10.2 ● Facilitated Diffusion

The transport of many substances across simple lipid bilayer membranes via passive diffusion is far too slow to sustain life processes. On the other hand, the transport rates for many ions and small molecules across actual biological membranes is much higher than anticipated from passive diffusion alone. This difference is due to specific proteins in the membrane that facilitate transport of these species across the membrane. Similar proteins capable of effecting facilitated diffusion of a variety of solutes are present in essentially all natural membranes. Such proteins have two features in common: (a) they facilitate net movement of solutes only in the thermodynamically favored direction (that is,G 0), and (b) they display a measurable affinity and specificity for the transported solute. Consequently, facilitated diffusion rates display saturation behavior similar to that observed with substrate binding by enzymes (Chapter 14). Such behavior provides a simple means for distinguishing between passive diffusion and facilitated diffusion experimentally. The dependence of transport rate on solute concentration takes the form of a rectangular hyperbola (Figure 10.3), so that the transport rate approaches a limiting value, Vmax, at very high solute concentration. Figure 10.3 also shows the graphical behavior exhibited by simple passive diffusion. Because passive diffusion does not involve formation of a specific solute:protein complex, the plot of rate versus concentration is linear, not hyperbolic.

Glucose Transport in Erythrocytes Occurs by Facilitated Diffusion

Many transport processes in a variety of cells occur by facilitated diffusion. Table 10.1 lists just a few of these. The glucose transporter of erythrocytes illustrates many of the important features of facilitated transport systems. Although glucose transport operates variously by passive diffusion, facilitated diffusion, or active transport mechanisms, depending on the particular cell, the glucose transport system of erythrocytes (red blood cells) operates exclusively by facilitated diffusion. The erythrocyte glucose transporter has a molecular mass of approximately 55 kD and is found on SDS polyacrylamide electrophoresis gels (Figure 10.4) as band 4.5. Typical erythrocytes contain around 500,000 copies of this protein. The active form of this transport protein in the erythrocyte membrane is a trimer. Hydropathy analysis of the amino acid sequence of the erythrocyte glucose transporter has provided a model for the structure of the protein (Figure 10.5). In this model, the protein spans the membrane 12 times, with both the N- and C-termini located on the cytoplasmic side. Transmembrane segments M7, M8, and M11 comprise a hydrophilic transmembrane channel, with segments M9 and M10 forming a relatively hydrophobic pocket adjacent to the glucose-binding site. Cytochalasin B, a fungal metabolite (Figure 10.6), is a competitive inhibitor of glucose transport. The mechanism of glucose transport is not well understood. An alternating conformation model, in

● A model for the arrangement of the glucose transport protein in the erythrocyte membrane. Hydropathy analysis is consistent with 12 transmembrane helical segments.

+

H3N

Inside

Helices 9 and 10 form

a hydrophobic pocket

COO

CH2

HN O

OH

OO

●The structure of cytocha-

The Anion Transporter of Erythrocytes Also

Operates by Facilitated Diffusion

The anion transport system is another facilitated diffusion system of the erythrocyte membrane. Chloride and bicarbonate (HCO3 ) ions are exchanged across the red cell membrane by a 95-kD transmembrane protein. This protein is abundant in the red cell membrane and is represented by band 3 on SDS electrophoresis gels (Figure 10.4). The gene for the human erythrocyte anion transporter has been sequenced and hydropathy analysis has yielded a model for the arrangement of the protein in the red cell membrane (Figure 10.7). The model has 14 transmembrane segments, and the sequence includes 3 regions: a hydrophilic, cytoplasmic domain (residues 1 through 403) that interacts with numerous cytoplasmic and membrane proteins; a hydrophobic domain (residues 404 through 882) that comprises the anion transporting channel; and an acidic, C-terminal domain (residues 883 through 911). This transport system facilitates a one-for-one exchange of chloride and bicarbonate, so that the net transport process is electrically neutral. The net direction of anion flow through this protein depends on the sum of the chloride and bicarbonate concentration gradients. Typically, carbon dioxide is collected

then carried in the blood to the lungs, where bicarbonate diffuses out of the erythrocytes in exchange for Cl ions.