LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOLUME 1. THE BASIS OF BIOCHEMISTRY, STRUCTURE, AND CATALYSIS - 2011

PART I. STRUCTURE AND CATALYSIS

11. BIOLOGICAL MEMBRANES AND TRANSPORT

Questions and Problems

Determination of the cross-sectional area of a lipid molecule.

When Phospholipids are carefully layered onto the Water surface, they orient themselves at the water-air interface such that their polar "HEAD groups" face the water, while their hydrophobic "tails" point toward the air. An apparatus (a) was designed to reduce the surface area accessible to the lipid layer. By measuring the force required to compress the Lipids together, it is possible to determine when the molecules are tightly packed into a continuous monolayer. Once this state is reached, the force required to further decrease the surface area increases sharply (b). How would you use this apparatus to determine the average area occupied by a single lipid molecule within the monolayer?

Evidence supporting the existence of Cell/29.html">The Lipid Bilayer.

In 1925, E. Gorter and F. Grendel used an apparatus similar to the one described in Problem 1 to determine the surface area of a lipid monolayer formed by lipids extracted from the erythrocytes of various animal species. They used a Microscope to measure the dimensions of individual Cells and calculated the average surface area of a single erythrocyte. Their findings are summarized in the table. Were these researchers justified in concluding that "chromocytes (erythrocytes) are covered by a layer of fatty substances that is two molecules thick" (i.e., a lipid bilayer)?

	Volume of packed cells	Number of cells	Total surface area of the lipid monolayer	Total surface area of a single cell
Animals	(mL)	(per mm3)	of cells (m2)	(µm2)
Dog	40	8,000,000	62	98
Sheep	10	9,900,000	6.0	29.8
Human	1	4,740,000	0.92	99.4

Number of detergent molecules in a micelle.

When a small amount of sodium dodecyl sulfate (SDS; Na⁺CH₃(CH₂)₁₁OSO₃^-) is dissolved in water, the detergent ions enter the solution as monomeric species. Upon further addition of detergent, a threshold concentration is reached (the critical micelle concentration) at which monomers associate to form micelles. The critical micelle concentration of SDS is 8.2 mM. Micelles have an average particle molecular weight (the sum of the molecular weights of the constituent monomers) of ~18,000. Calculate the average number of detergent molecules in a micelle.

Properties of lipids and lipid bilayers.

Lipid bilayers formed between two aqueous phases possess a remarkable property: they form two-dimensional sheets whose edges are kept very close to one another, and they undergo self-sealing to form Liposomes. (a) What lipid properties account for this characteristic of bilayers? Explain, (b) What are the implications of this property for The Structure of Introduction/36.html">Biological Membranes?

Length of a fatty acid molecule.

The carbon-carbon bond distance for a single bond (as in the acyl chain of a saturated fatty acid) is 1.5 Å. Estimate the length of an individual palmitic acid molecule in its fully extended conformation. If two palmitic acid molecules are placed end-to-end, how does their total length compare with the thickness of a lipid bilayer in a biological membrane?

Temperature dependence of lateral diffusion.

The experiment described in Fig. 11-17 was conducted at 37 °C. What would be the effect on the diffusion rate if the experiment were performed at 10 °C?

Gastric juice synthesis: energetics.

Gastric juice (pH 1.5) is produced by pumping H⁺ and Cl^- ions from Blood Plasma (pH 7.4) into The Stomach. Calculate The amount of Free energy required to concentrate H⁺ in 1 L of gastric juice at 37 °C. How many ATP molecules must be hydrolyzed to supply this amount of free energy under cellular conditions? The free-energy change for ATP Hydrolysis under intracellular conditions is approximately -58 kJ/mol (as shown in Chapter 13). Disregard any effects of the transmembrane electrical potential.

Energetics of the Na⁺/K⁺-ATPase.

What is the free-energy change associated with The transport of 1 mol of Na⁺ out of The Cell and into the blood at 37 °C for a typical vertebrate cell with a transmembrane potential of -0,07 V (inside negative)? Assume the intracellular Na⁺ concentration is 12 mM, and the blood plasma concentration is 145 mM.

Effect of ouabain on renal tissue.

Ouabain specifically inhibits The activity of the Na⁺/K⁺-ATPase in animal Tissues. No other enzyme is known to be inhibited by ouabain. When ouabain is added to thin slices of living Kidney tissue, it inhibits oxygen consumption by 66%. Why? What does this observation tell us about how renal tissue utilizes respiratory energy?

Energetics of symport.

Suppose you determine experimentally that a cellular glucose transport system driven by Na⁺ symport can accumulate glucose to concentrations 25 times higher than those in the extracellular medium, whereas the extracellular [Na⁺] is only 10 times greater than the intracellular [Na⁺]. Does this violate the Laws of Thermodynamics? If not, how would you account for this observation?

Localization of a membrane protein.

The following observations have been made regarding an unknown membrane protein X. It can be extracted from disrupted Erythrocyte membranes using a concentrated salt solution, and it can also be cleaved into fragments by Proteolytic Enzymes. Treatment of intact erythrocytes with proteolytic enzymes, followed by membrane disruption and extraction, yields intact protein X. However, treatment of erythrocyte ghosts (which consist of The Plasma Membrane obtained by cell lysis and Hemoglobin removal) with proteolytic enzymes, followed by disruption and extraction, yields extensively fragmented X. What do these observations indicate

regarding the localization of X within the plasma membrane? Are these properties characteristic of an integral or a peripheral membrane protein?

Membrane self-sealing.

Biological membranes are self-sealing—if punctured or mechanically disrupted, they rapidly and spontaneously "heal the wound." What membrane properties are responsible for this vital feature?

Lipid melting points.

Membrane Lipids from tissue samples obtained from different parts of a reindeer's leg exhibit varying fatty acid compositions. Membrane lipids from tissue near the hoof have a higher proportion of Unsaturated Fatty acids than lipids from tissue higher up in the leg. What is the physiological significance of this fact?

Transbilayer (flip-flop) diffusion.

The inner monolayer of the human erythrocyte membrane consists predominantly of phosphatidylethanolamine and phosphatidylserine. The outer monolayer is composed primarily of phosphatidylcholine and sphingomyelin. Although the membrane phospholipid components are capable of lateral diffusion within the fluid bilayer, this Asymmetry is maintained continuously. How?

Membrane permeability.

At pH 7, Tryptophan crosses a lipid bilayer at roughly one-thousandth The rate of the closely related compound indole:

Propose an explanation for this observation.

Water transport by Aquaporins.

Each human erythrocyte contains 2 × 10⁵ AQP-1 monomers. If water molecules flow across the plasma membrane at a rate of 5 × 10⁸ per AQP-1 tetramer per second, and the volume of an erythrocyte is 5 × 10^-11 mL, how rapidly will the erythrocyte volume decrease by half upon exposure to the high osmolarity (1 M) encountered in the renal medullary interstitium? Assume the erythrocyte consists entirely of water.

Labeling of the lactose transporter.

The bacterial lactose transporter, which is highly specific for its substrate lactose, contains a Cys residue essential for its transport activity. Covalent Modification of this Cys residue by N-ethylmaleimide (NEM) irreversibly inactivates the transporter. A high concentration of lactose in the medium prevents this inactivation, presumably due to steric hindrance protecting the Cys residue located at or near the lactose-binding site. Assuming you know nothing further about this transporter protein, propose an experiment that would allow you to determine the M_r of the Cys-containing polypeptide of the transporter.

Predicting membrane protein topology from the Amino Acid Sequence.

You have cloned the Gene for a human erythrocyte protein suspected of being a membrane protein. From The nucleotide sequence of the gene, you deduce its amino acid sequence. How would you evaluate, solely from the sequence, the likelihood that this protein is an integral membrane protein? Assuming the protein turns out to be a type I or type II integral protein, propose biochemical or chemical experiments to determine its specific type.

Intestinal leucine uptake.

You are studying the uptake of L-leucine by epithelial Cells of the mouse Small Intestine. Measurements of the uptake rates for L-leucine and several of its analogs in the presence and absence of Na⁺ yield the results shown in the table. What Conclusions can you draw about the properties and MECHANISM OF ACTION of the leucine transporter? Would you expect L-leucine uptake to be inhibited by ouabain?

	Uptake in the presence of Na+	Uptake in the absence of Na+
Substrate	Vmax        Kt (mM)	Vmax       Kt (mM)
L-leucine	420        0.24	23        0.2
D-leucine	310        4.7	5        4.7
L-valine	225        0.31	19        0.31

Effect of an ionophore on Active Transport.

Consider the leucine transporter described in Problem 19. Would V_max and/or K_t change if you added a Na⁺ ionophore to the assay solution containing Na⁺? Explain.

Surface density of a membrane protein.

An E. coli cell can be induced to produce about 10,000 copies of the lactose transporter (M_r = 31,000) per cell. Assume E. coli is a cylinder with a diameter of 1 µm and a length of 2 µm. What fraction of the plasma membrane surface area is occupied by lactose transporter molecules? Explain how you arrived at your Conclusion.

Helical wheel plot.

A helical wheel plot is a two-dimensional representation of an α Helix viewed along its central axis (see Fig. 11-29b, as well as Fig. 4-4d). Use the diagram provided below to determine the distribution of amino acid residues in a helical segment with the following sequence:

-Val-Asp-Arg-Val-Phe-Ser-Asn-Val-Cys-Thr-His-Leu-Lys-Thr-Leu-Gln-Asp-Lys-.

What can you infer about The properties of this helix's surface? How should this helix be oriented within the three-dimensional structure of an integral membrane protein?

Molecular COMPOSITION OF THE E. coli membrane.

The plasma membrane of E. coli is approximately 75% protein and 25% phospholipid by mass. How many membrane lipid molecules are there per membrane protein molecule? Assume an average protein Molecular Weight of M_r = 50,000 and an average phospholipid mass of M_r = 750. What additional information is needed to estimate the fraction of the membrane surface covered by lipids?

Biochemistry on the Internet

Membrane protein topology.

The adrenaline hormone receptor in animal cells is an integral membrane protein (M_r = 64,000) generally believed to possess seven transmembrane segments.

a) Demonstrate that a protein of this size is capable of spanning the membrane seven times.

b) Given The amino acid sequence of this protein, how would you predict which regions form the transmembrane helices?

c) Visit the Protein Data Bank (www.rcsb.org). Use the PDB identifier 1DEP to retrieve the data page for a portion of the turkey β-adrenergic receptor (one type of adrenaline receptor). Using Jmol to examine the structure, predict whether this region of the receptor is localized within the membrane or on the membrane surface. Explain.

d) Retrieve the data for a portion of another receptor—the neuronal and myocytic Acetylcholine Receptor—using the PDB identifier 1A11. As in (c), predict the localization of this receptor portion and explain your answer.

If you have not used the PDB before, consult the detailed instructions in Box 4-4 (p. 193).

Analysis of Experimental Data

Fluid-mosaic model of Biological Membrane Structure.

Figure 11-3 illustrates the widely accepted fluid-mosaic model of biological membrane structure. This model was described in detail in a 1971 review article by S. J. Singer. The article originally discussed three models of membrane structure prevalent at the time:

A. The Davson-Danielli-Robertson model.

In 1971, this was the most popular model. According to this proposal, phospholipids form a lipid bilayer, with Proteins positioned on both surfaces of the bilayer and attached via ionic interactions between the charged phospholipid head groups and charged protein groups. Crucially, this model posits no proteins penetrating the interior of the bilayer.

B. The Benson model (lipoprotein subunit model). In this view, proteins are considered globular, and the membrane is treated as a protein-lipid mixture. The hydrophobic lipid tails insert into the hydrophobic Regions of the proteins, while the lipid head groups face the aqueous phase. There is no continuous lipid bilayer.

The mosaic model of a membrane composed of lipids and Globular proteins. This model is depicted in Fig. 11-3. Lipids form a bilayer into which proteins are embedded, some protruding past the bilayer boundaries and others entirely submerged. Proteins are anchored within the bilayer through hydrophobic interactions between the hydrophobic lipid tails and the hydrophobic domains of the proteins. Examine the data below and evaluate how well they support each membrane structure model. Which model or models are corroborated, and which are refuted? What caveats should be kept in mind regarding these data and their interpretation? Explain your reasoning.

a) When cells were fixed with osmium tetroxide, stained, and analyzed by Electron Microscopy, they exhibited a "railroad track" appearance: two dark electron-dense lines separated by a light intermediate region (see Fig. 11-1).

b) The thickness of cell membranes fixed and stained in this manner ranged from 5 to 9 nm. The thickness of a pure phospholipid bilayer without embedded proteins ranged from 4 to 4.5 nm, and the thickness of a single protein monolayer is ~1 nm.

c) Singer noted: "The average Amino Acid Composition of Membrane Proteins is indistinguishable from that of soluble proteins. In particular, a significant fraction consists of hydrophobic residues" (p. 165).

d) As described in Problems 1 and 2 of this chapter, researchers extracted cell membranes, isolated the lipids, and compared the area of the lipid monolayer with the surface area of the original cell membrane. Interpreting these results was complicated by the phenomenon illustrated in the graph for Problem 1: the monolayer area depends on the applied lateral pressure. At very low pressure, The ratio of monolayer area to cell membrane surface area was 2.0. At higher pressures, more representative of physiological conditions within the cell, this ratio was substantially lower.

e) Circular dichroism spectroscopy, based on UV light polarization changes, provides insights into Protein Secondary structure (see Fig. 4-9). This technique revealed that membrane proteins contain a relatively high proportion of α helices and very few β sheets. These findings are consistent with the globular structure of most membrane proteins.

f) Phospholipase C is an enzyme that removes polar head groups (including phosphate groups) from phospholipids. Several studies reported that treating intact membranes with phospholipase C cleaved about 70% of the polar head groups without disrupting the "railroad track" morphological pattern.

g) Singer described a study in which "a glycoprotein with a molecular weight of 31,000 in human erythrocyte membranes was digested with Trypsin to yield soluble glycopeptides with a molecular weight of about 10,000, while the remaining portion of the glycoprotein remained highly hydrophobic" (p. 199). Trypsin treatment did not cause major structural alterations in the membranes, which remained intact. Singer's review also discussed other studies on this topic. However, the data available in 1971 did not conclusively prove the validity of model C; this model gained widespread acceptance only later, as additional research data accumulated.