Biochemistry - Chemical Reactions in Living Cells, Volume 1 - D. Metzler 1980

Membranes and Cell Walls
Metabolism and Membrane Functions
Transport of Molecules Across Membranes

The ability of Cells to absorb nutrients and excrete various compounds is of exceptional importance. As already mentioned in Sec. A.4, small neutral molecules can cross membranes simply by ordinary diffusion. The rate of diffusion of a substance is determined by its solubility in the membrane, its diffusion coefficient within the membrane (ch. 6, Sec. A.7), and the concentration gradient between the outside and the inside of The Cell. In The transport of charged particles, a significant influence on the diffusion process is also exerted by the electrical potential difference resulting from the accumulation of an excess of negatively charged ions within the cell.

Water, carbon dioxide, oxygen, and anesthetic molecules penetrate cells via ordinary diffusion. The process of Facilitated Diffusion is significantly more widespread [40]. Like ordinary diffusion, facilitated diffusion depends on a concentration gradient—during this process, molecules always move from a region of higher concentration to a region of lower concentration. Facilitated diffusion is characterized by a saturation effect, which means that as the concentration of the diffusing substance (on the high-concentration side) increases, the rate of flux through the membrane approaches a certain maximum. The saturation effect is also observed in enzymatic reactions (ch. 6, Sec. A.2), indicating certain common features in the mechanisms of these two processes. Evidently, in facilitated diffusion, the transported substance binds to a mobile carrier (most often a protein). The carrier then diffuses a short distance to the opposite side of the membrane, where it releases the bound molecule or ion. If the rates of association and dissociation of the substance with the carrier exceed the rate of diffusion, the transport of molecules can be described by the Michaelis–Menten Equation (ch. 6, Sec. A.5), and the “kinetic parameters” of the diffusion process—namely, Vmax (maximum velocity) and Km (Michaelis constant)—are determined using equation (6-15).

Of greatest interest is Active Transport, in which a substance is transported across the membrane against a concentration gradient, i.e., from a region of lower concentration to a region of higher concentration. This process is accompanied by an increase in Free energy amounting to 5.71 lg c₂/c₁ kJ·mol^-1 [equation (3-25)], where c₂ and c₁ are the higher and lower concentrations, respectively. This circumstance necessitates the coupling of active transport with some spontaneously occurring exergonic reaction. Such coupling can be accomplished in at least two ways. In primary active transport, There is a direct coupling with a reaction of the ATP Hydrolysis type and the “pumping” of the solute across the membrane, whereas in secondary active transport, the energy of the electrochemical gradient generated for another solute is utilized. In the second case, one solute is “pumped” against its concentration gradient, and then the second is transported across the membrane As a result of exchange with the first. Another variation of active transport is known as group translocation [41]. In this process, the transported substance first undergoes covalent modification, and the resulting product enters the cell.

Transport processes, whether facilitated or active, appear to be highly complex and proceed with the participation of several Membrane Proteins. Sometimes the term permease is used to describe the transport system. Because the amounts of proteins involved in substance transport are insignificant, genetic analysis Methods have been employed to study transport systems. It is hoped that these methods will help determine the number of genes determining the proteins that participate in The transfer of specific compounds across membranes.

a. Binding Proteins

Significant progress has been made recently in the isolation of binding proteins, which are generally considered to be components of permease systems. Most of these proteins have been extracted from the bacterial surface by various methods, notably by a sudden change in the Osmotic Pressure of the medium (osmotic Shock) [42–44].

For example, if E. coli cells suspended in 0.5 M sucrose are treated with an EDTA solution (10^-4 M) for 10 min and then diluted with cold water, proteins that bind sugars, Amino Acids, Metal Ions, and other substances are extracted from them. One of the proteins with a Molecular Weight of ~35,000 specifically binds galactose. The localization of binding proteins in bacterial cells has not been precisely established. Binding proteins are usually classified as “periplasmic” (Sec. D), but they may be loosely bound to The Plasma Membrane.

Binding proteins would be well-suited to act as mobile carriers in facilitated diffusion; however, most of the isolated proteins apparently belong to active transport systems, and their function in transfer processes is still not definitively established. According to one hypothesis, a binding protein has a high affinity for the transported substance (substrate) and binds firmly to it on the outer surface of the cell. The resulting protein-substrate complex then diffuses to the inner side of the membrane. Here, as a result of a process coupled with a spontaneously occurring exergonic reaction, such as ATP hydrolysis, the conformation of the protein changes in such a way that its affinity for the substrate decreases. Consequently, the transported substance enters the cell, and the binding protein diffuses back to the outer surface. There, its conformation returns to the initial state, likely under METABOLISM/18.html">The Influence of Chemical factors.

What classes of molecules and ions are transported across membranes? Inorganic ions enter the cell from the environment, and as a result of this transfer, they are sometimes heavily concentrated (ch. 2, Sec. E.2). For example, the roots of green plants can extract essential substances from very dilute solutions. A similar capability is possessed by microorganisms, such as Yeasts and Bacteria, which have systems capable of selectively concentrating many ions, including K⁺, Ca²⁺, sulfates, and phosphates. Frog Skin is able to absorb Na⁺ ions from a medium with an NaCl concentration of 10^-5 M and transfer them to the internal environment, where the NaCl concentration exceeds 0.1 M. Gastric mucosa cells can concentrate hydrogen ions in gastric juice to approximately 0.16 M.

Amino acids are actively transported into animal and bacterial cells [38, 39]. E. coli possess specific transport systems for almost every amino acid, and for Some amino acids, there are even multiple such systems. Typically, alongside a system characterized by high affinity for The amino acid and The ability to “pump” it from regions of very low concentration, there are parallel-functioning systems with receptors that do not possess such a high affinity for the substrate. Amino Acid and sugar transport systems are quite well studied in bacteria [38, 45, 46]. In one such system, studied in detail using chemical and Genetic Methods, the penetration of various sugars (including aldohexoses) into the cell is coupled with The breakdown of phosphoenolpyruvate (Table 3-5). Apparently, when this system Functions, sugars cross the inner membrane in the form of phosphate esters (group translocation) [46a, 46b]. In another system, the Transport of Amino acids and lactose is coupled with the electron transfer system (ch. 10) in a membrane-bound redox chain. This system is believed to be independent of ATP synthesis.

Intracellular Organelles have their own ion-concentrating systems. For instance, Mitochondria can concentrate K⁺, Ca²⁺, Mg²⁺, and other divalent metal ions, as well as dicarboxylic acids (ch. 10). In mitochondria, the transport of many substances occurs most likely via exchange diffusion, i.e., through secondary active transport.

b. GENETIC ASPECTS OF Transport

A vast number of Mutations affecting nutrient uptake have been identified in microorganisms [38]. Here we will limit ourselves to considering the system responsible for potassium transport in E. coli [45, 47]. One E. coli mutant lives normally in a 0.1 M K⁺ solution, but cannot survive at significantly lower concentrations of this ion, although most other strains easily tolerate such conditions. At least 6 genes required for the functioning of three different potassium uptake systems have been discovered in E. coli strain K 12. Two such systems transport potassium into the cell (against the concentration gradient) at relatively high concentrations of K⁺ ions in the surrounding medium. The third system is capable of “pumping” K⁺ ions into the cell from a medium with a very low concentration: the half-saturation constant (Km) is approximately 10^-6 M. It is interesting to note that if the bacterium grows in a medium with a high K⁺ content, the system characterized by high affinity for K⁺ ions is inactive, meaning the corresponding Gene is “turned off” (repressed). However, if this bacterium is cultured in a medium with a very low concentration of K⁺ ions, Gene Expression occurs and the transport system begins to function.

A large number of defects in transport systems have been noted not only in bacteria. A whole range of DISEASES ASSOCIATED WITH membrane transport defects has been described in humans [48]. In some of these diseases, substance reabsorption in the renal tubules and Absorption in the Small Intestine are impaired. For instance, cystinuria involves The formation of cystine stones in the Kidneys and bladder. Such patients excrete up to 1 g of cystine per day, compared to a normal value of approximately 0.05 g. Cases of excretion of large amounts of Lysine, Arginine, and Ornithine are also known. The existence of such Hereditary diseases indicates that human cells, like bacterial ones, possess the ability to concentrate various amino acids (see also ch. 14, Sec. B.3) and other substances. In renal tubular cells, substances are absorbed at one side of the cell (the lower part of the cell in Fig. 1-3) and released into the bloodstream from the other side. Another well-studied, yet very rare disorder of human absorption processes leads to The Development of renal glycosuria. The proximal renal tubules are also involved in this process. Such an autosomal dominant mutation can be misdiagnosed as Diabetes Mellitus. In reality, individuals with this defect generally feel well, and this condition is not considered a disease.

c. Sodium, Potassium, and Calcium Pumps

In almost all cells, the sodium concentration is relatively low, whereas the potassium concentration is quite high (Table 5-2, Addendum 5-3). According to one hypothesis [49], The properties of the Cytoplasm resemble those of ion-exchange resins bearing fixed charges. Ion-exchange resins with a high degree of cross-linking selectively bind specific ions. For example, resins containing sulfonic acid residues bind potassium, while resins containing phosphinic acid residues bind sodium. This hypothesis is supported by NMR data indicating Changes in the relaxation times of intracellular water protons and ³⁹K⁺ and ²³Na⁺ ions within cells [50]. However, experimental measurements of this kind are very complex, and the evidence obtained from them is disputed [51].

Class="center">Table 5-2 Content of major ions (in millimoles per kg of H₂O) in human Blood Plasma and Skeletal Muscle intracellular fluid^a

^a Based on Muntwyler E., Water and Electrolyte Metabolism and Acid-Base Balance, p. 14, Mosby, St. Louis, Missouri, 1968; White A., Handler P., Smith E. L., Principles of Biochemistry, 5th ed., p. 802, McGraw-Hill, New York, 1973; Long C., Biochemist’s Handbook, p. 670, Van Nostrand, Princeton, New Jersey, 1961. It should be kept in mind that the data given for certain ions may vary within very wide limits.

^b Milliequivalents per kg of H₂O.

^c Phosphates and other non-protein anions.

Currently, the ion-pump hypothesis—which posits that cells possess an ion pump that expels Na⁺ ions and pumps in K⁺ ions—is more widely accepted than the ion-exchange hypothesis. Various methodological approaches have been used to study this process. For example, the entire cytoplasm can be removed from a giant squid axon, and the remaining cell envelope can be filled with various ionic solutions. Erythrocyte ghosts can be filled in a similar manner. The active transfer of ions into and out of cells has been observed both in the aforementioned objects and in various intact cells of other types. It has been found that ion transport is blocked by inhibitors such as cyanide, which is known to disrupt nearly all oxidative metabolic processes in cells. However, cyanide blockage is reversed by The addition of ATP or other phosphate compounds characterized by a high group-transfer potential to the cells.

Since ATP is the most active compound of this kind in Eukaryotic cells, it is presumed to serve as the natural energy source during ion concentration. However, as indicated in Sec. a, bacteria (and mitochondria) can utilize energy from other sources as well.

The uptake of K⁺ ions and the release of Na⁺ ions by cells are specifically blocked by certain cardiac Glycosides. Ouabain (Fig. 12-18) is most commonly used for this purpose. Tritium-labeled ouabain binds to the outer surface of cells, and data characterizing this binding make it possible to calculate the number of ion-pumping sites per cell [62]. In the case of erythrocytes, this amounts to 100–200 per cell (or approximately 1 site per 1 µm²), whereas in HeLa cells (a human Cancer cell line widely used in laboratories worldwide, grown in culture for many years), the number of such sites is 10⁵–10⁶ (i.e., ~10³ sites per 1 µm²). Further research established that in the presence of excess Na⁺ ions inside the cell and K⁺ ions in the external environment, ATP hydrolysis occurs. The hydrolysis rate is determined by the concentrations of both alkali metal ions and the number of ouabain-binding sites; magnesium ions are required for this process.

The facts discussed above led to the concept that (Na⁺ + K⁺)-dependent ATPase is, essentially, the membrane ion pump. To activate the enzyme system, K⁺ and Na⁺ ions must be located on opposite sides of the membrane. At the same time, the purified enzyme hydrolyzes ATP in vitro in the presence of Na⁺ + K⁺ + Mg²⁺. This protein has been successfully isolated in purified form [53–56]. Upon gel Electrophoresis in the presence of sodium dodecyl sulfate, the purified (Na⁺ + K⁺)-dependent ATPase separates into two subunits. The larger one is a polypeptide chain with a molecular weight of ~95,000–100,000, while the smaller one is a glycoprotein with a molecular weight of ~50,000. Antibodies against the isolated large subunit bind to membrane fragments that apparently belong to Regions of the inner surface of the plasma membrane [57]. It is logical to assume that the glycoprotein subunit of the enzyme is located on the outer surface of the membrane.

Appendix 5-B

Alkali Metal Ions

Despite the fact that sodium and potassium are present in roughly equal amounts in the Earth's crust, all living cells accumulate exclusively potassium ions^a-c. As it turns out, sodium ions are required only by certain marine organisms and Multicellular animals to regulate the composition of their Body Fluids. Most non-marine plants show no apparent need for sodium.

The tendency to accumulate potassium ions is particularly interesting given that in seawater the concentration of Na⁺ is ~0.46 M, whereas that of K⁺ is only 0.01 M. Other alkali metals are present in seawater in even smaller amounts; for instance, the concentration of Li⁺ is 0.026 mM, Rb⁺ is 0.001 mM, and Cs⁺ is present only in trace amounts. In groundwater, the concentration of K⁺ is approximately 0.1 mM and that of Na⁺ is 0.65 mM, yet plants are known to exhibit a clear preference for potassium as well.

The intracellular concentration of K⁺ ranges from 200 mM in E. coli and 150 mM in mammalian muscle to ~30 mM in freshwater invertebrates (bivalves, hydras) and certain Protozoa, the latter exhibiting the lowest concentrations of this ion. K⁺ cannot be replaced by Na⁺, although a partial substitution of K⁺ by Rb⁺ and, to a lesser extent, by Cs⁺ is generally possible. In some microorganisms, rubidium can almost completely replace potassium, and even rats can survive for short periods when K⁺ is almost entirely replaced by Rb⁺. In brown Algae, the majority of K⁺ ions are replaced by protons^d. Human requirements for potassium are quite high, reaching approximately 2 g/day. It has been suggested that modern humans suffer from a chronic potassium deficiency resulting from food preparation and the boiling of vegetables^e.

Sodium is also essential for higher animals; rats die on a sodium-free diet. Intracellular sodium content varies among animal species, but it is typically 5 to 10 times lower than that of potassium. In blood, the ratio is reversed: the concentration of Na⁺ in human blood plasma is 0.15 M, while that of K⁺ is 0.005 M. Interestingly, the taste of salt in food is among the most essential^b.

The reasons behind the cellular selectivity for K⁺ are far from obvious; it may well be related to the differing degrees of Hydration of K⁺ and Na⁺ ions (Chapter 4, Section B.8.b). These ions may play a paramount role in maintaining membrane excitability (Section B.3), even in bacteria. The contrasting concentrations of these two ions on opposite sides of the membrane provide a readily available source of energy for many membrane-associated processes.

K⁺ ions are required for the function of numerous intracellular Enzymes^b,c. These include enzymes that catalyze the phosphorylation of carboxyl groups or enolate anions, as well as elimination reactions yielding enols.

^a Kernan, R. P., Cell K, Butterworth, London, 1965.

^b Suelter, C. H. In: Metal Ions in Biological Systems (Sigel, H., ed.), Vol. 3, pp. 201–251, Dekker, New York, 1974.

^c Suelter, C. H., Science, 168, 789–795 (1970).

^d Steinbach, H. B., Comp. Biochem. Physiol., 4, pp. 677–720 (1962).

^e Weber, C. E., J. Theor. Biol., 29, 327–328 (1970).

^f Kaunitz, H., Nature (London), 178, 1141–1144 (1956).

The stoichiometric relationships in the sodium-potassium pump are quite remarkable. For the breakdown of every ATP molecule, 3 sodium ions are pumped out of the cell, while 2 potassium ions are pumped in from the outside. Since more positively charged ions are pumped out of the cell than enter it, an excess negative charge is generated internally. The presence of an intracellular negative charge was established long ago through measurements of the electrical Membrane Potential (Section B.3). Because The cell membrane remains permeable to K⁺ ions, the Development of the membrane potential drives the diffusion of these ions back into the cell through the membrane, leading to partial neutralization of the negative membrane charge. A steady state is established when the rate of passive diffusion balances the membrane potential generated by active transport.

It is well established that the transport of Na⁺ and K⁺ by the ion pump is powered by the energy of ATP hydrolysis¹. The function of the (Na⁺+K⁺)-dependent ATPase is not merely to hydrolyze ATP, and in this sense, the term ATPase is somewhat misleading. The ATP Cleavage process must be coupled to a specific mechanism responsible for ion translocation, which remains elusive to this day. A purely hypothetical model of such a mechanism is shown in Fig. 5-3. It is postulated that the ion pump proteins exist in two Conformations. In conformation A, the protein tightly binds 3 Na⁺ ions, whereas in conformation B, it binds 2 K⁺ ions. ATP acts as the "motor" driving these conformational changes and may also participate directly in forming the ion-binding sites. The pump illustrated in Fig. 5-3 is embedded in the membrane such that the large enzyme subunits face the cell interior, while the glycoprotein subunits project outward. At the center (which may lie at the interface between the two dimer subunits, as shown in Fig. 4-9) is a narrow cavity into which chelating groups project (such as the C=O group of the peptide chain). These groups form three binding sites for Na⁺ ions, with a diameter of 0.095 nm. Spontaneous binding of sodium ions triggers a phosphorylation reaction, in which the phosphoryl group from the Mg—ATP^2- complex is transferred to group Y. Phosphorylation, in turn, induces a transition of the protein to conformation B, in which the channel opens to the outside and closes to the inside. Concurrently, the affinity for Na⁺ ions decreases, and these ions are released into the extracellular medium.

¹ The pumping of sodium and potassium ions is one of the most energetically demanding cellular processes. It is estimated that in resting muscle, half of the generated ATP is consumed for this purpose, and even more in Nerve Cells. Thus, this process accounts for a very substantial fraction of the total metabolic energy budget (Chapter 3, Section A.5).

FIG. 5-3. Purely hypothetical model of the (Na⁺+K⁺) pump. The opening and closing of the channel on opposite sides of the membrane, along with the alternating changes in Na⁺ and K⁺ binding efficiency, are driven by the energy of ATP hydrolysis. A. Conformation A, which binds Na⁺. Protruding groups form binding sites measuring 0.1 nm within the central cavity. B. Three Na⁺ ions are bound. C. Group Y is phosphorylated. Na⁺ ions may be bound more tightly due to the negative charge. D. Conformational transition occurs. The phosphoryl group is transferred to another acceptor, X. E. Conformation B, which binds K⁺. K⁺-binding sites measuring 0.13 nm are formed; Na⁺ ions diffuse outward. F. Two potassium ions are bound. G. Hydrolysis of the bond between X and the phosphoryl group induces a return to the initial conformation A. H. Potassium ions diffuse into the cell interior

In the next step, 2 K⁺ ions are bound. Conformation B is characterized by a high affinity for K⁺ ions. The reverse transition to conformation A, accompanied by the release of K⁺ ions into the intracellular space, is triggered by the hydrolytic Cleavage of the phosphoryl group as inorganic phosphate (P_i).

One might wonder how the opening and closing of the channel are synchronized with changes in the number and Specificity of the ion-binding sites. However, we may recall the structural changes that occur during the Oxygenation of Hemoglobin (Fig. 4-19). Although the relative rotation of subunits causes only minor geometric shifts in the groups projecting into the central channel, it results in profound changes in 2,3-diphosphoglycerate binding. Similarly, in our case, minor displacements can render the Na⁺-binding sites inaccessible while generating new binding sites for the larger K⁺ ions, potentially utilizing the exact same chelating groups involved in Na⁺ binding.

The question of how phosphorylation induces conformational changes is discussed in Chapter 7, Section E.5. Since the transfer of a phosphoryl residue between various side chains of the protein is entirely feasible, it likely drives the A→B conformational transition, as depicted in Fig. 5-3 (phosphoryl transfer from Y to X).

The Chemical Nature of the Na⁺ and K⁺ binding sites in the ion pump remains unknown. However, valuable insights are provided by studies on peptide-type Antibiotics, many of which bind metal ions and catalyze their diffusion across membranes [58]. An example of such a compound is valinomycin, a cyclic depsipeptide (a peptide containing ester bonds alongside amide bonds). This antibiotic is composed of residues of D- and L-valine, L-lactic acid, and D-oxyisovaleric acid.

The incorporation of such an ionophore into an artificial membrane during incubation in a K⁺-containing medium leads to a dramatic increase in membrane conductance¹. The Structure of valinomycin has been thoroughly investigated [58, 60–63a]. It has been shown that upon binding a potassium ion, the molecule adopts a more compact conformation. This conformational change results from the disruption of two Hydrogen Bonds and the formation of new ones, accompanied by the "wrapping" of the molecule around the potassium ion (Fig. 5-4). The transport of potassium by valinomycin occurs passively, yet it involves cyclic transitions between two conformations of the carrier as it undergoes ion binding, transmembrane diffusion, and ion release on the opposite side of the membrane. The rate of this transport is extraordinarily high—each valinomycin molecule is capable of translocating approximately 10⁴ potassium ions across the membrane per second. Consequently, even extremely small amounts of the ionophore can induce profound Changes in membrane permeability and conductance.

FIG. 5-4. Structure of free valinomycin (A) and its complex with K⁺ (B). It is hypothesized that the exposed carbonyl oxygen atoms P, P', M, and M' form an initial complex with K⁺. Subsequently, hydrogen bonds 1 and 2 are broken, allowing oxygen atoms R and R' to also participate in binding K⁺ ions (resulting in the formation of 6 coordinate covalent bonds). The "folding" of the molecule induced by K⁺ binding is accompanied by minor conformational rearrangements. As a result, oxygen atoms Q and Q' complete the formation of intramolecular hydrogen bonds in the K⁺-containing complex ion. (Duak et al., Science, 176, 911, 1972, copyright 1972 by the American Association for the Advancement of Science.)

¹ It should be noted that there is no evidence suggesting these antibiotics play a similar role in the sporulating cells that synthesize them. It is hypothesized that bacitracin enhances the rate of Mn²⁺ ion diffusion across membranes [59].

Box 5-C

Antibiotics

Many organisms produce chemical substances that are toxic to other organisms. The roots or leaves of certain plants secrete compounds that inhibit the growth of other plants. More familiar to us are the medically useful antibiotics produced by Fungi and bacteria. Although the ability of certain organisms to inhibit the growth of others was well known in the past century, widespread interest in this problem arose only in 1929, when Alexander Fleming discovered that Penicillium notatum inhibits the growth of staphylococci. This observation directly led to the isolation of penicillin, which was first used to treat patients in 1941. A few years later, Waksman isolated actinomycin (Box 15-B) and streptomycin (Box 12-A) from soil actinomycetes (streptomycetes); he also proposed the term "antibiotics" for these compounds. The Use of streptomycin proved effective in the Treatment of tuberculosis, stimulating the search for other substances with similar properties. Since then, approximately 50 new antibiotics have been discovered every year, and The production of more than 60 antibiotics has already been established.

The following main classes of antibiotics are known: 1) Peptides such as gramicidins and tyrocidines (more than 200 are known); 2) penicillin and Cephalosporins (Box 7-D); 3) Tetracyclines (Fig. 12-10); 4) macrolides, macrocyclic lactones such as erythromycins (Fig. 12-10); 5) polyene antibiotics (Fig. 12-10).

How do antibiotics work? Some, like penicillin, block the action of specific enzymes (Box 7-D). Peptide antibiotics (Section B.2.c) frequently form complexes with metal ions and apparently disrupt The regulation of ion permeability in bacterial membranes. Polyene antibiotics affect proton and ion transport in fungal membranes. Tetracyclines, like many Other Antibiotics, directly interfere with Protein Synthesis (Chapter 15, Section V.2.z). Certain other antibiotics intercalate into DNA molecules (Chapter 2, Section G.9; Box 15-B). Thus, there is no single MECHANISM OF ACTION common to all antibiotics. The search for a necessary antibiotic involves finding a compound that exhibits high toxicity toward the infectious agent while remaining minimally toxic to human cells.

Do we need new antibiotics? Yes. More effective antibiotics are needed against Gram-negative bacteria such as Salmonella, which occasionally cause severe infectious diseases. Current antifungal agents do not meet our requirements, and more effective antibiotics against protozoa are urgently needed. The search for antibiotics among mixed microbial populations of soils, swamps, and lakes will undoubtedly continue. However, according to Perlman, chemical modification of already known antibiotics will play an increasingly important role in obtaining new, more effective antibiotics. Today, Semisynthetic Penicillins play a vital role in medicine, and the chemical modification of rifamycin-type antibiotics (Box 15-A) has led to the development of a new series of effective therapeutic agents.

^a Perlman D. In: Medicinal Chemistry (A. Burger, ed.), 3rd ed., Part I, pp. 305–370. Wiley, New York, 1970.

A unique property of valinomycin is its ability to form significantly more stable complexes with potassium than with sodium, making it a specific ionophore for potassium. When added to a mitochondrial suspension, it incorporates into the membranes and specifically accelerates the uptake of potassium ions¹. Unlike valinomycin, antamanide—a peptide isolated from fungi (Box 15-B)—selectively binds sodium ions rather than potassium ions, owing to differences in the geometry of their binding sites. The STRUCTURE OF THE Na⁺-antamanide complex is shown in Fig. 5-5. Another ionophore discussed in Chapter 2, enterobactin (Fig. 2-44), serves as an iron carrier. Notably, in this case as well, Metal ion binding is accompanied by significant Conformational Changes in the peptide. Certain Phospholipids are also thought to function as ionophores [64a].

Calcium Ions are also actively pumped out of most cells by a system that in many respects resembles the (Na⁺+K⁺)-dependent ATPase [65, 66]. A vitamin D-dependent calcium-binding protein has been isolated from the intestinal mucosa [14, 67] (Box 5-D). This protein has a high molecular weight and is similar to the muscle calcium-binding protein (Chapter 4, Section V.8.c).

It is quite possible that transport systems exist for many other ions as well. The only ion that apparently is not actively transported is the chloride ion. Membranes are relatively permeable to these ions (Section A.6), which are the principal anions of the plasma (Table 5-2).

¹ Upon the addition of valinomycin to a suspension of Streptococcus faecalis cells, the ratio [K⁺]_in/[K⁺]_out rapidly decreases [64]. It is believed that the antibiotic activity of valinomycin is due to the efflux of K⁺ (or Rb⁺) ions from the cells.

In most cases, chloride ions are distributed passively in accordance with the Donnan equation [68, 69]:

[K⁺]_in[Cl^-]_in = [K⁺]_out[Cl^-]_out.       (5-1)

The subscripts "in" and "out" denote that the concentrations pertain to the inner and outer sides of the cell membrane, respectively. It follows from equation (5-1) that since the intracellular potassium ion concentration is maintained at a high level through the action of the (Na⁺ + K⁺)-pump, in the presence of non-diffusible anions, the intracellular chloride ion concentration must be low. In accordance with the constancy of the ion concentration product on the outside of the cell, where the K⁺ concentration is low, the Cl^- concentration must be high.

FIG. 5-5. Proposed structure of the Na⁺-antamanide complex [60].