Transport of Molecules across Cell Membrane

The following points highlight the five processes involved in transport of molecules across cell membrane. The processes are: 1. Passive Diffusion 2. Facilitated Diffusion 3. Active Transport 4. Group Translocation 5. Ion Transport through Ionophores.

Process # 1. Passive Diffusion:

By passive diffusion, molecules move across the membrane without interacting with any specific carrier protein in the cell membrane. The movement is always from a higher to a lower concentration and such movement continues till the concentration of the solute on both sides of the membrane is the same.

At that concentration, an equilibrium is reached. However, if the molecule inside the cell is consumed, the inward movement continues, e.g. oxygen transported into the cell is continuously consumed for respiration, or carbon dioxide for photosynthesis. The molecules that can enter into cells by passive diffusion are generally small in size and are non-polar in nature.

Process # 2. Facilitated Diffusion:

This process of transport differs from passive diffusion in having a requirement of a transporter or carrier protein. These proteins — also known as permeases — are located in the membrane and they are believed to span across the entire width of the lipid-bilayer.

The transporter proteins are specific for a particular kind of molecule, or sometimes a group of closely related molecules. The function of the transporters is to bind the transportable molecule on one side of the membrane and release it on the other side. Presumably, this function is associated with conformational change of the transporter protein molecule.

This is schematically shown in Fig. 8.81:

Three types of transporter proteins have been recognized. These are uniporters which transport only one kind of molecules in a single direction, either from outside inwards or in the opposite direction. Symporters can transport two different kinds of molecules in the same direction; and anti-porters carry two different molecules in opposite directions.

The functions of these three types of transporters are schematically shown in Fig. 8.82:

The transport proteins resemble the enzymes to certain extent. Both transporters and enzymes show specificity or selectivity regarding the kind of molecule or a group of molecules with which they can interact. Also, both exhibit a saturation kinetics which means that by gradual increase of the concentration of the molecules with which they react, a point of saturation is reached, beyond which further increase results in no increase of the velocity. But there is one important difference. While enzymes transform the substrate molecules into products, transporter proteins deliver the molecules unchanged across the membrane.

The saturation kinetics observed in case of facilitated diffusion is absent in passive diffusion. In passive diffusion, the deciding factor is the concentration gradient and the rate of transport increases with the difference in the concentrations of the transportable molecules on the two sides of the membrane.

As equilibrium is reached, the concentrations are the same on two sides. In case of facilitated diffusion, increase in the concentration of the transportable molecules on one side of the membrane also results in an increase in the rate of transport, till all the transporter proteins are saturated with such molecules.

At this point, the rate of diffusion is maximum and this rate is maintained till the concentrations on both sides are equal. Thus, by both passive diffusion and facilitated diffusion, the final concentration is the same. But as facilitated diffusion is a more rapid process, equilibrium is reached more quickly.

This is diagrammatically shown in Fig. 8.83:

Process # 3. Active Transport:

Active transport of nutrient molecules differs basically from the diffusion processes, either passive or facilitated. In the active process, transport across membrane occurs against concentration gradient i.e. from a lower concentration to a higher concentration of the solute. Such transport can only occur with expenditure of energy. In other words, active transport is always an energy-consuming (endergonic) process. In biological systems, active transport is often much more important than diffusion processes.

The reason why active transport of solute molecules from a lower to a higher concentration requires input of energy is understandable from thermodynamic principles. Due to increase in concentration of solute molecules in the cell, free energy (G) increases because entropy (S) decreases.

The relation between the changes of free energy (∆G) and entropy is expressed by the equation, ∆G = ∆H – T∆S, where H represents enthalpy (total energy of the system) and T stands for absolute temperature. As concentration increases in the cell, entropy falls because the solute molecules are brought closer to each other resulting in their loss of freedom of movement.

Other factors, i.e. H and T, remaining the same, decrease in S means increase in the value of G i.e. free-energy. Thus, active transport leads to gain in free energy (G). A process which leads to increase in free energy cannot occur spontaneously and can only occur through input of energy. This energy is supplied by either ATP hydrolysis or by proton motive force (PMF) generated by unequal concentration of protons (H⁺) on either side of the membrane.

The quantity of energy (expressed in calories) required in active transport can be calculated using the equation AG = RT In C₂/C₁, where ∆G represents difference in free energy, R the gas constant (= 1.98), T the temperature expressed as absolute temperature (273+°C), In the natural logarithm (2.303 x log₁₀), C₂ the concentration of the solute inside the cell and Q the concentration of the solute outside.

Now, in active transport C₂ is greater than C₁ and, hence, the factor C₂/C₁ is always a positive integer. This means that ∆G has a positive value. A system having a positive ∆G cannot operate spontaneously. From the above equation, the quantity of energy required for transport of 1 mole of a non-ionized solute across a membrane which is permeable to the solute molecule, from a concentration of 0.001 M to 0.1 M at a given temperature, say 30°C, can be calculated;

∆G = RT In C₂/C₁ = 1.98 x (273+30) x 2.303 log (0.1/0.001)

= 1.98 x 303 x 2.303 x 2

= 2,763.3 cal

= 2.76 Kcal.

For charged molecules, i.e. ions and radicals, the above equation has to be modified to accommodate another gradient besides the concentration gradient, i.e. the electro-potential gradient, because the latter also contributes to free energy change in the system.

Like facilitated diffusion, active transport also involves mediation of transporter proteins present in the cell membrane. These proteins presumably function in the same way as those involved in facilitated diffusion. They, by virtue of having a high affinity for the transportable solute, combine with it and thereby undergo a conformational change losing affinity for the solute molecules resulting in release on the other side of the membrane.

When a particular solute has to be actively transported using proton motive force, the transporter protein binds both H⁺ and the solute molecules. One example of this type of transport is provided by uptake of lactose by E. coli. The lactose-transporter (lactose- permease) binds both H⁺ and lactose molecules and transport them simultaneously into the cell (symport).

Another example is found in case of glutamate transport in E. coli. The transporter in this case binds glutamate on the outside and transports it inside, and binds H⁺ inside and expels them outside (anti-port), so that glutamate uptake is linked with H⁺ expulsion.

(i) Active transport of lactose in E. coli:

Lactose transport in E. coli is an inducible system. Bacteria grown in absence of lactose are unable to transport the sugar into the cells. But when lactose is added in the medium replacing other sugars (like glucose), the bacteria quickly develop the ability to transport lactose due to new synthesis of lactose permease. This enzyme is a membrane integral protein which acts as lactose transporter.

It has been observed that transport of lactose into the cell is accompanied by a rise in pH value of the external medium due to depletion of H⁺ ions. According to the chemiosmotic theory, oxidation of respiratory substrates leads to expulsion of H⁺ out of the cell creating a proton gradient across the cell membrane which is utilized for ATP synthesis. The proton gradient so created is utilized by E. coli also for lactose uptake.

The lactose permease molecule contains a particular site for binding lactose and another site for binding H⁺, and both sites must be engaged for the enzyme to be functional. The permease protein spans across the membrane and it binds both lactose and proton on the outer face.

This binding causes a change in the conformation of the protein and results in release of both lactose and H⁺ inside the cell (symport). After the release, the permease returns to its original conformation, allowing the cycle to be repeated. The proton gradient is maintained by the respiratory H⁺-expulsion.

Function of lactose permease in E. coli is diagrammatically shown in Fig. 8.84:

(ii) Transport of Na⁺ and K⁺:

In most biological organisms, the intracellular concentration of K⁺ is higher than that of the external medium. Reverse is the situation in case of Na⁺. The ability of cells to maintain such a differential concentration of Na⁺ and K⁺ vis-a-vis the external medium has been found to be due to an enzyme, Na⁺K⁺-ATPase present in the membrane.

This enzyme requires the presence of Na⁺ inside the cell and K⁺ outside for becoming functional. It has an ATP binding site and another site for binding Na⁺ towards its inner face. On its outer face, the enzyme has a K⁺-binding site. It has been suggested that as the enzyme binds Na⁺ and ATP, it induces an ATP hydrolysis resulting in its conformational change.

The altered enzyme conformation loses affinity for Na⁺ which is released outside; but, at the same time the altered conformation develops affinity for K⁺ which is transported inside the cell. Binding of K⁺ with the enzyme causes dephosphorylation (release of inorganic phosphate) which brings back the original conformation of the enzyme protein and allows binding of Na⁺ and ATP. A purified Na⁺ K⁺– ATPase is a glycoprotein consisting of two large and two small subunits.

The small subunits have carbohydrate side chains on their outer face (Fig. 8.85):

The active expulsion of Na⁺ by the Na⁺ K⁺-ATPase activity creates a Na⁺ gradient across the membrane which is utilized by many microorganisms for active uptake of amino acids and sugars. The inward transport of these compounds is accompanied by simultaneous uptake of Na⁺ which moves down a concentration gradient, whereas amino acids and sugars move against a concentration gradient i.e. from a lower (outside) to a higher (inside) concentration.

The higher outside concentration of Na⁺ is maintained by active expulsion of Na⁺ through Na⁺ K⁺-ATPase activity. The transporter proteins specific for amino acids or sugars have two binding sites, one for Na⁺ and the other for the amino acid or sugar. Only when both sites are occupied by the respective ligands, can transport occur (symport).

Process # 4. Group Translocation:

Group translocation is a type of transport in which the transportable molecule is chemically altered to an impermeable form during its passage through the cytoplasmic membrane. Due to the transformation, the molecule is trapped in the cytoplasm and cannot escape.

The best known example of group translocation is the phosphotransferase system (PTS) occurring in a wide variety of bacteria for transport of sugars. The system has been found to operate in E. coli, Salmonella, Vibrio, Staphylococcus, Clostridium, Fusobacterium etc. However, in some bacteria, like Azotobacter, Mycobacterium, Nocardia and Micrococcus, PTS is absent.

At least three different proteins have been found to be involved in group translocation system of different sugars. For some sugars, like glucose, an additional protein is required. These proteins are enzyme I, enzyme II and a heat-stable small protein, called HPr. The additional protein for glucose is enzyme III.

All these proteins except enzyme II are cytoplasmic. Enzyme II is a membrane-integral protein. It is also different for transport of different sugars, while enzyme I and HPr are common to all sugar transport systems. These proteins serve as a chain of carriers in the PTS.

The operation of this translocation system (PTS) starts with transfer of the high-energy phosphate group of phosphoenol pyruvic acid (PEP) to enzyme I (E-I) in the cytoplasm of the bacterial cell. Next, the phosphate group of the phosphorylated E-I (E-I-P) is transferred to HPr, also a cytoplasmic protein, to produce HPr-(P). The phosphate group is then transferred to the membrane-bound enzyme II (E-II) directly, or, in case of glucose transport, via enzyme III (E-III).

Finally, phosphorylated E-II or E-III donates its phosphate group to the sugar being transported and the sugar is released in the cytoplasm as its phosphate ester. For example, in case of glucose, it is glucose 6-phosphate. The negatively charged sugar phosphate in prevented from escaping through the cytoplasmic membrane.

In case of mannitol, the phosphorylated form in which it is released is mannitol-1-phosphate. Since the membrane is generally impervious to charged molecular, the PTS can lead to high concentration of the sugar phosphate in the cytoplasm, while the concentration of sugars is much lower in the external medium. Thus, in PTS, sugars are translocate against a concentration gradient. It is therefore, a type of active transport in which the energy is supplied by the energy-rich compound PEP.

Enzyme II is a membrane-integral protein which is specific for the sugar being transported. Many different kinds of sugars can be transported by PTS. These include glucose, galactose, fructose, pentose’s, sugar alcohols like mannitol, ribitol, and galactosides. For each of these compounds, there appears to be a different enzyme II.

The small heat-stable protein (HPr) of the PTS has a molecular weight of 9,400 Daltons. When HPr is phosphorylated by E-I~(P), the phosphate group is attached through a N-atoms of a histidine residue of the HPr.

A schematic representation of the chain of carries in the operation of phosphotransferase system of bacteria is shown in Fig. 8.86:

Process # 5. Ion Transport through Ionophores:

Certain antibiotic compounds elaborated by micro

nisms can act as ionophores when they are integrated into the membrane. These agents are not, however, natural transport components of the membrane. Two of such agents are valinomycin produced by a species of Streptomyces and gramicidin produced by Bacillus brevis. Transport of ions by these ionophores has been well-studied in isolated mitochondria.

Valinomycin is a cyclic molecule consisting of 12 alternating molecules of D- or L-valine and L-lactate or D-hydroxyisovalerate. The structure of valinomycin is shown in Fig. 8.87A. The circular valinomycin molecule has a hydrophobic periphery which matches well with the hydrbphobic lipid bilayer of the membrane where valinomycin is inserted.

The central core of the valinomycin molecille is hydrophilic and negatively charged. The negatively charged core can bind positively charged cations. Valinomycin can specifically transport K⁺, because the size is such that it fits snugly in the central core. Na⁺ or Li⁺ are less efficiently transported by valinomycin, because their size does not match with the size of the central core of the molecule.

Another antibiotic compound, non-actin, has also a circular structure and is known to transport K⁺ across mitochondrial membrane.

Its structure is shown in Fig. 8.87B:

According to one hypothesis, valinomycin acts as a carrier and the molecule diffuses back and forth across the membrane lipid bilayer transporting K⁺ across the membrane.

The positive charge of the ion is masked by the negatively charged core of the valinomycin molecule (Fig. 8.88):

Gramicidin A is a polypeptide antibiotic consisting of a linear chain of 15 amino acid residues. Two gramicidin molecules joined by H-bonds at their N-terminal ends form a helix spanning across the membrane. Through the central channel of the helix, monovalent cations like H⁺, NH₄⁺, K⁺, Na⁺ and Li⁺ may be transported into the cell.

The order of preference of transportable ions is as mentioned i.e. H⁺ has the highest preference and Li⁺ the lowest.

The structure of gramicidin A molecule and the postulated helix formed by a pair of gramicidin molecules in the lipid bilayer of the membrane are shown in Fig. 8.89 A and B: