Concentration Gradient; Within a System, Different Substances in the Medium Will Each Diffuse at Different Rates According to Their Individual Gradients

Home , Facilitated diffusion, Potocytosis, Uniporter

Biomolecules Biological Macromolecules

• Life depends on four types of organic macromolecules: 1. Carbohydrates 2. Lipids 3. Proteins 4. Nucleic acids 1. Carbohydrates

• Contain carbon, hydrogen and oxygen in a ratio of 1:2:1 • Account for less that 1% of body weight • Used as energy source • Called saccharides Carbohydrates

• Compounds containing C, H and O

• General formula : Cx(H2O)y • All have C=O and -OH functional groups. • Classified based on • Size of base carbon chain • Number of sugarunits • Location of C=O • Stereochemistry Types of carbohydrates

• Classifications based on number of sugarunits in total chain. • Monosaccharides - single sugarunit • Disaccharides - two sugarunits • Oligosaccharides - 2 to 10 sugarunits • Polysaccharides - more than 10units • Chaining relies on ‘bridging’ of oxygenatoms • glycoside bonds

Monosaccharides • Based on location of C=O

Aldose Ketose - aldehyde C=O - ketone C=O

triose tetrose pentose hexose Can be either aldose or ketose sugar. Stereoisomers • Stereochemistry • Study of the spatial arrangement ofmolecules.

• Stereoisomers have • the same order and types of bonds. • different spatial arrangements. • different properties. • Many biologically importantchemicals, like sugars, exist as stereoisomers. Your body can tell the difference. Chiral center

Asymmetric carbon - 4 different things are attached to it. • Cl • | • I - C - F • | • Br Chiral center • You must have at least one asymmetric carbon to havestereoisomers

Some important monosaccharides

• D-glyceraldehyde Simplest sugar • D-glucose Most important indiet • D-fructose Sweetest of allsugars • D-galactose Part of milk sugar • D-ribose Used in RNA

• note that each is a D-enantiomer

Hemiacetal & hemiketal formation

H H An aldehyde can C O react with an + R' OH R' O C OH alcohol to form R R a hemiacetal. aldehyde alcohol hemiacetal

A ketone can R R react with an C O + "R OH "R O C OH alcohol to form R' R' a hemiketal. ketone alcohol hemiketal

Pentoses and 1CHO H C OH hexoses can cyclize 2 as the ketone or HO C H 3 D-glucose aldehyde reacts H C OH (linear form) with a distalOH. 4 H C OH Glucose forms an 5 CH OH intra-molecular 6 2 hemiacetal, as the 6CH2OH 6 CH2OH C1aldehyde & C5 5 5 H O H H O OH OHreact, to form H H a 6-member 4 OH H 1 4 OH H 1 pyranose ring, OH OH OH H 3 2 3 2 named after pyran. H OH H OH -D-glucose -D-glucose These representations of the cyclic sugars are called Haworth projections.

6CH2OH 6 CH2OH 5 5 H O H H O OH H H 4 OH H 1 4 OH H 1 OH OH OH H 3 2 3 2 H OH H OH -D-glucose -D-glucose

Cyclization of glucose produces a new asymmetriccenter at C1. The 2 stereoisomers are called anomers,  & . Haworth projections represent the cyclic sugars ashaving essentially planar rings, with the OH at theanomeric C1:   (OH below the ring)   (OH above the ring). Monosaccharides can be oxidized by relatively mild oxidizing agents such as ferric (Fe3+) or cupric (Cu2+) ion . The carbonyl carbon is oxidized to a carboxyl group. Glucose and other sugars capable of reducing ferric or cupric ion are called reducing sugars. Glycosidic Bonds

The anomeric hydroxyl and a hydroxyl of another sugar or some other compound can join together, splitting out water to forma glycosidic bond:

R-OH + HO-R'  R-O-R' + H2O E.g., methanol reacts with the anomeric OH onglucose to form methylglucoside (methyl-glucopyranose).

H OH H OH

H O H2O H O HO HO HO H + CH3-OH HO H H OH H OH

H OH H OCH3 -D-glucopyranose methanol methyl--D-glucopyranose Glycoside formation

•  or  -OH group of cyclicmonosaccharide can form link with another one (ormore).

CH2OH CH2OH H O OH H O H H H • glycosidic bond OH H H OH OH H OH OH H OH OH H

• sugar -O- sugar CH2OH CH2OH H O H O H H H OH H o OH H OH • oxygen bridge OH H OH H H OH + H2 O

Types Of (O-Glycosidic) Bonding

In O-Glycosidic Bonding There are Two Types of Linkages

1) Alpha Linkage 2) Beta Linkage • Maltose is a disaccharide, formed by two glucose units. It occurs in cells as a breakdown product of starch. It is also formed in the seeds during germination. It is commonly called malt sugar. • The glycosidic bond of maltoseis formed between the OH of carbon 1 and carbon 4 of 2 glucose monomers. Therefore it forms an alpha (1–> 4) glycosidic bond. Lactose is a disaccharide found in milk and hence commonly called milk sugar. It is formed by condensation of a glucose molecule and a galactose molecule.

composed of a glucose and a galactose monomer. They form a beta (1–> 4) glycosidic bond. Sucrose is disaccharide found extensively in plants. It is commonly called cane sugar. It is formed by a condensation of a molecule of glucose and a molecule of fructose.

Sucrose links the anomeric hydroxyls of glucose and fructose to form an alpha (1–>2) glycosidic bond. Cellulose

The most abundant natural polymer found in the world. Found in the cell wall of almost all plants, it provides physical structure and strength.

Cellulose is a straight chain polysaccharide with beta (1–>4) linkages. STARCH

 Amylose is a straight chain with alpha (1–>4) linkages

 Amylopectin is an alpha (1–>4) chain with alpha (1–>6) .

Reference books:-

1. Campbell, MK (2012) Biochemistry, 7th ed., Published by Cengage Learning.

2. Campbell, PN and Smith AD (2011) Biochemistry Illustrated, 4th ed., Published by Churchill Livingstone.

3. Tymoczko JL, Berg JM and Stryer L (2012) Biochemistry: A short course, 2nd ed., W.H.Freeman.

4. Berg JM, Tymoczko JL and Stryer L (2011) Biochemistry, W.H.Freeman and Company.

5. Nelson DL and Cox MM (2008) Lehninger Principles of Biochemistry, 5th Edition., W.H. Freeman

6. Willey MJ, Sherwood, LM & Woolverton C J (2013) Prescott, Harley and Klein’s Microbiology by. 9th Ed., McGrawHill.

7. Voet,D. and Voet J.G (2004) Biochemistry 3rd edition, John Wiley and Sons. Carbohydrate Representations Based on a Fall 2005 Chemistry 14D honors project

Some Useful Vocabulary:

 Aldose: A polyhydroxy aldehyde, i.e., a carbohydrate containing an aldehyde functional group.

 Ketose: A polyhydroxy ketone, i.e., a carbohydrate containing a ketone functional group.

 Furanose: A five-member closed chain form of a monosaccharide.

 Pyranose: A six-member cyclic form of a monosaccharide.

 Fischer Projection: A way of representing an acyclic (open chain) carbohydrate structure. Vertical lines point away from the viewer and horizontal lines point toward the viewer.

 Haworth Projection: A way of representing a cyclic (closed chain) carbohydrate. Substituents can either point up or down on this ring.

 Chair Conformation: The most stable conformation of cyclohexane that resembles a chair.

 Monosaccharide: A single sugar. A carbohydrate that cannot be broken down into a simpler carbohydrate.

 Anomeric carbon: The carbon in a cyclic sugar that is the carbonyl carbon in the open- chain (acyclic) form.

Carbohydrates are the most abundant class of bioorganic compounds in the biological world. They constitute most of Earth’s biomass, from tiny structural components of cells, to food we eat for metabolic energy. To better understand the role carbohydrates play in the biological world, a basic chemical understanding of how carbohydrates are formed and represented in their simplest form is essential.

In organic chemistry, monosaccharides, the simplest carbohydrates are represented in three ways: the Fischer projection, Haworth projection, and the chair conformation of D-glucose (Figure 1). By the time you are finished reading this tutorial, you will have learned how to represent monosaccharides in these three ways. Figure 1

Monosaccharides, also known as saccharides, are carbohydrates that cannot be broken down into simpler carbohydrates. They can be polyhydroxy aldehydes or polyhydroxy ketones because they have either an aldehyde or a ketone group, along with –OH substituted carbons in a chain.

Polyhydroxy aldehydes are called aldoses. Polyhydroxy ketones are called ketoses. The suffix “ose” means sugar, while the prefixes “ald” and “ket” are used for aldehyde and ketone, respectively.

Classification of monosaccharides is based on the number of carbons they contain (Table 1).

Table 1

Number of Name Aldose Form Ketose Form carbons in monosaccharide 3 Triose

aldotriose ketotriose 4 Tetrose

aldotetrose ketotetrose

Tutorial: Carbohydrate Representations 2 5 Pentose

aldopentose ketopentose 6 Hexose

aldohexose ketohexose

Monosaccharides can exist in an open chain (acyclic) form, or in closed chain (cyclic) form. The open chain form of monosaccharides is illustrated with Fischer projections. A Haworth projection can be used to represent the cyclic form of monosaccharides. The five-member closed chain form of a monosaccharide is known as a furanose, while the six-member cyclic form of a monosaccharide is known as a pyranose. Often six-member monosaccharide rings can also be represented in chair conformation.

Figure 2 shows pyranose and furanose rings. Substituents have been omitted so that the ring structure can be emphasized.

Figure 2

Imagine we are asked to show the Fischer, Haworth, and chair conformation of _ and _ D-glucose. How do we go about doing this? There are many things to consider; however, there are also some rules that will simplify the process. Let’s walk through how this problem can be approached.

A Fischer projection shows the skeleton of the acyclic monosaccharide (Figure 3). Glucose is an aldohexose. This means that the top of the Fischer projection of glucose contains an aldehyde group and that there are six carbons in the polyhydroxy chain. In Figure 3, the structure on the right shows wedges and dashes to indicate how the sugar looks in three dimensions.

Tutorial: Carbohydrate Representations 3 Figure 3

D and L Notation:

We need to make sure that the glucose is a D-glucose. We do this by looking at the monosaccharide with the ketone or aldehyde group on top; if the –OH group on the bottom-most asymmetric carbon is on the left side, the notation is L; if the –OH group on the bottom-most asymmetric carbon is on the right side, the notation is D (Figure 3).

Figure 4 This oxygen becomes protonated H O and singly bonded to the carbon.

H OH

HO H

H OH

This oxygen loses its hydrogen and H OH attacks the carbon of the carbonyl (C=O)

CH2OH

Next, we rotate the C-5 carbon’s substituents once over to put –OH on the vertical plane. This shift shows us what direction the –CH2OH group with point in the Haworth projection. A substituent on the right side of the vertical line in a Fischer projection will be pointing down in a Haworth projection. Conversely, a substituent on the left side in a Fischer projection will be pointing up in a Haworth projection. (Figure 5)

Figure 5

Tutorial: Carbohydrate Representations 4 Therefore, the –CH2OH group will be pointing up in the Haworth projection. Moreover, the–OH group on the C-2 will be pointing down and its –H substituent will be pointing up. And, the –OH group on the C-3 will be pointing up while its –H substituent points down, and so on.

In the next step, the carbonyl oxygen becomes protonated and singly bonded to C-1. The –OH group on C-5 becomes deprotonated and forms an O-C bond with C-1, resulting in a six- membered ring. At this point, it is important to recognize the difference between alpha () and beta () glucose (Figure 6).

The  or  configuration is determined by looking at the anomeric carbon which is the carbon that is between the two oxygen atoms of the hemiacetal or acetal. In a cyclic sugar, the anomeric carbon is the carbonyl carbon in the acyclic form. (C-1 in our case)

In an  monosaccharide, the –OH group attached to the anomeric carbon is on the right side of the Fischer projection, points down in the Haworth projection, and is axial in the chair conformation.

In a  monosaccharide, the –OH group attached to the anomeric carbon is on the left side of the Fischer projection, points up in the Haworth projection, and is equatorial in the chair conformation.

The table below summarizes the translation between Fischer projections, Haworth projections, and chair conformation. The translations for the chair conformation are used to determine  and  configurations and only concern the anomeric carbon’s substituent. The Fischer and Haworth projection translations apply to all substituents on the carbon chain.

Figure 6

Table 2

Fischer Projection Haworth Projection Configuration Chair Conformation

Right Down  Axial

Left Up  Equatorial

Tutorial: Carbohydrate Representations 5

In -D-glucose the anomeric carbon’s –OH group is on the left. In the Haworth projection this alcohol group points up. The other substituents point up in the Haworth projection if they are on the left side in the Fischer projection, and point down if they are on the right side in the Fischer projection. (Figure 7)

Figure 7

In -D-glucose the anomeric carbon’s –OH group is on the right. In the Haworth projection of -D-glucose illustrated below the –OH group points down. Once again, the rest of the substituents also follow the translation rule from Fischer to Haworth. (Figure 7)

Take a moment to compare the Fischer and Haworth projections and notice how one form is translated to the other form.

When going from the Haworth projection to the chair conformation, the anomeric carbon’s substituent that points down in the Haworth projection is going to be axial, and the substituent that points up in the Haworth projection is going to be equatorial. An axial –OH on the anomeric carbon makes the sugar an  sugar, while an equatorial –OH on the anomeric carbon makes the monosaccharide a  sugar. Besides the substituents on the anomeric carbon, everything else is drawn relative to the Haworth projection. In other words, all the other substituents are drawn pointing up if they were pointing up in the Haworth projection, and pointing down if they were pointing down in the Haworth projection. (Figure 8)

It helps to number the carbons in the monosaccharide in the Haworth projection and chair conformation to prevent any careless mistakes.

Tutorial: Carbohydrate Representations 6 Figure 8

There is a shortfall in the method we have been using to determine the difference between an  and  sugar in the chair conformation. (This is also the method used in Bruice.) For example, it is true that in the chair conformational isomer that we drew that the axial –OH group of the anomeric carbon was  while the equatorial –OH group of the anomeric carbon was . But a ring flip would make this fact untrue (Figure 9). An -D-glucose would still be an -D-glucose after a ring flip, but the –OH group attached to the anomeric carbon would no longer be axial! (To learn more about ring flips, refer to your textbook and class notes)

Figure 9

Tutorial: Carbohydrate Representations 7 The method we have been using works, but only when the chair conformational isomer is drawn so that the anomeric carbon is at the bottom right corner. However, a less restricted method that is always true exists. As long as the primary alcohol, CH2OH, and the anomeric –OH group are on opposite sides of the ring, as in the trans isomer, it is . Moreover, if the primary alcohol and the anomeric –OH group are on the same side of the ring as in the cis isomer, it is . For example, in the illustration below, notice that when CH2OH points up, the anomeric –OH group points down (opposite side of the pyranose). This is the trans isomer, and thus an -pyranose. (Figure 10)

Figure 10

In -glucose, or any  monosaccharide, the pyranose would be the cis isomer. Thus, when CH2OH points above the plane of the ring, the anomeric –OH group will point up as well. Conversely, when the CH2OH points down, the anomeric –OH group will also point down. (Figure 11)

Figure 11

To get a better understanding of this concept, build models with your molecular model building kit.

The cyclic monosaccharides in this example are pyranoses because they have a six-membered ring. -D-glucose can also be called -D-glucopyranose. You may be asked specifically to form another type of cyclic sugar, such as a furanose. In that case the –OH that becomes deprotonated and attacks the carbonyl carbon may be different than the one from the last stereocenter. (Figure 12)

Tutorial: Carbohydrate Representations 8 Figure 12

As long as all the rules are applied consistently, the transition from Fischer projection to Haworth projection to chair conformation will be a simple one. The chair conformation can only be done with pyranoses. The following are some practice problems to help you strengthen this skill.

Practice Problems Solutions start on page 10.

1. Make a table with the Fischer, Haworth, and chair conformation (when applicable, i.e. when a six-membered ring exists) for each of the following sugars: (a) b-D-galactose, (b) a-D- ribose, and (c) b-D-fructose.

1. Draw the Haworth projection of as a -pyranose anomer of the sugar given below:

1. Draw the chair conformation of -D-glucopyranose.

1. The following questions apply to the sugar A. (a) Is this sugar a ketose or an aldose? (b) Draw an arrow pointing to the anomeric carbon. (c) Box the OH group attached to C-4. (d) Is this the  or  anomer? Sugar A

Tutorial: Carbohydrate Representations 9 Practice Problem Solutions

1. Fischer projection Haworth projection Chair conformation

(a)

(b) Furanoses do not have chair

HO OH conformations HO OH HOCH2 OH O (c) HO Furanoses do not have chair

conformations CH2OH HO

Hydrogens can be omitted from Haworth projections to avoid cluttering, but do not draw a stick instead of a hydrogen atom. As in all bond-line or “stick” structures, a stick that is only attached to one other atom represents a methyl group.

HOCH2 OH HOCH2 OH O O HO HO

CH2OH CH2OH HO HO Correct Haworth projection Too many methyl groups!

CH2OH 1. HO O OH

CH2OH

OH OH

1. (a) Ketose.

Tutorial: Carbohydrate Representations 10

(b)-(d)

Works Cited 1. Lecture Notes and practice problems from Professor William Nguyen. (Summer 2004, 14C)

2. Bruice, Organic Chemistry, Fourth Edition.

3. 14C Thinkbook for Fall 2004, Professor Hardinger.

4. I want to thank Professor Hardinger and Mark Smuckler for helping me edit this tutorial.

Tutorial: Carbohydrate Representations 11

Transport Across Membranes

Diffusion

Diffusion is a process of passive transport in which molecules move from an area of higher concentration to one of lower concentration.

Key Points

 Substances diffuse according to their concentration gradient; within a system, different substances in the medium will each diffuse at different rates according to their individual gradients.  After a substance has diffused completely through a space, removing its concentration gradient, molecules will still move around in the space, but there will be no net movement of the number of molecules from one area to another, a state known as dynamic equilibrium.  Several factors affect the rate of diffusion of a solute including the mass of the solute, the temperature of the environment, the solvent density, and the distance traveled.

Key Terms

 diffusion: The passive movement of a solute across a permeable membrane  concentration gradient: A concentration gradient is present when a membrane separates two different concentrations of molecules.

Examples

When someone is cooking food in a kitchen, the smell begins to waft through the house, and eventually everyone can tell what‟s for dinner! This is due to the diffusion of odor molecules through the air, from an area of high concentration (the kitchen) to areas of low concentration (your upstairs bedroom).

Diffusion is a passive process of transport. A single substance tends to move from an area of high concentration to an area of low concentration until the concentration is equal across a space. You are familiar with diffusion of substances through the air. For example, think about someone opening a bottle of ammonia in a room filled with people. The ammonia gas is at its highest concentration in the bottle; its lowest concentration is at the edges of the room. The ammonia vapor will diffuse, or spread away, from the bottle; gradually, more and more people will smell the ammonia as it spreads. Materials move within the cell „s cytosol by diffusion, and certain materials move through the plasma membrane by diffusion. Diffusion expends no energy. On the contrary, concentration gradients are a form of potential energy, dissipated as the gradient is eliminated.

Diffusion: Diffusion through a permeable membrane moves a substance from an area of high concentration (extracellular fluid, in this case) down its concentration gradient (into the cytoplasm).

Each separate substance in a medium, such as the extracellular fluid, has its own concentration gradient independent of the concentration gradients of other materials. In addition, each substance will diffuse according to that gradient. Within a system, there will be different rates of diffusion of the different substances in the medium.

Factors That Affect Diffusion

Molecules move constantly in a random manner at a rate that depends on their mass, their environment, and the amount of thermal energy they possess, which in turn is a function of temperature. This movement accounts for the diffusion of molecules through whatever medium in which they are localized. A substance will tend to move into any space available to it until it is evenly distributed throughout it. After a substance has diffused completely through a space removing its concentration gradient, molecules will still move around in the space, but there will be no net movement of the number of molecules from one area to another. This lack of a concentration gradient in which there is no net movement of a substance is known as dynamic equilibrium. While diffusion will go forward in the presence of a concentration gradient of a substance, several factors affect the rate of diffusion:

 Extent of the concentration gradient: The greater the difference in concentration, the more rapid the diffusion. The closer the distribution of the material gets to equilibrium, the slower the rate of diffusion becomes.  Mass of the molecules diffusing: Heavier molecules move more slowly; therefore, they diffuse more slowly. The reverse is true for lighter molecules.  Temperature: Higher temperatures increase the energy and therefore the movement of the molecules, increasing the rate of diffusion. Lower temperatures decrease the energy of the molecules, thus decreasing the rate of diffusion.  Solvent density: As the density of a solvent increases, the rate of diffusion decreases. The molecules slow down because they have a more difficult time getting through the denser medium. If the medium is less dense, diffusion increases. Because cells primarily use diffusion to move materials within the cytoplasm, any increase in the cytoplasm‟s density will inhibit the movement of the materials. An example of this is a person experiencing dehydration. As the body‟s cells lose water, the rate of diffusion decreases in the cytoplasm, and the cells‟ functions deteriorate. Neurons tend to be very sensitive to this effect. Dehydration frequently leads to unconsciousness and possibly coma because of the decrease in diffusion rate within the cells.  Solubility: As discussed earlier, nonpolar or lipid-soluble materials pass through plasma membranes more easily than polar materials, allowing a faster rate of diffusion.  Surface area and thickness of the plasma membrane: Increased surface area increases the rate of diffusion, whereas a thicker membrane reduces it.  Distance travelled: The greater the distance that a substance must travel, the slower the rate of diffusion. This places an upper limitation on cell size. A large, spherical cell will die because nutrients or waste cannot reach or leave the center of the cell. Therefore, cells must either be small in size, as in the case of many prokaryotes, or be flattened, as with many single-celled eukaryotes. A variation of diffusion is the process of filtration. In filtration, material moves according to its concentration gradient through a membrane; sometimes the rate of diffusion is enhanced by pressure, causing the substances to filter more rapidly. This occurs in the kidney where blood pressure forces large amounts of water and accompanying dissolved substances, or solutes, out of the blood and into the renal tubules. The rate of diffusion in this instance is almost totally dependent on pressure. One of the effects of high blood pressure is the appearance of protein in the urine, which is “squeezed through” by the abnormally high pressure. Osmosis

Osmosis is the movement of water across a membrane from an area of low solute concentration to an area of high solute concentration.

Key Takeaways

Key Points

 Osmosis occurs according to the concentration gradient of water across the membrane, which is inversely proportional to the concentration of solutes.  Osmosis occurs until the concentration gradient of water goes to zero or until the hydrostatic pressure of the water balances the osmotic pressure.  Osmosis occurs when there is a concentration gradient of a solute within a solution, but the membrane does not allow diffusion of the solute.

Key Terms

 solute: Any substance that is dissolved in a liquid solvent to create a solution  osmosis: The net movement of solvent molecules from a region of high solvent potential to a region of lower solvent potential through a partially permeable membrane  semipermeable membrane: A type of biological membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally by specialized facilitated diffusion

Osmosis and Semipermeable Membranes

Osmosis is the movement of water through a semipermeable membrane according to the concentration gradient of water across the membrane, which is inversely proportional to the concentration of solutes. Semipermeable membranes, also termed selectively permeable membranes or partially permeable membranes, allow certain molecules or ions to pass through by diffusion.

While diffusion transports materials across membranes and within cells, osmosis transports only water across a membrane. The semipermeable membrane limits the diffusion of solutes in the water. Not surprisingly, the aquaporin proteins that facilitate water movement play a large role in osmosis, most prominently in red blood cells and the membranes of kidney tubules.

Mechanism of Osmosis

Osmosis is a special case of diffusion. Water, like other substances, moves from an area of high concentration to one of low concentration. An obvious question is what makes water move at all? Imagine a beaker with a semipermeable membrane separating the two sides or halves. On both sides of the membrane the water level is the same, but there are different concentrations of a dissolved substance, or solute, that cannot cross the membrane (otherwise the concentrations on each side would be balanced by the solute crossing the membrane). If the volume of the solution on both sides of the membrane is the same but the concentrations of solute are different, then there are different amounts of water, the solvent, on either side of the membrane. If there is more solute in one area, then there is less water; if there is less solute in one area, then there must be more water. To illustrate this, imagine two full glasses of water. One has a single teaspoon of sugar in it, whereas the second one contains one-quarter cup of sugar. If the total volume of the solutions in both cups is the same, which cup contains more water? Because the large amount of sugar in the second cup takes up much more space than the teaspoon of sugar in the first cup, the first cup has more water in it.

Osmosis: In osmosis, water always moves from an area of higher water concentration to one of lower concentration. In the diagram shown, the solute cannot pass through the selectively permeable membrane, but the water can.

Returning to the beaker example, recall that it has a mixture of solutes on either side of the membrane. A principle of diffusion is that the molecules move around and will spread evenly throughout the medium if they can. However, only the material capable of passing through the membrane will diffuse through it. In this example, the solute cannot diffuse through the membrane, but the water can. Water has a concentration gradient in this system. Thus, water will diffuse down its concentration gradient, crossing the membrane to the side where it is less concentrated. This diffusion of water through the membrane—osmosis—will continue until the concentration gradient of water goes to zero or until the hydrostatic pressure of the water balances the osmotic pressure. In the beaker example, this means that the level of fluid in the side with a higher solute concentration will go up. Tonicity

Tonicity, which is directly related to the osmolarity of a solution, affects osmosis by determining the direction of water flow.

Key Points

 Osmolarity describes the total solute concentration of a solution; solutions with a low solute concentration have a low osmolarity, while those with a high osmolarity have a high solute concentration.  Water moves from the side of the membrane with lower osmolarity (and more water) to the side with higher osmolarity (and less water).  In a hypotonic solution, the extracellular fluid has a lower osmolarity than the fluid inside the cell; water enters the cell.  In a hypertonic solution, the extracellular fluid has a higher osmolarity than the fluid inside the cell; water leaves the cell.  In an isotonic solution, the extracellular fluid has the same osmolarity as the cell; there will be no net movement of water into or out of the cell.

Key Terms  osmolarity: The osmotic concentration of a solution, normally expressed as osmoles of solute per litre of solution.  hypotonic: Having a lower osmotic pressure than another; a cell in this environment causes water to enter the cell, causing it to swell.  hypertonic: having a greater osmotic pressure than another  isotonic: having the same osmotic pressure

Examples

Tonicity is the reason why salt water fish cannot live in fresh water and vice versa. A salt water fish‟s cells have evolved to have a very high solute concentration to match the high osmolarity of the salt water they live in. If you place a salt water fish in fresh water, which has a low osmolarity, water in the environment will flow into the cells of the fish, eventually causing them to burst and killing the fish.

Tonicity describes how an extracellular solution can change the volume of a cell by affecting osmosis. A solution‟s tonicity often directly correlates with the osmolarity of the solution. Osmolarity describes the total solute concentration of the solution. A solution with low osmolarity has a greater number of water molecules relative to the number of solute particles; a solution with high osmolarity has fewer water molecules with respect to solute particles. In a situation in which solutions of two different osmolarities are separated by a membrane permeable to water, though not to the solute, water will move from the side of the membrane with lower osmolarity (and more water) to the side with higher osmolarity (and less water). This effect makes sense if you remember that the solute cannot move across the membrane, and thus the only component in the system that can move—the water—moves along its own concentration gradient. An important distinction that concerns living systems is that osmolarity measures the number of particles (which may be molecules) in a solution. Therefore, a solution that is cloudy with cells may have a lower osmolarity than a solution that is clear if the second solution contains more dissolved molecules than there are cells.

Hypotonic Solutions

Three terms—hypotonic, isotonic, and hypertonic—are used to relate the osmolarity of a cell to the osmolarity of the extracellular fluid that contains the cells. In a hypotonic situation, the extracellular fluid has lower osmolarity than the fluid inside the cell, and water enters the cell. (In living systems, the point of reference is always the cytoplasm, so the prefix hypo- means that the extracellular fluid has a lower concentration of solutes, or a lower osmolarity, than the cell cytoplasm. ) It also means that the extracellular fluid has a higher concentration of water in the solution than does the cell. In this situation, water will follow its concentration gradient and enter the cell, causing the cell to expand.

Changes in Cell Shape Due to Dissolved Solutes: Osmotic pressure changes the shape of red blood cells in hypertonic, isotonic, and hypotonic solutions.

Hypertonic Solutions As for a hypertonic solution, the prefix hyper- refers to the extracellular fluid having a higher osmolarity than the cell‟s cytoplasm; therefore, the fluid contains less water than the cell does. Because the cell has a relatively higher concentration of water, water will leave the cell, and the cell will shrink.

Isotonic Solutions

In an isotonic solution, the extracellular fluid has the same osmolarity as the cell. If the osmolarity of the cell matches that of the extracellular fluid, there will be no net movement of water into or out of the cell, although water will still move in and out.

Blood cells and plant cells in hypertonic, isotonic, and hypotonic solutions take on characteristic appearances. Cells in an isotonic solution retain their shape. Cells in a hypotonic solution swell as water enters the cell, and may burst if the concentration gradient is large enough between the inside and outside of the cell. Cells in a hypertonic solution shrink as water exits the cell, becoming shriveled. Facilitated transport

Facilitated diffusion is a process by which molecules are transported across the plasma membrane with the help of membrane proteins.

Key Takeaways

Key Points

 A concentration gradient exists that would allow ions and polar molecules to diffuse into the cell, but these materials are repelled by the hydrophobic parts of the cell membrane.  Facilitated diffusion uses integral membrane proteins to move polar or charged substances across the hydrophobic regions of the membrane.  Channel proteins can aid in the facilitated diffusion of substances by forming a hydrophilic passage through the plasma membrane through which polar and charged substances can pass.  Channel proteins can be open at all times, constantly allowing a particular substance into or out of the cell, depending on the concentration gradient; or they can be gated and can only be opened by a particular biological signal.  Carrier proteins aid in facilitated diffusion by binding a particular substance, then altering their shape to bring that substance into or out of the cell.

Key Terms

 facilitated diffusion: The spontaneous passage of molecules or ions across a biological membrane passing through specific transmembrane integral proteins.  membrane protein: Proteins that are attached to, or associated with the membrane of a cell or an organelle.

Examples

Channel-mediated facilitated diffusion functions much like a bridge over a river that must raise and lower in order to allow boats to pass. When the bridge is lowered, boats cannot pass through to the other side of the river. Similarly, a gated channel protein often remains closed, not allowing substances into the cell until it receives a signal (like the binding of an ion) to open. When this signal is received, the bridge (gate) opens, allowing the boats (substance) to pass through the bridge and into the other side of the river (cell).

Facilitated transport is a type of passive transport. Unlike simple diffusion where materials pass through a membrane without the help of proteins, in facilitated transport, also called facilitated diffusion, materials diffuse across the plasma membrane with the help of membrane proteins. A concentration gradient exists that would allow these materials to diffuse into the cell without expending cellular energy. However, these materials are ions or polar molecules that are repelled by the hydrophobic parts of the cell membrane. Facilitated transport proteins shield these materials from the repulsive force of the membrane, allowing them to diffuse into the cell.

The material being transported is first attached to protein or glycoprotein receptors on the exterior surface of the plasma membrane. This allows the material that is needed by the cell to be removed from the extracellular fluid. The substances are then passed to specific integral proteins that facilitate their passage. Some of these integral proteins are collections of beta-pleated sheets that form a channel through the phospholipid bilayer. Others are carrier proteins which bind with the substance and aid its diffusion through the membrane.

Channels

Channel Proteins in Facilitated Transport: Facilitated transport moves substances down their concentration gradients. They may cross the plasma membrane with the aid of channel proteins.

The integral proteins involved in facilitated transport are collectively referred to as transport proteins; they function as either channels for the material or carriers. In both cases, they are transmembrane proteins. Channels are specific for the substance that is being transported. Channel proteins have hydrophilic domains exposed to the intracellular and extracellular fluids; they additionally have a hydrophilic channel through their core that provides a hydrated opening through the membrane layers. Passage through the channel allows polar compounds to avoid the nonpolar central layer of the plasma membrane that would otherwise slow or prevent their entry into the cell. Aquaporins are channel proteins that allow water to pass through the membrane at a very high rate.

Channel proteins are either open at all times or they are “gated,” which controls the opening of the channel. The attachment of a particular ion to the channel protein may control the opening or other mechanisms or substances may be involved. In some tissues, sodium and chloride ions pass freely through open channels, whereas in other tissues, a gate must be opened to allow passage. An example of this occurs in the kidney, where both forms of channels are found in different parts of the renal tubules. Cells involved in the transmission of electrical impulses, such as nerve and muscle cells, have gated channels for sodium, potassium, and calcium in their membranes. Opening and closing of these channels changes the relative concentrations on opposing sides of the membrane of these ions, resulting in the facilitation of electrical transmission along membranes (in the case of nerve cells) or in muscle contraction (in the case of muscle cells).

Carrier Proteins Another type of protein embedded in the plasma membrane is a carrier protein. This protein binds a substance and, in doing so, triggers a change of its own shape, moving the bound molecule from the outside of the cell to its interior; depending on the gradient, the material may move in the opposite direction. Carrier proteins are typically specific for a single substance. This adds to the overall selectivity of the plasma membrane. The exact mechanism for the change of shape is poorly understood. Proteins can change shape when their hydrogen bonds are affected, but this may not fully explain this mechanism. Each carrier protein is specific to one substance, and there are a finite number of these proteins in any membrane. This can cause problems in transporting enough of the material for the cell to function properly.

Carrier Proteins: Some substances are able to move down their concentration gradient across the plasma membrane with the aid of carrier proteins. Carrier proteins change shape as they move molecules across the membrane.

An example of this process occurs in the kidney. Glucose, water, salts, ions, and amino acids needed by the body are filtered in one part of the kidney. This filtrate, which includes glucose, is then reabsorbed in another part of the kidney. Because there are only a finite number of carrier proteins for glucose, if more glucose is present than the proteins can handle, the excess is not transported; it is excreted from the body in the urine. In a diabetic individual, this is described as “spilling glucose into the urine.” A different group of carrier proteins called glucose transport proteins, or GLUTs, are involved in transporting glucose and other hexose sugars through plasma membranes within the body.

Channel and carrier proteins transport material at different rates. Channel proteins transport much more quickly than do carrier proteins. Channel proteins facilitate diffusion at a rate of tens of millions of molecules per second, whereas carrier proteins work at a rate of a thousand to a million molecules per second. The Role of Passive Transport

Passive transport, such as diffusion and osmosis, moves materials of small molecular weight across membranes.

Key Points

 Plasma membranes are selectively permeable; if they were to lose this selectivity, the cell would no longer be able to sustain itself.  In passive transport, substances simply move from an area of higher concentration to an area of lower concentration, which does not require the input of energy.  Concentration gradient, size of the particles that are diffusing, and temperature of the system affect the rate of diffusion.  Some materials diffuse readily through the membrane, but others require specialized proteins, such as channels and transporters, to carry them into or out of the cell.

Key Terms

 concentration gradient: A concentration gradient is present when a membrane separates two different concentrations of molecules.  passive transport: A movement of biochemicals and other atomic or molecular substances across membranes that does not require an input of chemical energy.  permeable: Of or relating to substance, substrate, membrane or material that absorbs or allows the passage of fluids.

Introduction: Passive Transport

Plasma membranes must allow or prevent certain substances from entering or leaving a cell. In other words, plasma membranes are selectively permeable; they allow some substances to pass through, but not others. If they were to lose this selectivity, the cell would no longer be able to sustain itself, and it would be destroyed. Some cells require larger amounts of specific substances than other cells; they must have a way of obtaining these materials from extracellular fluids. This may happen passively, as certain materials move back and forth, or the cell may have special mechanisms that facilitate transport. Some materials are so important to a cell that it spends some of its energy (hydrolyzing adenosine triphosphate (ATP)) to obtain these materials. Red blood cells use some of their energy to do this. All cells spend the majority of their energy to maintain an imbalance of sodium and potassium ions between the interior and exterior of the cell.

The most direct forms of membrane transport are passive. Passive transport is a naturally-occurring phenomenon and does not require the cell to exert any of its energy to accomplish the movement. In passive transport, substances move from an area of higher concentration to an area of lower concentration. A physical space in which there is a range of concentrations of a single substance is said to have a concentration gradient.

Passive Transport: Diffusion is a type of passive transport. Diffusion through a permeable membrane moves a substance from an area of high concentration (extracellular fluid, in this case) down its concentration gradient (into the cytoplasm).

The passive forms of transport, diffusion and osmosis, move materials of small molecular weight across membranes. Substances diffuse from areas of high concentration to areas of lower concentration; this process continues until the substance is evenly distributed in a system. In solutions containing more than one substance, each type of molecule diffuses according to its own concentration gradient, independent of the diffusion of other substances. Many factors can affect the rate of diffusion, including, but not limited to, concentration gradient, size of the particles that are diffusing, and temperature of the system. In living systems, diffusion of substances in and out of cells is mediated by the plasma membrane. Some materials diffuse readily through the membrane, but others are hindered; their passage is made possible by specialized proteins, such as channels and transporters. The chemistry of living things occurs in aqueous solutions; balancing the concentrations of those solutions is an ongoing problem. In living systems, diffusion of some substances would be slow or difficult without membrane proteins that facilitate transport. Primary Active Transport

The sodium-potassium pump maintains the electrochemical gradient of living cells by moving sodium in and potassium out of the cell.

Key Points

 The sodium-potassium pump moves K+ into the cell while moving Na+ at a ratio of three Na+ for every two K+ ions.  When the sodium-potassium- ATPase enzyme points into the cell, it has a high affinity for sodium ions and binds three of them, hydrolyzing ATP and changing shape.  As the enzyme changes shape, it reorients itself towards the outside of the cell, and the three sodium ions are released.  The enzyme‟s new shape allows two potassium to bind and the phosphate group to detach, and the carrier protein repositions itself towards the interior of the cell.  The enzyme changes shape again, releasing the potassium ions into the cell.  After potassium is released into the cell, the enzyme binds three sodium ions, which starts the process over again.

Key Terms

 electrogenic pump: An ion pump that generates a net charge flow as a result of its activity.  Na+-K+ ATPase: An enzyme located in the plasma membrane of all animal cells that pumps sodium out of cells while pumping potassium into cells.

Primary Active Transport

The primary active transport that functions with the active transport of sodium and potassium allows secondary active transport to occur. The secondary transport method is still considered active because it depends on the use of energy as does primary transport.

Active Transport of Sodium and Potassium: Primary active transport moves ions across a membrane, creating an electrochemical gradient (electrogenic transport). One of the most important pumps in animals cells is the sodium-potassium pump ( Na+-K+ ATPase ), which maintains the electrochemical gradient (and the correct concentrations of Na+ and K+) in living cells. The sodium- potassium pump moves two K+ into the cell while moving three Na+ out of the cell. The Na+-K+ ATPase exists in two forms, depending on its orientation to the interior or exterior of the cell and its affinity for either sodium or potassium ions. The process consists of the following six steps:

 With the enzyme oriented towards the interior of the cell, the carrier has a high affinity for sodium ions. Three sodium ions bind to the protein.  ATP is hydrolyzed by the protein carrier, and a low-energy phosphate group attaches to it.  As a result, the carrier changes shape and re-orients itself towards the exterior of the membrane. The protein‟s affinity for sodium decreases, and the three sodium ions leave the carrier.  The shape change increases the carrier‟s affinity for potassium ions, and two such ions attach to the protein. Subsequently, the low-energy phosphate group detaches from the carrier.  With the phosphate group removed and potassium ions attached, the carrier protein repositions itself towards the interior of the cell.  The carrier protein, in its new configuration, has a decreased affinity for potassium, and the two ions are released into the cytoplasm. The protein now has a higher affinity for sodium ions, and the process starts again.

Several things have happened as a result of this process. At this point, there are more sodium ions outside of the cell than inside and more potassium ions inside than out. For every three ions of sodium that move out, two ions of potassium move in. This results in the interior being slightly more negative relative to the exterior. This difference in charge is important in creating the conditions necessary for the secondary process. The sodium-potassium pump is, therefore, an electrogenic pump (a pump that creates a charge imbalance), creating an electrical imbalance across the membrane and contributing to the membrane potential. Electrochemical Gradient

To move substances against the membrane‟s electrochemical gradient, the cell utilizes active transport, which requires energy from ATP.

Key Points

 The electrical and concentration gradients of a membrane tend to drive sodium into and potassium out of the cell, and active transport works against these gradients.  To move substances against a concentration or electrochemical gradient, the cell must utilize energy in the form of ATP during active transport.  Primary active transport, which is directly dependent on ATP, moves ions across a membrane and creates a difference in charge across that membrane.  Secondary active transport, created by primary active transport, is the transport of a solute in the direction of its electrochemical gradient and does not directly require ATP.  Carrier proteins such as uniporters, symporters, and antiporters perform primary active transport and facilitate the movement of solutes across the cell‟s membrane.

Key Terms

 adenosine triphosphate: a multifunctional nucleoside triphosphate used in cells as a coenzyme, often called the “molecular unit of energy currency” in intracellular energy transfer  active transport: movement of a substance across a cell membrane against its concentration gradient (from low to high concentration) facilitated by ATP conversion  electrochemical gradient: The difference in charge and chemical concentration across a membrane.

Electrochemical Gradients

Electrochemical Gradient: Electrochemical gradients arise from the combined effects of concentration gradients and electrical gradients.

Simple concentration gradients are differential concentrations of a substance across a space or a membrane, but in living systems, gradients are more complex. Because ions move into and out of cells and because cells contain proteins that do not move across the membrane and are mostly negatively charged, there is also an electrical gradient, a difference of charge, across the plasma membrane. The interior of living cells is electrically negative with respect to the extracellular fluid in which they are bathed. At the same time, cells have higher concentrations of potassium (K+) and lower concentrations of sodium (Na+) than does the extracellular fluid. In a living cell, the concentration gradient of Na+ tends to drive it into the cell, and the electrical gradient of Na+ (a positive ion) also tends to drive it inward to the negatively-charged interior. The situation is more complex, however, for other elements such as potassium. The electrical gradient of K+, a positive ion, also tends to drive it into the cell, but the concentration gradient of K+ tends to drive K+ out of the cell. The combined gradient of concentration and electrical charge that affects an ion is called its electrochemical gradient.

Moving Against a Gradient

To move substances against a concentration or electrochemical gradient, the cell must use energy. This energy is harvested from adenosine triphosphate (ATP) generated through the cell‟s metabolism. Active transport mechanisms, collectively called pumps, work against electrochemical gradients. Small substances constantly pass through plasma membranes. Active transport maintains concentrations of ions and other substances needed by living cells in the face of these passive movements. Much of a cell‟s supply of metabolic energy may be spent maintaining these processes. For example, most of a red blood cell‟s metabolic energy is used to maintain the imbalance between exterior and interior sodium and potassium levels required by the cell. Because active transport mechanisms depend on a cell‟s metabolism for energy, they are sensitive to many metabolic poisons that interfere with the supply of ATP.

Two mechanisms exist for the transport of small-molecular weight material and small molecules. Primary active transport moves ions across a membrane and creates a difference in charge across that membrane, which is directly dependent on ATP. Secondary active transport describes the movement of material that is due to the electrochemical gradient established by primary active transport that does not directly require ATP.

Carrier Proteins for Active Transport An important membrane adaption for active transport is the presence of specific carrier proteins or pumps to facilitate movement. There are three types of these proteins or transporters: uniporters, symporters, and antiporters. A uniporter carries one specific ion or molecule. A symporter carries two different ions or molecules, both in the same direction. An antiporter also carries two different ions or molecules, but in different directions. All of these transporters can also transport small, uncharged organic molecules like glucose. These three types of carrier proteins are also found in facilitated diffusion, but they do not require ATP to work in that process. Some examples of pumps for active transport are Na+-K+ ATPase, which carries sodium and potassium ions, and H+-K+ ATPase, which carries hydrogen and potassium ions. Both of these are antiporter carrier proteins. Two other carrier protein pumps are Ca2+ ATPase and H+ ATPase, which carry only calcium and only hydrogen ions, respectively.

Uniporters, Symporters, and Antiporters: A uniporter carries one molecule or ion. A symporter carries two different molecules or ions, both in the same direction. An antiporter also carries two different molecules or ions, but in different directions. Secondary Active Transport

In secondary active transport, a molecule is moved down its electrochemical gradient as another is moved up its concentration gradient.

Key Points

 While secondary active transport consumes ATP to generate the gradient down which a molecule is moved, the energy is not directly used to move the molecule across the membrane.  Both antiporters and symporters are used in secondary active transport.  Secondary active transport brings sodium ions into the cell, and as sodium ion concentrations build outside the plasma membrane, an electrochemical gradient is created.  If a channel protein is open via primary active transport, the ions will be pulled through the membrane along with other substances that can attach themselves to the transport protein through the membrane.  Secondary active transport is used to store high-energy hydrogen ions in the mitochondria of plant and animal cells for the production of ATP.  The potential energy in the hydrogen ions is translated into kinetic energy as the ions surge through the channel protein ATP synthase, and that energy is used to convert ADP into ATP.

Key Terms

 secondary active transport: A method of transport in which the electrochemical potential difference created by pumping ions out of the cell is used to transport molecules across a membrane.

Secondary Active Transport (Co-transport)

Unlike in primary active transport, in secondary active transport, ATP is not directly coupled to the molecule of interest. Instead, another molecule is moved up its concentration gradient, which generates an electrochemical gradient. The molecule of interest is then transported down the electrochemical gradient. While this process still consumes ATP to generate that gradient, the energy is not directly used to move the molecule across the membrane, hence it is known as secondary active transport. Both antiporters and symporters are used in secondary active transport. Co-transporters can be classified as symporters and antiporters depending on whether the substances move in the same or opposite directions across the cell membrane.

Secondary active transport brings sodium ions, and possibly other compounds, into the cell. As sodium ion concentrations build outside the plasma membrane because of the action of the primary active transport process, an electrochemical gradient is created. If a channel protein exists and is open, the sodium ions will be pulled through the membrane. This movement is used to transport other substances that can attach themselves to the transport protein through the membrane. Many amino acids, as well as glucose, enter a cell this way. This secondary process is also used to store high-energy hydrogen ions in the mitochondria of plant and animal cells for the production of ATP. The potential energy that accumulates in the stored hydrogen ions is translated into kinetic energy as the ions surge through the channel protein ATP synthase, and that energy is used to convert ADP into ATP.

Secondary Active Transport: An electrochemical gradient, created by primary active transport, can move other substances against their concentration gradients, a process called co-transport or secondary active transport. Endocytosis

Endocytosis takes up particles into the cell by invaginating the cell membrane, resulting in the release of the material inside of the cell.

 Phagocytosis is the taking in of large food particles, while pinocytosis takes in liquid particles.  Receptor-mediated endocytosis uses special receptor proteins to help carry large particles across the cell membrane.

Key Terms

 endosome: An endocytic vacuole through which molecules internalized during endocytosis pass en route to lysosomes  neutrophil: A cell, especially a white blood cell that consumes foreign invaders in the blood.

Endocytosis Endocytosis is a type of active transport that moves particles, such as large molecules, parts of cells, and even whole cells, into a cell. There are different variations of endocytosis, but all share a common characteristic: the plasma membrane of the cell invaginates, forming a pocket around the target particle. The pocket pinches off, resulting in the particle being contained in a newly-created intracellular vesicle formed from the plasma membrane.

Phagocytosis

Phagocytosis: In phagocytosis, the cell membrane surrounds the particle and engulfs it.

Phagocytosis (the condition of “cell eating”) is the process by which large particles, such as cells or relatively large particles, are taken in by a cell. For example, when microorganisms invade the human body, a type of white blood cell called a neutrophil will remove the invaders through this process, surrounding and engulfing the microorganism, which is then destroyed by the neutrophil.

In preparation for phagocytosis, a portion of the inward-facing surface of the plasma membrane becomes coated with a protein called clathrin, which stabilizes this section of the membrane. The coated portion of the membrane then extends from the body of the cell and surrounds the particle, eventually enclosing it. Once the vesicle containing the particle is enclosed within the cell, the clathrin disengages from the membrane and the vesicle merges with a lysosome for the breakdown of the material in the newly-formed compartment ( endosome ). When accessible nutrients from the degradation of the vesicular contents have been extracted, the newly-formed endosome merges with the plasma membrane and releases its contents into the extracellular fluid. The endosomal membrane again becomes part of the plasma membrane.

Pinocytosis

Pinocytosis: In pinocytosis, the cell membrane invaginates, surrounds a small volume of fluid, and pinches off.

A variation of endocytosis is called pinocytosis. This literally means “cell drinking” and was named at a time when the assumption was that the cell was purposefully taking in extracellular fluid. In reality, this is a process that takes in molecules, including water, which the cell needs from the extracellular fluid. Pinocytosis results in a much smaller vesicle than does phagocytosis, and the vesicle does not need to merge with a lysosome.

Potocytosis, a variant of pinocytosis, is a process that uses a coating protein, called caveolin, on the cytoplasmic side of the plasma membrane, which performs a similar function to clathrin. The cavities in the plasma membrane that form the vacuoles have membrane receptors and lipid rafts in addition to caveolin. The vacuoles or vesicles formed in caveolae (singular caveola) are smaller than those in pinocytosis. Potocytosis is used to bring small molecules into the cell and to transport these molecules through the cell for their release on the other side of the cell, a process called transcytosis.

Receptor-mediated Endocytosis

Receptor-Mediated Endocytosis: In receptor-mediated endocytosis, uptake of substances by the cell is targeted to a single type of substance that binds to the receptor on the external surface of the cell membrane.

A targeted variation of endocytosis, known as receptor-mediated endocytosis, employs receptor proteins in the plasma membrane that have a specific binding affinity for certain substances. In receptor-mediated endocytosis, as in phagocytosis, clathrin is attached to the cytoplasmic side of the plasma membrane. If uptake of a compound is dependent on receptor-mediated endocytosis and the process is ineffective, the material will not be removed from the tissue fluids or blood. Instead, it will stay in those fluids and increase in concentration. Some human diseases are caused by the failure of receptor-mediated endocytosis. For example, the form of cholesterol termed low- density lipoprotein or LDL (also referred to as “bad” cholesterol) is removed from the blood by receptor-mediated endocytosis. In the human genetic disease familial hypercholesterolemia, the LDL receptors are defective or missing entirely. People with this condition have life-threatening levels of cholesterol in their blood, because their cells cannot clear LDL particles from their blood.

Although receptor-mediated endocytosis is designed to bring specific substances that are normally found in the extracellular fluid into the cell, other substances may gain entry into the cell at the same site. Flu viruses, diphtheria, and cholera toxin all have sites that cross-react with normal receptor-binding sites and gain entry into cells. Reference:- https://courses.lumenlearning.com/boundless- ap/chapter/transport-across-membranes/

Reference:- Genetics, Crash course Cell Biology and Matt Stubbs,Narin Suleyman 4th edition, 2013, Mosby Elsevier.

Keto- and Enol Tautomerism in Sugars Keto• and Enol Tautomerism in Sugars

Defining Keto• and Enol Tautomerism

Ketones are in equilibrium with a form known as an enol. The name enol derives from the fact that enols are a combination of a carbonyl (C=O) containing group, such as an aldehyde or ketone and an alcohol hydroxyl (OH) group. The interconversion between these two forms arises from a process called tautomerism.

Ketones and enols are isomers. Isomers are molecules that are made of the same atoms, but differ in their connectivity.

These two isomers are related by a change in the position of the hydrogen and the double bond in the molecule. Thus, tautomerism describes the equilibrium between keto and enol forms interconverted through a change in the position of bonding electrons and hydrogen to produce two isomers.

This process is typical of ketones, aldehydes and esters, and in general, the interconversion is slow. The isomer containing the carbonyl compound is favoured over the enol form, with the keto form dominating at >99%. Manipulation of these proportions can be achieved through catalysis, specifically by acids or bases.

The Formation of Cyclic Sugars

Sugars are polyhydroxylated (containing many OH groups) chains of carbon atoms that additionally feature an aldehyde or ketone functional group.

In general, all sugars feature a 1:2:1 ratio of carbon: hydrogen: oxygen. Subsequently, carbohydrates possess the ability to undergo internal, or intramolecular, reactions between the reactive hydroxyl group and the carbonyl carbon.

The hydroxyl is nucleophilic; it possesses a lone electron pair that it can donate to the carbonyl carbon and thus form a bond. The carbonyl carbon is

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amenable to such bond formation as its electrons experience a pulling effect by the doubly•bonded oxygen. Oxygen can attract the bonding pair of electrons more strongly than carbon; it is said to be more electronegative.

When a single nucleophilic hydroxyl group in a sugar attacks an aldehyde or ketone, a product called a hemiacetal or hemiketal, respectively, is produced. The prefix hemi• denotes the reversibility of the reaction. They are also referred to as acetals or pyranoses.

Figure 1 Example of a cyclisation reaction with D•glucose. When an aldose cyclizes, the hydroxyl group on C5 undergoes an intramolecular reaction with the C1 carbonyl group of the aldehyde. The product formed is a hemiacetal. Note the two•dimensional representation of the sugar D•glucose on the left• hand side is called a Fischer Projection, the resultant 3D representation of the hemiacetal is called a Haworth Projection.

These are cyclic, due to the ring•closing effect produced by chemical bond formation. Note that an attack on an aldehyde to produce a hemiacetal results in a 6•membered ring. In sugars, this is referred to an aldose. Alternatively, if the hydroxyl attacks a ketone to produce a hemiketal, the resultant product is a 5•membered ring called a ketose, or furanose. Another convention to note is ‘D ‘and ‘L ‘.

The prefix ‘D ‘is used to refer to the direction that an optical isomer (one with asymmetric groups surrounding a central atom) rotates plane polarised light. ‘D ‘isomers have the OH of the chiral (C5) centre pointing to the right; ‘L ‘isomers have the OH of the chiral (C5) centre pointing to the left.

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Figure 2 Haworth projection of αDGlucopyranose. Note the OH is on the opposite side of CH2OH

Figure 3 Haworth projection of βDGlucopyranose. Note the OH is on the same side as CH2OH.

Keto•Enol Intermediates in Sugar Interconversion and Epimerization

In sugars, the linear and cyclic hemiketal or hemiacetal of the sugar exist in equilibrium; in the linear form, sugars can undergo keto•enol tautomerism. This takes place during the interconversion between the aldose and ketose forms. Recall that tautomers are readily interconvertible isomers that differ in the position of the protons and electrons.

To convert between a 6•membered aldose, the sugar must tautomerise, to give an intermediate called and ene–diol, so•called because there is an alcohol group adjacent to the carbonyl (diol, two alcohols). This requires the presence of a base.

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The base removes the proton adjacent to the anomeric, carbonyl carbon. This is referred to as the alpha hydrogen. In doing so, a double bond between the alpha carbon and carbonyl carbon is formed, as one of the C=O carbonyl bonds break, and the liberated electron pair on oxygen picks up a proton from an acid. This amounts to the movement of a proton from the alpha position to the carbonyl oxygen. Figure 4 is an example of D•glucose tautomerization.

The collapse to a ketose sugar requires the abstraction (removal) of the C2 (alpha) hydroxyl hydrogen by an acid, which subsequently results in the movement of the electron pair to the C2 carbon to form a carbonyl group.

One bond of the C=C bond opens to accommodate a hydrogen, provided by a base. The intramolecular reaction that occurs between the C5 carbon and C1 ketone results in the cyclic ketose.

The reaction is reversible; the proton is transferred from the C1 OH group along the C=C bond to the C2 alpha position. Figure 4 illustrates the mechanism of D•glucose tautomerization.

Figure 4 An enediol rearrangement. In the presence of a base, D•glucose may be converted to D•fructose. note how the position of the carbonyl has moved from C1 in D•glucose to C2 in D•fructose (movement of a bonding pair of electrons), and the alpha hydrogen has moved to C1 in the fructose/ ketose sugar.

The conjugate base of the enediol, called an enolate may also serve as an intermediate for another reaction called epimerisation. Epimers are optical isomers, differing in the arrangement of the same atoms about the anomeric

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carbon.

In this process, the enediol, in its deprotonated form called an enolate (the C1 hydroxyl lacks a proton), abstracts a proton from the neighbouring C2 hydroxyl.

The resulting deprotonated C2 oxygen donates a lone electron pair to the C2 carbon and thus form a carbonyl at this position. This is concurrent with breakage of one of the C=C bonds; the electron pair is used to accept a proton. This is illustrated in Figure 5.

Figure 5 An Epimerisation reaction. This reaction intermediate is an enolate, the conjugate base of the enediol. In this example, D•glucose may be converted into D•mannose via the removal of hydrogen at C2 carbon followed by addition of hydrogen across the C=C of the enolate.

Keto•enol tautomerism is an important process in sugar biochemistry. The interconversion of an aldose to a ketose, such as D• glucose to D•fructose, occurs via their common enolate isomer. This is also true of epimerisation reactions, that allow interconversion of two aldoses, such as D•glucose and D• mannose or two ketoses, such as D•psicose and D•fructose. Epimers differ only in the arrangement of atoms or groups around the optically active, asymmetric C1.

Sources

http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch18/ch18•1.html http://plaza.ufl.edu/tmullins/BCH3023/carbohydrates.html

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https://nptel.ac.in/courses/104103071/module11/lec31/4.html chem.libretexts.org/.../22.05%3A_The_Reactions_of_Monosaccha rides_in_Basic_Solutions

Further Reading

All Carbohydrate Content

What are Carbohydrates?

Carbohydrate Structure

Carbohydrate Monosaccharides

Carbohydrate Metabolism

6/6 ) contents  Etymology and definition  Classification of ionophores  Mechanism of action  Examples of ionophores  Applications of ionophores ( importance) IONOPHORES Etymology  ι ό ν = ion (Greek) Φέρω( phero) = carry ( Greek)  ionophores can be literally called as “Charge carriers / charge bearers” .  T h e word ionophore was coined by Pressman in 1964. Ionophores, initially called ion complexers were discovered in 1950’s and first used as anti- coccidial agents. • Ionophores are specific molecules that complex /carry specific cations and facilitate their transport through biological membranes. • They are molecules that act as membrane shuttles for particular ions across the lipid membranes with out expenditure of energy Ionophores contain hydrophilic centres that bind specific ion(s), and a hydrophobic portion that interacts with the lipid interior of the membrane Most ionophores adopt a cyclic ring formation by concentrating oxygen/ nitrogen functional groups at the centre of their structure to associate with the cations. With the hydrophobic groups in contact with the acyl groups of the membrane , the ionophore is able to “ dissolve” and diffuse to the opposite side of the membrane Classification  ionophores can be classified based on; Mechanism of their action Chemical structure Based on the mechanism of action a) Mobile carrier ionophores : These bind to a particular ion and shield its charge from the hydrophobic environment of the membrane . They form a lipid soluble complex with the cation which then diffuses across the membrane. Eg Valinomycin – K+ By reversibly binding to the ion to form lipid soluble complexes which rapidly diffuse across the membrane, they catalyse passive transfer of cations across the otherwise impermeable hydrophobic membrane. Generally;  three steps are involved ; Complexation of the ionophore with the ion Diffusion of the complex via the membrane interface to the other side of the membrane. Reverse complexation process illustration ion ionophore (b) Channel- forming ionophores  These introduce a hydrophilic pore into the membrane allowing specific cations to pass through with out coming into contact with the hydrophobic interior of the membrane ,eg Gramicidin. Channel forming ionophores are usually large molecules ion

Channel of the ionophore Based on the chemical structure; i. polyether ionophores eg monensin and maduracin ii. Peptide ionophores iii. Cyclodepsipeptide ionophores eg Valinomycin iv. Macrotetrolides ( macrocyclic compounds containing tetrahydrofuranyl carboxylic acid residues linked together) v. Cryptates ( synthetic bi and polycyclic multidentate ligands for variety cations) vi. Crown ethers eg a crown ether

cation associate region Example of Ionophores  The biologically significant classes of ionophores are the channel forming and the mobile carrier ionophores; 1. Valinomycin These is a circular depsipeptide molecule which contains ester and amide linkages ( A depsipeptide is a molecule containing both peptide and ester bonds ). It contains D- valine, L- valine, L- Lactic acid and hydroxyisovaleric acid.  Valinomycin is specific to K+ ions, which it transfers in complexed and uncomplexed state. It has a pluckered ring , stabilized by hydrogen bonds which therefore suits it to surround single un hydrated K+ ions representation Valinomycin O O O K + O O O Hydrophobic Chemical structure How it works,  T h e six oxygen atoms of the ionophore interractwith the bound K+ ion replacing the O-atoms of water of hydration.  Each valinomycin molecule is ableto carry about 10000 K+ ions per second – Very rapid transport rate!

NB. Valinomycin can not carry sodium ions because they are small and there fore can not simultaneously interact with six O-atoms – thus being energetically unfavourable 2. Gramicidin A  This is a linear 15-amino acid -peptide with alternating D- and L- amino acids . The structure is double helical in organic solvents and is an end-to- end dimer in water.  In lipid bilayer membrane, gramicidin dimerises and folds as a right hand β-helix. The dimer spans the bilayer . The hydrophobic outer surface of gramicidin dimer interacts with the core of the lipid bilayer while the ions pass through the more polar lumen of the helix. cont;  Gating (opening and closure ) of gramicidin channels is thought to involve reversible dimerisations . An open ion channel forms when gramicidin molecules join end to end to span the membrane . Cations there fore move through the channel in a single file along with a single file of water molecules. linear structure Folding of gramicidin model of gramicidin

Channel for the passage of ions

Carbon atom

Nitrogen atom

Oxygen atom 3. Ionomycin  Has a hydrophobic periphery.  It carries Ca2+ ions into the cells and organelles.  It was first isolated from bacterium Streptomyces conglobatus. Calcimycin (ionophore A23187) is also a calcium ionophore that was first isolated from the fermentation reactions of Streptomyces chartreuses. chemical structures

ionomycin calcimycin ( ionophore –A23187) 4. Monensin  It was first isolated from Streptomyces cinnamonensis

 I t complexes with Na+ and H+ ions . It is apolyether anti-biotic. Siderophores These are bacterial ionophores specific for carriage of Fe3+ ions . Complexation of Fe3+ ions solubises it for its uptake . The binding ligands are catechols ( orthohydroxyphenol). 6. Nigericin- exchanges H+ and K + ions across the membrane 3,5-dinitrophenol (DNP)  It is a hydrogen ion ionophore and a chemical uncoupler. It rapidly transports protons from the cytosolic side to the matrix side of the inner Carbonylcyanide –m- mitochondrial membrane chlorophenylhydrazone (CCP) also acts in similar way leading to non-voltage dependent proton DNP Inner mitochondrial transfer H+ membrane H

- High hydrogen ions on the Low hydrogen ion cytosolic side concentration in the matrix causes DNP to causes DNP to dissociate be protonated releasing protons 7. Beauvericin Beauvericin is a cyclic hexadepsipeptide with alternating methyl- phenylalanyl and hydroxy-iso-valeryl residues. Its ion complexing capability allows beauvericin to transport alkaline earth metal and alkali- metal ions across cell membranes.

Region where a cation is bound( polar region of the molecule) 8. Enniatins  These are mixture of depsipeptides that bind and transfer ammonium ion across the membrane other ionophores; salinomycin, lasolacid, octadecadienic acid (first isolated in the mitochondria of the beef heart)  a n d manyothers… Importance of ionophores  They creat electrochemicalgradient resulting in changes in the physiological state of the cell such as; Oxidative phosphorylation, Osmotic balance, Neurotransmission,  Cell signalling. Hormone Actionetc. exploiting the effects of ionophores……….  As antibiotics and treatment of coccidiosis in poultry.  Coccidia parasites do not have osmoregulatory organelles . Treatment with ionophores therefore disrupt the osmotic balance resulting in the influx of water and subsequent vacuolarisation of the cell resulting in bursting of the cell. effects on bacteria……..  In bacteria , ionophores disrupts the electrochemical gradient there by causing cell death particularly in gram-positive bacteria.  This is because, theyare surrounded by peptidoglycan layer which is porous and therefore allow the lipophilic ionophores to pass through. However , gram –negative bacterial cells are surrounded by a lipopolysaccharide layer which does not allow the ionophore to pass through thus they are passive to ionophores. Feed additives in live stock Ionophores are used in therapeutic levels to improve the feed efficiency in livestock. Ionophores target ruminant microbial population thus altering their ecology.  This subsequentlyresults into carbon and nitrogen nutrient retention by the animal Example case example of monensin…  Monensin is used as a methane inhibitor and a propionate enhancer ( more efficiently utilised) ruminants. It also reduces dietary protein deamination resulting in less ammonia in urinary excretion. This increases energy availability and nitrogen retention thus improving animal productivity.  By reducing the population of fermenting bacteria (methanogens) , monensin reduces methane production by 30% and amino acid degradation by 50% . However, error in administering may result in toxicity, to the animal neuropathy and muscle biomedical science Research  Many ionophore have been utilized in manipulating the physiological state of the cell during research. example;  Monensin action disrupts the Golgi functioning . Calcium and ionomycin are used to introduce calcium in the cells and organelles. Many physiological processes that are normally triggered by the binding of hormones to cell surface receptors can be elicited by use of calcium ionophores to raise cytosolic calcium level. Cont; Enniatin inhibits acyl-COA cholesterol transferase Valinomycin. Inhibits phytohemagglutin (PHA)stimulated blastogenesis and proliferation in human lymphocytes. Gramicidin A and nigericin exchange H+ and K+ across the mitochondrial membrane and uncouples oxidative phosphorylation. They combine with K+ ions and transfer it to the cytoplasm where they are protonated as they diffuse to the outer membrane surface . This induces non-voltage dependent H+/K+ exchange in the mitochondria cont;  D N P and CCP are chemical uncouplers .Uncoupling results into non- shivering thermogenesis.  Many ionophores have also been used to makethe membrane selective electrodes in electrochemistry Conclusion  Ionophores are molecules that are able to shuttle ions across the otherwise hydrophobic lipid layer of the cell membranes  The major two classes of ionophores ( channel forming and mobile career ) particularly Ionomycin / calcimycin and valinomycin ;  A r e applied to increase calcium levels in pharmacological research  Are used as antibiotics  A r e used to design membrane selectiveelectrodes Reference:- https://www.slideshare.net/ManirihoHillar y/ionophores Thank you

CONTENTS 1.INTRODUCTION

2.STEADY STATE DIFFUSION

3.MECHANISM

4.FACTOR INFLUENCE THE DIFFUSION

5.TRANSPORT ACROSS THE MEMBRANE

6.METHODS & PROCEDURES

7.APPLICATIONS

2 8.REFERENCES INTRODUCTION DEFINITION: Diffusion:

 “The movement of particles in a solid from an area of high concentration to an area of low concentration, resulting in the uniform distribution of the substance.”

 During diffusion molecules move from an area of high concentration to an area of low concentration.

 They are said to move down a concentration gradient.

 The material that undergoes the transport is known as diffusant or permeant or penetrant. 3 INTRODUCTION

 Diffusion is a passive process which means that no energy is needed.  Molecules diffuse until they are evenly spaced apart and equilibrium is reached.

high low concentration concentration

4 INTRODUCTION

 In which states are molecules able to diffuse?

solid (e.g. ice) liquid (e.g. water) gas (e.g. steam) Molecules in liquids and gases are constantly moving and bumping into each other. This means that they tend to spread out. By contrast, solids cannot diffuse. 5 STEADY STATE DIFFUSION

 Diffusion is direct result of Brownian motion.  Molecule diffuse spontaneously from region of higher concentration to region of lower concentration until diffusion equilibrium is established.  Rate of diffusion independent of time.  Flux proportional to concentration gradient =

C C1 1 Fick’s first law of diffusion

C dC C2 J  D 2 dx x1 x2 6 x D = diffusion coefficient STEADY- STATE DIFFUSION

 The flux J is expressed as either in number of atom per unit time.  Steady state diffusion means that J does not depend upon time.  In this case fick„s 1st law hold that flux along direction “x” is-

dC J  D dx

Where, J is equal to rate of mass transfer across unit surface area of barrier.

7 STEADY-STATE DIFFUSION

SINK CONDITION:  State in which concentration in receptor compartment is maintained at lower level compaired to its concentration in donor compartment.

 During diffusion donor compartment act as source & receptor compartment act as sink.

 Useful for maintaining concentration gradient nearly constant.

8 STEADY-STATE DIFFUSION Fick´s 1st law:  Mass get transported from 1 compartment to another over period of time is Flux.  Flux (J) = Rate of mass transfer across unit surface area of barrier. J= 1/S (dm/dt) 1) Where, dm = change in mass of material. S = barrier surface area. dt = change in time.

9 STEADY-STATE DIFFUSION Acc.to Fick‘s law,flux is directly proportional to conc.gradient.

dC J  D 2) dx

D=diffusion coefficient of penetrant. dx=change in distance. Combine equation 1) & 2) gives, dm/dt= -DS (dc/dx) * This equation represent rate of mass transfer as per fick‘s law.

10 MECHANISM

Mechanisms:  Gases & Liquids – random (Brownian) motion.

 Solids – vacancy diffusion or interstitial diffusion.

I. Vacancy diffusion:

 Only adjacent atoms can move into a vacancy.  Vacancy moves in opposite direction of atomic motion.  Rate depends on concentration of vacancies.

11 MECHANISM • atoms exchange with vacancies .  rate depends on:  -- number of vacancies  -- activation energy to exchange.

increasing elapsed time 12 MECHANISM Inter diffusion: Atoms tend to migrate from regions of high conc. to regions of low concentration.  – smaller atoms can diffuse between atoms.

Initially After some time

•Atom can move into any adjacent empty interstitial position (usually smaller atoms). •Rate depends on concentration of interstitial atoms. 13 •(Usually faster than vacancy diffusion). Factors that Influence Diffusion I. Diffusing Species:

 Magnitude of diffusion coefficient, D – indicates the rate at which atoms diffuse.

 Both diffusing species and host material influence the coefficient:  Relative sizes of atoms.  Openness of lattice.  Ionic charges.

14 Factors that Influence Diffusion II. Temperature:  Very strong effect on the diffusion coefficient:

• Diffusion coefficient increases with increasing T.

 Qd  D  D exp  o  RT 

D = diffusion coefficient

Do = pre-exponential

Qd = activationenergy R = gas constant T = absolute temperature 15 DIFFUSION ACROSS MEMBRANE

Diffusion across membrane:

There are two ways in which substances can enter or leave a cell: 1) Passive a) Simple Diffusion b) Facilitated Diffusion c) Osmosis 2) Active a) Molecules b) Particles

16 DIFFUSION ACROSS MEMBRANE

This is the movement of specific molecules down a concentration gradient, passing through the membrane via a specific carrier protein

Selection is by size; shape; charge.

Common molecules entering/leaving cells include glucose & amino- acids.

17 DIFFUSION ACROSS MEMBRANE

It is passive and requires no energy from the cell.

If the molecule is changed on entering the cell (glucose + ATP → glucose phosphate + ADP), then the concentration gradient of glucose will be kept high.

18 DIFFUSION ACROSS MEMBRANE Active Transport :  Active transport is the energy-demanding transfer of a substance across a cell membrane against its concentration gradient, i.e., from lower concentration to higher concentration.  Special proteins within the cell membrane act as specific protein „carriers‟. The energy for active transport comes from ATP.

19 DIFFUSION ACROSS MEMBRANE

Passive Transport: • Passive diffusion is the process by which molecules spontaneously diffuse from a region of higher concentration to a region of lower concentration.

• Lipid-soluble drugs penetrate the lipid cell membrane and can pass the cell membrane by passive diffusion.

• Also, large molecules, such as proteins and protein-bound drugs, cannot diffuse through the cell membrane.

20 METHODS & PROCEDURES Two types: A) horizontal transport cell:

a. wurester cell b. Viles chein permeation cell

B) vertical transport cell:

a) Aquair and weiner diffusion cell b) biber and rhodes cell c) franz diffusion cell

21 Horizontal Transport Cell wurester cell: Receptor and donor compartment made of pyrex glass material. Animal or human skin acts as semi permeable cell and barrier may be supported on a perforated plate. Drug sample solution taken in donor compartment and solvent in the receptor compartment. Whole set up placed in constant temperature bath to maintain the temp of 37±0.2°C. The liquid in receptor stirred by using magnetic beads to obtain uniform distribution. 22 VERTICAL TRANSPORT CELL Aquair and weiner diffusion cell: Receptor and donor compartment made of pyrex glass or plastic material. Animal or human skin acts as semi permeable cell and barrier may be supported on a perforated plate. Drug sample solution taken in upper compartment and solvent in the lower compartment. Whole set up placed in constant temperature bath to maintain the temp of 37±0.2°C. The liquid in receptor stirred by using magnetic beads to obtain uniform distribution. 23 APPLICATIONS

1) The release of drug from dosage form is diffusion controlled,such dosage form available in market as sustained & controlled release product. 2) The molecular weight of polymers can be estimated from diffusion process. 3) The transport of drugs from GI tract,skin,etc., can be understood & predicted from the principles of diffusion. 4) The diffusion of drugs into tissues & their excretion through kidneys can be anticipated through diffusion studies. 5) The process such as dialysis, microfiltration, ultrafiltration, haemodialysis,osmosis,etc., use the principles of diffusion.

24 REFERENCES

1. C.V.S. Subrahamanyam,Textbook of physical pharmaceutics, 2nd Edition, Vallabh prakashan, pg.no.110-127

2. D.M. Brahmankar ,Biopharmaceutics and Pharmacokinetics-A Treatise”,2nd Edition, 2009,Vallabh Prakashan, pp- 10 to 22.

3. https://www.slideshare.net/Atishkhilari/diffusion-ppt- ak?from_action=save

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