Cell Biology Syllabus

Chapter 1. Introduction to Cell Biology The word cell comes from the Latin language, “cellulae”, which means “little rooms”. Cellulae was named by Robert Hooke in 1665. Robert Hooke was the first person to use the microscope. However, his microscope was only has 30-fold. Robert Hooke was examining the cork and he saw a network of tiny, box-like compartments, which were the dead plant tissue called parenchyma. Later, a Dutch scientist, Van Leeuwenhoek had improved the microscope to 300-fold, enabled him to observed living cells. Although microscope had been invented early during the Renaissance era, but the cell theory did not develop until 100 years later in 1830s. At that time, microscope had been improved to 1 micrometer of resolution, enabled the scientists to observe the detailed structures of eukaryotic cells as well as most bacteria. In 1838, a Germany botanist, Matthias Schleiden proposed that all plant tissues are consisting of cells and all embryonic plants are arises from a single cell. A year later, the similar conclusion was reported by Theodor Schwann concerning animal cells. In 1839, Schwann proposed the cell theory that all life are consisting of cell(s) and cell is the basic unit of all life. In 1855, Rudoff Virchow proposed that cell arose from the division of preexisting cell. The Origin of Life: How life starts is about the genesis of the first prokaryotes, the single-celled microorganisms that lack true nuclei. According to the old westerner traditional view, all life arises from the non-living matter through the spontaneous generation. However, this theory had been overturned by the Louis Pasteur in 1862. The spontaneous generation was then substituted by the biogenesis-“life-form-life” theory, which proposed that all life arises from the preexisting life through reproduction. The origin of life theory was proposed by A.I. Oparin and J.B.S. Haldane during 1920s. 4.6 billion years ago, the Earth was formed and the first life stars to form on Earth 3.9 billion years ago. When the Earth was still young, the oxygen level in the atmosphere was very low and probably thick with water vapor, nitrogen gas, methane gas, ammonia gas, hydrogen gas and hydrogen sulfide gas. As the Earth starts to cool, the water vapor condensed to form the oceans. At that time, the atmosphere was a highly reducing environment and the energy sources were the lightning and the ultraviolet radiation as well as the organic compounds that can formed from simpler molecules and atoms. The origin of life was started in four sequential steps:

1. The abiotic synthesis of small organic molecules such as amino acids and

nucleotides. 2. The joining of the monomer organic molecules into larger polymers. 3. The packaging of these polymers into a droplet called protobiont. 4. The origin of self-replicating molecules which made inheritance possible.

In 1953, Stanley Miller and Harold Urey tested the Oparin-Haldane hypothesis with a set of laboratory apparatus. Miller and Urey set up a closed system in their laboratory to simulate the conditions thought to have existed on early Earth. A warmed flask of water simulated the primeval sea; the strongly reducing atmosphere in the system consists of hydrogen gas, methane gas, ammonia gas, and the water vapor. Besides, sparks were discharged in the synthetic atmosphere to mimic lightning. A condenser was used to cool the atmosphere, raining water and dissolved compounds into the miniature sea. As the material circulated through the apparatus, they periodically collected the samples for analysis. They had identified a variety of organic compounds including amino acids such as alanine and glutamic acid that are common in proteins of organisms. They also found many other amino acids and complex, oily compounds. From the above results, organic molecules, the first step in the origin of life, can form in a strongly reducing atmosphere. It is unclear whether the young Earth’s atmosphere contained enough methane and ammonia to be reducing. Recent evidences suggest that the early atmosphere was made up of nitrogen and carbon dioxide, and was neither reducing nor oxidizing, therefore, less likely to produce organic molecules. Instead of forming in the atmosphere, the first organic compounds on Earth may have been synthesized near submerged volcanoes and deep-sea vents. These locations were the weak points on Earth crust where hot water and minerals gush into the ocean. These regions are also very rich in inorganic sulfur and iron compounds, which are important in ATP synthesis by present-day organisms. All cells are bounded by plasma membrane, which within the membrane is a semi fluid substances-cytosol, in which organelles are found. All cells also contain chromosomes which carrying genes in DNA form. Besides, one more important feature common to all cells today is the presence of ribosome.

Prokaryotic cells and Eukaryotic cells: Prokaryotic cells are different from the eukaryotic cells due to the absence of membrane-enclosed nucleus. In eukaryotic cells, DNA molecules are stored in nucleus which is surrounded by doubled-membrane called nuclear envelope. The DNA molecules of prokaryotic cells are concentrated in a region called nucleoid in cytoplasm; there is no membrane present to separate the nucleoid from the cytoplasmic environment. Besides, the prokaryotic cells also lack endomembrane system such as endoplasmic reticulum, mitochondria, chloroplasts, and Golgi apparatus present in eukaryotic cells. The prokaryotic cells also have an additional gel-like layer of capsule which is absence in most eukaryotic cells. Even though both the prokaryotic and eukaryotic cells have flagella and ribosomes, their ultra structures are significantly different from each other. The flagella of eukaryotic cells are made up of arrangement of microtubules while the flagella of prokaryotic cells lack microtubules. The sedimentation coefficient of ribosomes between the two types of cells also different. The sedimentation coefficient of eukaryotic ribosomes is 80S while the prokaryotic ribosomes are only 70S. Since the year of 2000, the tree of life consists of three main domains: Bacteria, Archaea, and Eukarya. Domain is the taxonomic level that is above the kingdom. The domain Bacteria includes most of the currently known prokaryotes, including the bacteria closely related to mitochondria and chloroplasts. The domain Archaea consists of a diverse group of prokaryotic organisms that inhabit a wide variety of environments. Some Archaea can use hydrogen as an energy source and some are capable of living in salty or hot environments. The domain Eukarya consists of organisms which are referred to as eukaryotes. This domain includes many groups of single-celled organisms as well as multicellular organisms such as plants, fungi, and animals. All life today shares a common ancestor. The origin of life starts about 4 billion years ago or even earlier. About 3 billion years ago, the last common ancestor of all living things starts to diverge into two categories: the bacteria and Archaea. About 2.5 billion years ago, the possible fusion Archaean and bacterium giving rise to the first ancestor of eukaryotes. About 1 to 2 billion years ago, the endosymbiosis of mitochondrial and chloroplast ancestors into the eukaryotic cells giving rise to the eukaryotic life like today.

Chapter 2. Microscopy and Cell Fractionation The microscope first used by the Renaissance scientists as well as the microscope often used by university students in laboratory, are all light microscopes (LMs). Light microscope uses visible light to observe on the specimens. Visible light is passed through the specimen and then through the glass lenses. The lenses refract the light radiation in such the way that the image of the specimen is magnified as it is projected into the eyes, onto photographic film or a digital sensor, or onto a video screen. Eye Ocular lens

Objective lens Specimen Condenser lens Light source

The two important parameters in microscopy are magnification and resolution. Magnification in microscopy is the ratio of an object’s image size to its real size. Resolution is measure of the clarity of the image; it’s the minimum distance two points can be distinguished as two points. Microscopy: There are two main types of microscopy: light microscopy and dark-field microscopy. In dark-field microscopy, light are directed from the side and only scattered light enters the lenses, resulting in the cells or the specimen appear as bright object against the dark background. The light microscopy can be further divided into several methods of microscopy: bright-field microscopy, phase contrast microscopy, differential-interference contrast microscopy, and fluorescence microscopy.

The specimens for bright-field microscopy can stain or unstained. The unstained specimens are usually naturally pigmented; otherwise, the images produced would be unclear. However, the stained specimens usually required the specimens to be fixed. In phase contrast microscopy, the velocity of the light rays are slowed down and refracted to varying extends due to different in density of different regions of the specimen. Through this microscopy, the internal structures of the cells can be better visualized. However, this microscopy is less sensitive. The differential-interference contrast microscopy or Normaski microscopy employs a pair of Wallaston prisms to splits the light rays into two separated rays and pass through the specimen. When the two rays recombined, any changes that occurred in any one of the two rays can cause it to interface with the second rays. Usually, the largest phase changes occurred at the edge of the cell, therefore, this region gives the strongest signal. The image appears 3D as the results of shadow casting illusion. However, this type of microscopy provides little about the locations of specific molecules in a cell. This limitation can be solved by the fluorescence microscopy. Fluorescence microscopy employs the fluorescence probes to detect on the specific molecules in cells. Examples of fluorescence probes usually used in cytology field are immunostaining, phalloidin, and green fluorescence proteins. Immunostaining is the fluorescence technique that is based on the ability of antibodies to recognize and bind to the specific molecules (antigens). However, antibodies themselves are nonfluorescent and need to be coupled with fluorescence dye. Examples of fluorescence dye are fluorescein which emits green fluorescence and rhodomine which emits red fluorescence. Phalloidin is a mushroom toxin that binds specifically to actin microfilaments and is used to study the cytoskeletons in the cells. Although the fluorescence microscopy is very efficient in detecting the presence of specific molecules in the cells, but the image requires the specimen to be extremely thin to avoid the overlapping of focal planes that makes the image blur. Electron Microscopes: Instead of using light, electron microscope focuses a beam of electrons through the specimen or onto its surface. The resolution is inversely proportional to the wavelength of the electromagnetic radiation a microscope uses for imaging. Electron beam has a shorter wavelength than light radiation wavelength. A modern electron microscope can has a resolution of 0.02nm but practically the electron microscope can only has resolution of about 2nm. Biologists use the terms cell ultrastructure to refer to the cell’s anatomy observed under electron microscope. There are two types of electron microscope: transmission electron microscope (TEM), and scanning electron microscope (SEM).

TEM is mainly used to study the internal ultrastructure of a cell. TEM requires the specimen to be very thin sectioned by using ultra microtome so that the electron beam can pass through the specimen. Besides, the specimen also needs to be fixed and rapidly frozen. Specimen to be observed under TEM requires multiples of preparation techniques before can be observed. Among the techniques involved are negative staining, shadowing, freeze fracturing and freeze etching. Negative staining technique is used to examine the shape and surface of a very small object without need to cut the specimen into thin section. The specimen is suspended into an electron-dense stain and the image produced is relief against dark background. The shadowing technique involves the spraying of thin layer of electron dense metal in a vacuum evaporator at side angle of the biospecimen. This would cause the accumulation of metal ions on only one side of the specimen and shadowing the other side. A metal replica is then generated, and is stabilized by the coating of carbon atoms. In freeze fracturing technique, the specimen is cryoprotected at temperature of -80oC using the liquid N2 and then placed in a vacuum evaporator of -100 oC. The Cryoprotected specimen is then strike with a sharp ultra microtome to create a fracture along the line of the hydrophobic core of plasma membrane. The fracture is then shadowed with a layer of platinum or gold and carbon to make a metal replica. Freeze etching technique is the method followed by the freeze fracturing technique. The ultra microtome is placed directly over the specimen for a short period of time to cause the small amount of water to evaporate from the surface to produce an etching effect so that the view over the outer membrane surface can be enhanced. This technique can minimize the formation of ice using volatile cryoprotectant such as methanol. SEM is very useful for detailed study of surface of specimens. The electron beam scans the surface of the specimen which is usually coated with a thin film of gold. The electron beam excites the gold surface to emit secondary electrons which are detected by a device that translates the electron beam pattern into electronic signal to a video screen. The images observed under SEM are appearing 3-D.

Electron source Condenser lens (electromagnetic lens) Specimen

Objective electromagnetic lens

Intermediate image

Projector lens

Eyes

Final image Although the EMs have a much higher resolution than the light microscope, EMs have several practical disadvantages over the light microscope. EMs have the money and space disadvantages over the light microscope. EMs are very expensive and require a big space like a block of building and therefore few institutions are able to own an EM. The specimen to be observed under EM has to undergo series of complex preparation techniques and this requires a lot of expertise. EMs also very sensitive to any vibrations and electromagnetic fields, making the images produced often not clear. Both the EMs and light microscopes provide little information about the functions of the organelles and other cellular structures in the cells. To achieve this, a biochemical technique is required, for example the cell fractionation.

Cell Fractionations: Cell fractionation is the technique used to separate and purify different components of the cell, so that they can be studied physically and chemically. The tissue of cells to be studied is subjected to homogenation to break the cell wall and the plasma membrane of the cells to produce a cell homogenate. The homogenate is then subjected to a centrifugal force by spinning the test tube containing the cell homogenate at various high speeds in an instrument called centrifuge. Under various high speeds, the different components in the cells, such as different organelles can be separated by the centrifugal forces based on their sizes and densities. Theodor Svedberg is the person who invents the ultracentrifuge which can applies high centrifugal forces on the cell homogenate up to more than 500000 times the force of Earth gravity. Ultracentrifuge is used to separate different organelles as well as macromolecules in cells. The rate of sedimentation is directly related to the sizes and densities of the macromolecules and organelles. The measurement for sedimentation rate during the sedimentation coefficient (unit: Svedberg, S). The centrifugal force separates the cell homogenate into two distinctive regions: the pellet and supernatant.

Supernatant

Pellet The pellet contains the nearly pure component targeted by the centrifugal force. The pellet is removed and the supernatant left can be further subjected to a higher centrifugal forces. This method is called differential centrifugation. Under centrifugal force of 1000g for 10 minutes, pellet rich in nuclei and cellular debris will be collected; under 20000g for 20 minutes, pellet rich in mitochondria or chloroplasts will be collected; 80000g for 60 minutes, pellet rich in microsomes and membranes pieces will be collected; 150000g for 3 hours, pellet rich in ribosomes will be collected.

One special type of differential centrifugation is called Density Gradient centrifugation (DGC). In DGC, the sample subjected for centrifugation is placed as a thin layer on the top surface of the gradients of solutes which consist of an increasingly concentration of solutes from top to bottom. After the sample is subjected to the centrifugal force, the particles or organelles moves downwards at different rates by sizes and densities, thus forming a series of discrete bands of pellets. The tubes used in most differential centrifugation are usually plastics that can be easily punctured to collect the pellet.

Thin layer of Sample

Gradients of solutes

Discrete bands

Another specific example of differential centrifugation is the equilibrium density centrifugation (EDC). EDC resembles the DGC but the solutes are highly concentrated so that the density gradient spans the range of densities of the organelles that are about to be separated. EDC also often used to separate the different forms of DNA and RNA molecules based on differences in density.

Chapter 3. Cell Chemistry In cells, large molecules or macromolecules can be formed from small organic molecules that are repeatedly joint together. Polymer is a large molecule that consists of many identical building blocks linked by covalent bonds. Monomers are the repeating units that serve as the building blocks of the polymer. In forming a polymer, monomers are connected by a condensation reaction or more specifically dehydration reaction. Through this dehydration reaction, these monomers are joint by covalent bonds. For each covalent bond that is formed, one molecule of water, H2O, is given up. In many cases, the monomers are not joint into a polymer simultaneously but the monomers are bonded to the polymer chain one by one. Dehydration reactions expend energy and they would only occur in the cell with the presence of specific enzymes. Each cell from each tissue, from each species of organism has thousands of various kinds of macromolecules. The inheritance different between organisms reflect the variations in polymers in their cells and these possible variety is limitless, for example, the DNA molecules and proteins. These macromolecules are unique to all organisms in this planet but their monomers are common to all of them. Examples of macromolecules most common in the cells are carbohydrates, lipids, proteins, and nucleic acids. Carbohydrates: Carbohydrates include the sugars and the polymers of sugars. Monosaccharide is the monomer sugar and it is the simplest carbohydrate. One most common monosaccharide is the glucose which has formula C6H12O6. Each monosaccharide molecule has two functional groups: the carbonyl group and the hydroxyl group. These hydroxyl groups are bonded to each of the carbon atoms in the monosaccharide molecule except to the carbon atom that involved in carbonyl group. For example, the glucose molecule:

H H OH H H H O =C C C C C C H OH H OH OH OH

The monosaccharides can be classified in many ways depending on the criteria taken account. The classification can be based on the locations of the carbonyl group, the sizes of the carbon skeleton and the spatial arrangement of their parts around the chiral carbon. Based on the location of the carbonyl group, monosaccharides can be divided into aldoses and ketoses. When the carbonyl group is located on the end of the carbon skeleton chain, C1, the monosaccharide is called the aldoses or aldehyde sugar. Examples of aldoses are glucose and galactose. If the carbonyl group is located on the positions other than C1, the monosaccharide is called the ketose or ketone sugar. Examples of ketoses are ribulose and fructose. Based on the sizes of the carbon skeletons, monosaccharides can be divided into trioses, pentoses, and hexoses. Trioses are the 3-carbon sugars (examples: glyceraldehydes, and dihydroxyacetone); pentoses are the 5-carbon sugars (examples: ribose, and ribulose); hexoses are the 6-carbon sugars (examples: glucose, galactose and fructose). For the optical isomerism to occur, chiral carbon or asymmetrical carbon must be present. Chiral carbon is the carbon atom in a carbon skeleton that is bonded to four different groups or atoms. Example of the isomer or enantiomer monosaccharides is glucose and galactose. They are the mirror image of each other. In aqueous solution, pentoses and hexoses such as glucose forms ring rather than extended straight chain. In aqueous solution, glucose favors the equilibrium towards the ring structure. C6H2OH

H

C5

C4

HO

O

H OH

H

C3

C2

H

H

C1

OH

OH

In the ring structure of glucose, two forms of glucose occur: α-glucose and β-glucose. The diagram above shows the structure of α-glucose where the hydroxyl group bonded to the C1 is facing downwards. The hydroxyl group bonded to the C1 is facing upwards for β-glucose. These two isomers seemed alike but they form a very different glycosidic bond that cannot be hydrolyzed by same enzyme.

Two monosaccharides can be joint together into disaccharide by the dehydration reaction. These two monosaccharides are joint into a disaccharide by the glycosidic bond. Examples of disaccharides are sucrose, maltose and lactose. Sucrose is the most abundant disaccharide in the biological world; it consists of glucose and fructose units joint together by glycosidic bond. Plants generally transport sugar in the form of sucrose from sugar source to sugar sink.

H

CH2OH

H2O

H

CH2OH

+ OH

HO

Maltose is formed by the glycosidic bond between 2 glucose units. Maltose is the ingredient used in brewing beer. Lactose is consists of glucose and galactose subunit and it is present in milk. Polysaccharides are the polymers of monosaccharides. Each polysaccharide molecule consists of hundreds to thousands of monosaccharide units joint together by glycosidic bonds. Polysaccharides can be a single long straight chain or branched chain. Polysaccharides are varying in their properties and functions. Polysaccharides can be categorized based on their functions. Polysaccharides such as starch and glycogen are storage polysaccharides while polysaccharides such as cellulose and chitin are structural polysaccharides. Starch is the storage polysaccharide of plants and it consists of entirely α-glucose monomers joint together by α-1,4-glycosidic bonds. Starch itself is not a pure substance but a mixture of amylose and amylopectin. Amylose is the form of starch which is a straight long chain without branches. Amylopectin is the form of starch that is more complex than amylose and branched with 1, 6-glycosidic bonds at each branch point. Amyloplast is the specialized plastid organelle that stores starch granule in plant cells. Most animals have enzymes that can hydrolyze plants’ starch into glucose, enable starch available as nutrient. Glycogen is the storage polysaccharide of animals. The structure of glycogen is resembles to amylopectin but it is more extensively branched and more soluble in water. In vertebrates, glycogen is stored in liver and muscle cells. When the demands for glucose in the blood increases, the cells can hydrolyze the stored glycogen into glucose released into the blood stream. However, glycogen in animal cells cannot sustain the animal for long; for example, the stored glycogen is depleted in about one day unless he or she is replenished by consumption of foods.

Cellulose is the most abundant carbohydrate on Earth. Cellulose is the major component of cell wall in plants. Cellulose is the polysaccharide consists of entirely glucose but different from starch, the units that make up cellulose are β-glucose units. These monomers are joint together by β-1,4-glycosidic bonds, these glycosidic bonds make the every other glucose units in cellulose chain upside down with respect to its neighbors:

β -glucose units

β-1,4-glycosidic bonds

The difference in glycosidic bonds in cellulose and starch or glycogen gives them distinct 3-D structures. Cellulose molecules are straight chain and never branched. Its hydroxyl groups are free to hydrogen bonded to other hydroxyl group from other parallel cellulose molecules. Through these hydrogen bonds, cellulose molecules can be grouped into a very strong microfibril. The enzymes that can hydrolyze the α linkages in starch are not able to hydrolyze the β linkages in cellulose due to their distinctive in shapes. Therefore, most animals cannot use cellulose as nutrients. Cellulose in our foods pass through the digestive tract eliminated as feces. Cellulose abrades the digestive tract wall and stimulates the lining wall to secrete mucus, thus smoothing the passage of foods through the tract. Most fruits, vegetables and whole grains are rich in cellulose and cellulose is referred to as insoluble fibers in food package labels. However, some microorganisms can digest cellulose into glucose. Some bacteria in rumen of cows and sheep enable them to obtain glucose from cellulose. Termites’ digestive system also contains microbes that enable them to make a meal of wood. Chitin is another polysaccharide found in cell wall of fungi and exoskeleton of arthropods, functioning as structural polysaccharide. In arthropods, pure chitin is leathery and it must be mineralized with calcium carbonate to hardening it. Chitin is a modified carbohydrate that formed from N-acetyl glucosamine subunits. N-acetyl glucosamine is similar to β-glucose except that the hydroxyl group bonded to C 2 is replaced by NH-CO-CH3 group. H OH

HO H

H N C O

CH3

Some of the carbohydrates are not entirely consist of carbon, oxygen and hydrogen alone, some of them are compounds in which the hydroxyl group is replaced by an amino group, forming important structural component and also lubricate the joints. Some examples of these complex and modified carbohydrates are galactosamnie, glucosamine, and chitin. Galactosamnie is the carbohydrate present in cartilage. Some of the carbohydrates are bonded to proteins or lipids, forming glycoproteins or glycolipids, respectively. Glycoproteins are formed in the cells through glycosylation process occurs in endoplasmic reticulum and Golgi apparatus. Most of these glycoproteins are embedded proteins in plasma membrane, functioning in cell-cell adhesions, cell-cell communications, and immunity responses. Glycolipids also occur on the cell surface and play similar roles as glycoproteins. Lipids: Lipids consists of a groups macromolecules that are not polymers. Most of the lipids do not similar to each other in terms of structures and functional groups, unlike carbohydrates, proteins, and nucleic acids. They are grouped together as lipids just because they have no or little affinity towards water, in other words, they are all hydrophobic, and they are only soluble in nonpolar solvents such as ether, and chloroform. However, many of them still have some polar bonds which are associated with oxygen but the major part of the molecules consists of hydrocarbons. Lipids consist of diverse of hydrophobic group but biologically we only focuses on fats, phospholipids, and steroids. Fats or triacylglycerol is the most abundant lipids in living world. Fats are constructed from two kinds of smaller molecules: glycerol and fatty acids. Glycerol is an alcohol with three carbons in their carbon skeletons, each carbon atom bearing a hydroxyl group. In other words, glycerol can also be called 1,2,3-propanetriol in chemistry context. Fatty acids are a group of carboxylic acids. Fatty acids have a carboxyl group as functional group connected to a long hydrocarbon chain, for example, palmitic acid: O C HO

C15H31

Three fatty acids are joining to glycerol by eater bonds into fats:

H H

C

O O

C

R1

C

R2

C

R3

O H

C

O O

H

C H

O

Ester Linkages

Fats are separated from water because of the hydrogen bonds formed between water molecules exclude fats molecules. The nonpolar hydrocarbon chains are the main reason for hydrophobic property of fats. Fats can be divided into saturated fats and unsaturated fats. The saturated fats are the fats in which their hydrocarbon chains from fatty acids do not have any double bond between the carbon atoms. Unsaturated fats are the fats in which their hydrocarbon chains of fatty acids have at least one double bonds between the carbon atoms. These double bonds in the hydrocarbon chains would make the hydrocarbon chains to bend or kink wherever the double bonds occur. Most animals’ fatty acids are saturated and they can tightly packed together side by side and solidify at room temperature. Examples of animal fats are lard and butter. Most plants and fishes fatty acids are unsaturated and have a kink where the double bonds occur. The kinks prevent the molecules to packed together and hardly solidify. Therefore, plants oils appear as liquid at room temperature. However, unsaturated fats don’t mean healthy fats. There are two types of double bonds: cis-double bonds, and trans-double bonds. The unsaturated fats with cis-double bonds are healthy fats that can prevent the atherosclerosis diseases. Saturated fats contribute to the cardiovascular diseases, for example cardial infarction. The hydrogenated vegetable oils on food labels are the unsaturated fats that had been saturated through the hydrogenation process, for industry purposes. However, the saturated fats and unsaturated fats produced in this process are contributed even more to cardiovascular diseases. The double bonds produced in this process are transdouble bonds. The bending occurs at trans-double bond is very different from the cisdouble bond. The bending making the unsaturated fats are even more easily solidified at room temperature.

Fats have many functions in biological world, such as energy storage, protection of vital organs, insulation of body from cool climate and provide buoyancy for marine mammals. Fats can store more than twice as much energy as a polysaccharide can such as starch. Most animals store energy in the form of fats. Plants are immobile and therefore can store energy as starch. Mammals also store fats in the form of adipose tissue. This tissue can cushions the vital organs such as kidneys and also insulates the body from cool climate. Phospholipids are also one type of lipids which are resemble to the fats but they are some structurally different from fats. Both of them are made up of glycerol and fatty acids but the hydroxyl groups of the glycerol in phospholipids are ester kinked to only two fatty acids. The third hydroxyl group is bonded to a phosphate group, PO4-. Another polar small molecule is bonded to the phosphate group, forming a phospholipids molecule. The polar small molecule can be choline, serine, ethanolamine, or inositol. Choline P

Hydrophilic head

Glycerol Hydrophobic tail

Due to the presence of the phosphate group and the polar small molecule, phospholipids show ambivalent property. The hydrophilic head has the strong affinity to water while the hydrophobic tail is excluded from water. When phospholipids are poured into the water or aqueous solution, they are self-assemble into phospholipids bilayers so that the hydrophilic heads are facing the aqueous environment while the hydrophobic tails are shielded from the water. In chemical context, phospholipids can be further into two major forms: phosphoglycerides, and sphingolipids. The structural properties of phospholipids described above are the properties of phosphoglycerides. Phosphoglycerides are the most prominent phospholipids in cellular membranes. It has a glycerol esterifies with two fatty acids and a phosphate group. The phosphate group usually has a small hydrophilic alcohol linked to it by an ester bond, for example, choline. The phosphoglycerides bonded to a choline molecule are called phosphatidyl choline. Except for inositol; serine, ethanolamine, and choline are all positively charged on their amino groups. The presence of positively charged amino group neutralized the negatively charged phosphate group, but it gives the hydrophilic head a higher degree of polarity.

Some of the membrane contains sphingolipids. Sphingolipids are based not on glycerol but on an amine alcohol-sphingosine. Sphingosine has a long hydrocarbon chain with a single unsaturated site near the polar end. Sphingosine can form an amide bond with a fatty acid. The hydrophobic tails of sphingolipids are derived from the hydrocarbon chain of the sphingosine and the hydrocarbon chain from fatty acid. The complete sphingolipids can be formed when any polar group is linked to the hydroxyl group of C1 of sphingosine. Steroids are the lipids that consist of four fused rings of carbon skeleton. Their structure are different from the other members of the lipids, the hydrophobic property is the only reason they are grouped as lipids. Different steroids have different functional groups bonded to the rings. Steroids only occur in eukaryotic cells. The most common steroids in animal cells is cholesterol. Cholesterol is the common component found in cell membrane, it is also the precursor from which other steroids are synthesized. Cholesterol plays an important function in membrane as temperature buffer. Examples of steroids hormones in the body are androgens, estrogens, progesterone, glucocorticoids, and mineralocorticoids. Proteins: Proteins are the most sophisticated molecules known in the world, resulting in their vast variety of functions. A protein might consist of one or more polypeptide chain(s) folded and coiled into a specific conformation with specific function. Polypeptide is a polymer of amino acids. Amino acids are the organic molecules that consist of both the amino group and the carboxyl group. Most amino acids have a chiral carbon which bonded to four different groups or atoms, except for the simplest amino acidglycine. Chiral carbon H N H

C*

R

O

H

OH

C

Note: In glycine, R is the hydrogen atom.

There are more than 60 types of amino acids known but only about 20 of them are involved in protein synthesis: Categories:

Amino acids

Nonpolar

1. Glycine (Gly) 2. 3. 4. 5.

Polar

Alanine (Ala) Valine (Val) Leucine (Leu) Isoleucine (Ile)

6. Methionine (Met) 7. Phenylalanine (Phe) 8. Tryptophan (Trp) 9. Proline (Pro)

1. Serine (Ser)

4. Tyrosine (Tyr)

2. Threonine (Thr) 3. Cysteine (Cys) Electrically charged

5. Asparagines (Asn) 6. Glutamine (Gln)

1. Aspartic acid (Asp)

4. Arginine (Arg)

2. Glutamic acid (Glu)

5. Histidine (His)

3. Lysine (Lys)

When two amino acids are positioned together with the carboxyl group of one amino acid is adjacent to the amino group of another amino acid, the enzymatic dehydration reaction is occurs, resulting in peptide linkage.

H

R1 N

H

C

C

H

O

H

R2

N

C H

OH C O Peptide linkage

When the amino acids are assed to the above structure one by one through peptide linkages, a polypeptide is formed. In polypeptide, the peptide linkages are the repeated constituents of the polymer. In polypeptide, the amino end is called Nterminus while the carboxyl end is called the C-terminus. The peptide linkages are formed one at a time, starting from the N-terminus to C-terminus.

Polypeptides don’t directly mean proteins. A functional protein isn’t a polypeptide chain alone. One or more polypeptide chain(s) can be folded and coiled into a molecule of unique 3-D conformation, the 3-D conformation is dependent on the unique sequences of amino acids in the polypeptide chain. The 3-D conformation is stabilized by the variety of bonds and interactions between parts of the polypeptide chain. Proteins can be categorized into monomeric proteins and multimeric proteins. Monomeric proteins are the proteins consist of only a single polypeptide chain which folded and coiled into the final 3-D conformation, for example ribonuclease. Multimeric proteins are the proteins consist of more than one polypeptide subunits. If the proteins consist of identical subunits, it is called homomeric proteins while if the proteins consist of different subunits, it is called heteromeric proteins. Example of multimeric proteins are hemoglobin and collagen. Hemoglobin is the protein that carries oxygen in human red blood cells which consists of four subunits. Hemoglobin is a multimeric protein as it consists of α-subunit and β-subunit. The structures of proteins can be divided into four different superimposed levels: primary structure, secondary structure, tertiary structure, and quaternary structure. Primary structure is the unique sequences of amino acids in the polypeptide chain. The primary structure is determined not by the random peptide linking between amino acids but by the inherited genetic information-the unique order of nucleotides in RNA molecules, encoded by the DNA molecules. Primary structure: +

H3N-Gly-Pro-Thr-Gly-Thr-Glu-Asn-……………………………..Pro-Lys-Glu-COO-

The secondary structure is the result of the coiling and folding of the polypeptide chain (primary structure) by the intramolecular hydrogen bonds formed between the repeating constituents of the polypeptide chain. Hydrogen bond: δ+

δ-

δ-

δ+

H

F

H

O

δ+

H

δ-

N

The hydrogen bonds formed in secondary structure:

C = O--------H - N Hydrogen bond Hydrogen bonds are weak but if they are repeated greatly, they can support a shape for that particular part of proteins.

There are two main types of secondary structure: α-helix and β-sheet. α-helix is the structure held together by the hydrogen bonds between every four amino acid repeating constituents, resulting in a delicate coil. Most globular proteins have multiple of α-helix separated by nonhelical regions. Some fibrous proteins for example α-keratin have α-helix formation over most of their length. β-sheet is the structure formed from the two or more regions in a polypeptide chain, lying side by side and connected through the hydrogen bonds between the repeating constituents of polypeptide chain. The β-sheet structure makes up the core of many globular proteins, for example the transthyretin. Motifs are the regions in proteins where small segment of α-helix and β-sheet can be connected to each other. The common motifs are β-α-β motif and α turn α motif. When same motif present in different proteins, these proteins serve the same purpose in each. For example, α turn α motif is common in DNA-binding proteins. The formations of the secondary structures are directly dependent on the sequences of amino acid residues in that segment. Leu, Met, and Glu are the strong inclinations to form α-helix while Ile, Val, and Phe are strong inclinations to form β-sheet. The Gly and Pro are the cyclic amino acids are the helix breakers. Tertiary structure is the overall conformation of a polypeptide chain resulting from the interactions between the side chains (R groups) of the amino acid residues. The bonds and interactions that involve the tertiary structure are the Disulfide Bridge, ionic bond, hydrogen bond, hydrophobic interaction, and Van der Waals forces. From these interactions, only the Disulfide Bridge and ionic bond are true bonds. The most common covalent bonds that contribute to the stabilization of a protein conformation are the Disulfide Bridges. Disulfide Bridge consists of a sulfhydryl group of two Cysteine residues react oxidatively. In monomeric proteins, Disulfide Bridges are intramolecular while in multimeric proteins, Disulfide Bridges might be intermolecular. Most globular proteins consist of numbers of segments called domains. Domains are the discrete, locally folded unit of tertiary structures, packed together compactly. Small globular proteins such as ribonuclease have only single domain while large globular proteins such as immunoglobulin has multiple of domains. Proteins with common functions usually have common domains. The quaternary structure is the result of the assembly and interactions of the subunits of tertiary structures. Quaternary structure only applies to the multimeric proteins. The bonds and interactions involve in quaternary structure are similar to the tertiary structure but these bonds and interactions are all intermolecular.

Base on the functions of different proteins, proteins can be categorized into enzymatic proteins, structural proteins, storage proteins, transport proteins, hormonal proteins, receptor proteins, cytoskeleton proteins and defensive proteins. Proteins

Functions and Examples

Enzymatic

Function in selective acceleration of chemical reactions; hydrolase enzymes speed up the hydrolysis reactions.

Structural

Provide supports for the body structures of animals; collagen and elastin provides the fibrous framework in animal connective tissues; keratin in hair, horn, feather, and other skin appendages.

Storage

Storage materials for amino acids; ovalbumin (egg white) is the amino acids source of the developing embryo; casein is the protein of milk providing the amino acids for mammal baby.

Transport

Transport a substance across membrane or carrying a substance between parts of body; hemoglobin is the globular protein that carries oxygen in red blood cells.

Hormonal

Involve in coordination organisms’ activities; insulin is the hormone that controls the glucose level in blood stream.

Receptor

Involve in response of cells to external chemical stimuli; G-proteinlinked receptors involve in receiving of many types of chemical stimuli.

Cytoskeleton Involve in movements of organelles in the cells and also the cell movements from place to place; microtubules consist of tubulin monomers (protein) involve in organelles and chromosomes movements; actin and myosin interactions also lead to the muscle contractions. Defensive

Antibodies in human immunity system also consist of proteins that protect the body against diseases.

Each protein involves in specific type of function and often these functions require the specific conformation of that particular protein. Any small changes in the conformation of the protein can make it malfunction. Even though a simple change in its primary structure can greatly affects the final conformation of the protein and its functional ability, for example, the sickle-cell disease due to the simple change in the primary structure of the hemoglobin: Normal hemoglobin Val-His-Leu-Thr-Pro-Glu-Glu

Sickle-cell hemoglobin Val-His-Leu-Thr-Pro-Val-Glu

Exposed βSubunit

Hydrophobic region

Hemoglobin The subunits do not interact with each other, therefore can carry oxygen efficiently.

The subunits interact interglobularly through the exposed hydrophobic regions into crystallized form, therefore reduce its ability to carry oxygen greatly.

Each protein in the cells undergoes many intermediates before the stable conformation is achieved. In the cells, chaperonins are the protein complex that assist the proper folding of other proteins but do not specifies the final shape of the proteins. Chaperonins only work by keeping the polypeptide chain away from the bad influences of the cytoplasmic environments. Chaperonins consist of the cap and a hollow cylinder with a cylinder space in it. When the unfolded polypeptide chain enters the cylinder space, the cap covers the opening of the space and the binding causes the cylinder to change shape. The change of shape provides hydrophilic environment for the proper folding of the polypeptide. When the folding is complete, the cap comes off and the protein is released.

Some specific example of proteins: insulin, hemoglobin, and transthyretin. Insulin is the hormonal protein that is secreted by the pancreas (β-cells). The main function of insulin is to decrease the level of blood glucose by promotes the uptake of glucose by the body cells so that they can be stored as glycogen in liver cells and muscle cells. Hemoglobin is the globular protein that carries oxygen in red blood cells. Hemoglobin is the protein that consists of four subunits (two α-subunits and two β-subunits). The protein hemoglobin itself is still not a functional protein; each of the subunit has a nonpolypeptide component called heme. Heme is a complexes or coordination compound which consists of more than one ligands compounds attached to a iron ion (transition metal ion) through the dative bond. The iron ion is the atom that binds to the oxygen.

β-subunit

Heme group Iron

α-subunit Transthyretin is a globular protein that found in blood stream. Transthyretin functions in transporting the vitamin A and thyroid hormone. Nucleic acids: The sequences of amino acid residues in polypeptide chains are programmed by the gene. Gene consists of DNA (Deoxyribonucleic acid). DNA provides the directions for its own replication, the synthesis of RNA and therefore indirectly controls the protein synthesis. Each chromosome contains one long DNA molecule which of thousands of genes. DNA is a nucleic acid. Nucleic acids are the macromoleculespolynucleotide. Nucleic acids consist of monomers called nucleotides. There are two types of nucleic acids in cells: DNA and RNA (Ribonucleic acid).

Each nucleotide consists of three parts: nitrogeneous base, pentose, and phosphate group. There are two types of pentose sugars: ribose and deoxyribose. Deoxyribose is the pentose sugar present in DNA while ribose sugar is present in RNA. Ribose: Deoxyribose: HOH2C5

OH

4

H

HOH2C5

OH

1

3

2

HO

H

4

H3

OH

HO

2

1

H

H

There are two types of nitrogeneous bases: purine and pyrimidine. Purine is a group of nitrogeneous bases which consists of adenine (A) and guanine (G). Purine is larger, with six-member ring of carbon and nitrogen atoms fused to a five-member ring. The pyrimidine is a group of smaller, with six-member ring of carbon and nitrogen atoms. Pyrimidine consists of cytosine (C), thymine (T), and uracil (U). Adenine, guanine, and cytosine are all present in both DNA and RNA while thymine is only present in DNA and uracil is only present in RNA. In each nucleotide, the portion that without the phosphate group is called nucleoside. Therefore, the whole nucleotide can also be called nucleoside monophosphate.

P

C5H2

Nitrogeneous base

The adjacent nucleotides are joining together by the covalent bonds called phosphodiester linkages. The phosphodiester linkages are formed between the hydroxyl groups on the 3’ carbon of one nucleotide with the phosphate group on 5’ carbon of the next nucleotide. A nucleic acid is a polynucleotide that consists of two ends: the 5’ end and 3’ end. The DNA strands are built from the 5’ to 3’ end during the replication process. The polynucleotide chains can be divided into two parts: the sugar phosphate backbone and nitrogeneous bases parts. The sugar phosphate backbone consists of the phosphate groups and the pentose sugars while the nitrogeneous bases part consists of different types of nitrogeneous bases. RNA molecule only consists of only single polynucleotide chain while DNA molecule consists of two polynucleotide chains that spiral around an imaginary axis by the Van der Waals forces, forming a double helix. The two sugar phosphate backbones are running in opposite 5’ to 3’ end direction from each other-the antiparallel arrangement. Between the sugar phosphate backbones are the nitrogeneous bases part, which is the interior of the double helix. The nitrogeneous bases are paired up to each other in the double helix by hydrogen bonds. The double strands of DNA do not formed randomly by the hydrogen bonds between the nitrogeneous bases. The nitrogeneous bases of the polynucleotides must be complimentary to each other. Only certain bases are compatible to each other: A always paired up with T; G always paired up with C. The complimentary of the double helix strands makes possible the precise copying of genes that are responsible for inheritance. DNA can serves as the template to order the nucleotides into new complementary strands, resulting in two identical copies of origin double helix DNA molecules during the replication process. The DNA molecules document the hereditary background of an organism and the information are passed from the parent to the offsprings. Besides carrying the heritable information, DNA also determines the amino acids sequences in proteins. The siblings have greater similarity in their DNA and proteins than other unrelated individuals of same species. If this evolutionary theory (the concept of molecular genealogy) is right, then the two species that appear to be closely related based on fossil records and anatomical evidences also share a greater proportion of their DNA and protein sequences. For example, the polypeptide chain in hemoglobin consists of 146 amino acid residues. The gorillas are similar greater to human and their polypeptide chain differ only one amino acid while mice which is distantly related to human and their polypeptide chain differ 27 amino acids. The frog even more distantly related to human and their polypeptide chain differ 67 amino acids.

Chapter 4. The Plasma Membrane and Its Functions Phospholipid Bilayers: Hydrophilic Head Hydrophobic Region

Plasma Membrane: Plasma membrane is the structure that controls the traffics of substances in and out of the cell it surrounds. Plasma membrane exhibits selective permeability that allows only certain substances to across it more easily than others. Therefore, plasma membrane can encloses the cytosol different from the surrounding solution but still permitting the uptake of nutrients and eliminations of waste products. However, the functions of the plasma membrane are not only that. The staple ingredients of plasma membrane are lipids and proteins, although carbohydrates also important. The most abundant lipid in plasma membrane is the phospholipids that make up the structure of the plasma membrane. The membrane proteins are the proteins that embedded in the plasma membrane or attached loosely on the surface of plasma membrane. Most of the specific functions of plasma membrane are determined by the membrane proteins. The carbohydrates also exist in plasma membrane as glycoproteins or glycolipids. Model building in biology: Many biologists as well as other fields of scientists might work with the models. Scientists often construct models as less abstract representations of ideas such as theories or natural phenomenon such as biological processes. Scientific models can take many forms, such as diagrams, graphs, 3-D objects, computer programs, or mathematical equations. The choice of model depends on how well it would be used to explain and communicate the object, idea, or process it represents. The acceptance and rejection of a model proposed depends on how well it fits the observations and explains the experimental results. Often, a new finding makes a model obsolete or become modified. Few models survive without modifications. In 1972, S.J. Singer and G. Nicolson proposed the fluid mosaic model regarding the structure of plasma membrane. According to the fluid mosaic model, plasma membrane is a fluid structure of lipids with a mosaic of membrane proteins embedded in or attached to the bilayers of phospholipids.

The Fluidity of Plasma Membrane: The plasma membrane is held together by the hydrophobic interactions between the hydrophobic tails of the phospholipids bilayers. Most of the lipids and proteins can drift about the plasma membrane laterally but it’s quite rare for them to flip-flop transversely across the membrane. This is because the hydrophilic parts of the molecules have to cross the hydrophobic core of the phospholipids bilayers. The lateral movements of phospholipid molecules are quite rapid-about 107 times per second while the proteins which are usually much larger, move laterally more slowly, may be once per month. Some of the membrane proteins move in highly directed manner, they are driven along the cytoskeleton fibers by the motor proteins connected to the cytoplasmic region of the membrane proteins. Some of the membrane proteins also remain immobile in the membrane most of the time as they are held by the cytoskeletons. Plasma membrane remains fluid as temperature decreases until finally the phospholipids settle into a closely packed arrangement (solidify). The plasma membrane can remains fluid to a lower temperature if it is rich in phospholipids with unsaturated hydrophobic tails. The presence of the unsaturated or double-bond kinks in the hydrophobic tails keep the molecules from packing together, thus enhancing the membrane fluidity. The cholesterol is the temperature buffer of the plasma membrane. At warm temperature, for example 37oC, cholesterol makes the membrane less fluid by restraining the movements of the phospholipids laterally. However, at low temperature, the cholesterol hinders the close packing of phospholipids, thus lowering the temperature required for the membrane to solidify. The Membrane Proteins: There are more than 50 types of membrane proteins found in the red blood cells’ plasma membrane. Although the phospholipids form the main fabric of plasma membrane, the membrane proteins are the components that determine most of the specific functions of plasma membrane. Different types of cells have different collection sets of membrane proteins and various membranes have also different collections of membrane proteins. However, these membrane proteins can be classified into two major types: the integral proteins and peripheral proteins. Integral proteins penetrate the hydrophobic core of phospholipid bilayers of the plasma membrane. Many of the integral proteins are transmembrane proteins that completely span the plasma membrane. The hydrophobic region of the integral

proteins usually consists of nonpolar amino acids such as Gly and Ala, and they are coiled into the α-helix structures. The hydrophilic region of the integral proteins is exposed to the surface of the plasma membrane. The peripheral proteins are the membrane proteins that attached loosely to the surface of the plasma membrane, often to the exposed part of the integral proteins. The Structure of the Plasma Membrane:

Integral proteins

Carbohydrate Chain Glycoproteins Peripheral proteins

Microfilament The Functions of Plasma Membrane (membrane proteins): The membrane proteins can carry out vast variety of functions. Some transmembrane proteins can be channel proteins, carrier proteins, or protein pump. For example the channel proteins can provide the hydrophilic channel for certain specific substances to cross the plasma membrane. The carrier proteins can shuttle the substances across the membrane by changing the conformation when the substance molecules attach to it. The protein pumps carry the substances across membrane by spending the energy in the form of ATP. The membrane proteins also function as the enzymes. These membrane proteins, like the other enzymes, have the active sites that can bind to and catalyze some biological reactions. The membrane proteins also function in signal transduction by detecting the specific chemical signals from outside the cells. The membrane proteins then transmit the signal to the interior of the cells. The signal molecules are also called ligands. When ligand molecule binds to the specific active site of the membrane proteins (receptors),

the membrane proteins change their conformation to trigger the specific chemical events in the interior of the cells. Membrane proteins also function in cell-cell recognitions. The membrane proteins involved in this ability usually are glycoproteins. The carbohydrate chains that bind to the proteins vary depending on the types of the cells and also the species of the organisms. For example, the human blood group designated A, B, AB, and O reflect the variations in the carbohydrate chains on the surface of red blood cells. This variety enables the cells to distinguish one type of cell from another. This ability is very crucial to the functioning of an organism as it is important in the sorting of cells into tissues and organs in an organism’s embryos. It is also important in rejections of the foreign cells by the immune systems. Membrane proteins also play an important role in intercellular joining. These intercellular junctions are provided by the tight junction, gap junction, and adhesive junction in animal cells and the plasmodesmata in plant cells. These junctions enable the joining of the cells together and some of them even allow the exchanges of cellular components from cell to cell. The junctions require the presences of the membrane proteins to occur. For example, the gap junctions in vertebrates require the membrane protein called connexins that join together into a ring of connexon. Besides, the membrane proteins also important in holding the extracellular matrix (ECM) and cytoskeletons together to the plasma membrane. The microfilaments may be bind to the membrane proteins to maintain and stabilize the cell shape and also to maintain the location of certain membrane proteins. The membrane proteins that bind to the ECM materials can coordinate the extracellular and intracellular changes. The Permeability of the Plasma Membrane: Plasma membrane is selectively permeable towards the substances that across it. It permits certain substances to across it more easily than other. The selective permeability of plasma membrane depends on both the discriminating barrier of the phospholipid bilayers and the specific transport proteins built into the plasma membrane. Hydrophobic molecules or nonpolar molecules such as CO2, O2, and hydrocarbon, dissolve in the lipid bilayers thus can across the plasma membrane easily by simple diffusion. The hydrophilic molecules, polar molecules and ions are impeded by the hydrophobic core of the lipid bilayers, thus cannot pass through the plasma membrane freely. The lowly polar molecules such as glucose can only pass through the plasma membrane very slowly, while the water molecules which are the extremely small polar molecules do not cross the plasma membrane rapidly. Such polar molecules are avoiding the lipid bilayers by pass through the transport

proteins. There are two types of transport proteins: the channel proteins and carrier proteins. Example the channel protein is the aquaporins. Carrier proteins are very specific, for example, the glucose transporter even rejects the fructose. Transports Across Plasma Membrane: There are two major types of transports of substances across plasma membrane: the passive transports and active transports. Passive transport is the spontaneous diffusions of a substance across plasma membrane down its concentration gradient, without the energy investment in the form of ATP. Passive transport can be further divided into simple diffusion and facilitated diffusion. Simple diffusion is the net diffusion of a substance from the region of high concentration to the region of lower concentration spontaneously, with or without the plasma membrane. Simple diffusion is the result of the thermal motion or kinetic energy of the molecules, leading the tendency for the molecules to spread out evenly into the available space. Each of the molecule move randomly, but the diffusion of a population of molecules may be overall directional. In the presence of plasma membrane, the simple diffusion does not simply means movement of the molecules across plasma membrane, but the net movement (the molecules move across the plasma membrane in both directions but there are more molecules that move across the plasma membrane to the side of lower concentration). The molecules diffuse across the plasma membrane until the equal concentration between both sides is achieved. At this moment, dynamic equilibrium between the both sides is achieved where there are equal rates of diffusion in both directions. Simple diffusion:

Facilitated diffusion involves the transport proteins such as channel proteins and carrier proteins. Channel proteins provide the hydrophilic channels that allow specific molecules or ions to diffuse through it. Example of these channel proteins are aquaporins and ion channels. Aquaporins are the water channel proteins that facilitate massive diffusion of water molecules across membrane. Iona channels may be gated channels that require chemical or electrical stimuli to trigger them to open or close. The chemical stimuli are the molecules other than the substance molecules to be transported, for example neurotransmitter molecules that trigger the sodium ion channels to open and allow the influx of sodium ions from the synaptic cleft. Channel Protein:

Carrier proteins in passive transports do not consume ATP. They have the binding sites that specific only to the substance molecules. Once the correct molecule binds to the bind site, the carrier protein changes conformation translocates the molecule across membrane. Carrier protein:

Osmosis is the net diffusion of water molecules across selectively permeable plasma membrane from the side of higher free water concentration to the side of lower free water concentration (down the concentration gradient of free water) without the ATP consumption. Osmosis only occurs when the dissolved substances are impeded by the plasma membrane. The side with lower free water concentration is the side with higher substance concentration, the water molecules cluster around the solute molecules thus there are fewer free water molecules, and vice versa. Tonicity is the ability of a solution to cause a cell to gain or lose water, considering both the concentration of solutes (penetrating or nonpenetrating) and the membrane permeability. There are three types of solutions/environments: hypotonic, hypertonic and isotonic solutions. Hypotonic solution is the solution that has higher free water concentration than the cytosol. Hypertonic solution is the solution that has lower free water concentration than the cytosol of the cells. Isotonic solution is the solution that has equal free water concentration as compared to that of the cytosol of the cells. Animal cells and plant cells or any cells with cell wall do not show the same result when they are immersed in the tonic solutions. In isotonic solution, cells do not gain or lose water to the environment due to there is no net movement of water molecules in or out of the cells, the volume of the cells remain constant. For animal cells, there is nothing changes in isotonic solution. For plant cells, the cells are in the form of flaccid/limp. In hypertonic solution, cells would lose water to the solution due to the net movement of water molecules out of the cells by osmosis. For animal cells, the excess loosing of water causes them to shrivel and finally die. For plant cells, the plasma membrane will pulled away from the cell wall. At this stage, the plant cells are said to be plasmolyzed and it can be lethal. In hypotonic solution, cells would gain water from the environment due to the net movement of water molecules into the cells by osmosis. For animal cells, excess uptake of water causes them to swell and finally burst (lyse). For plant cells, since there is rigid cell wall to give back pressure on the cells so that the further uptake of water is prevented, the plant cells are in turgid state. Turgid state is the healthy state for plant cells as it can provides mechanical support for nonwoody plants.

Since animal cells or any cells that do not have cell wall to prevent the lyse of the cells when immersed in hypotonic environment, these cells especially those that living in pond water, have certain mechanisms that prevent the excess uptake of water. These mechanisms are called osmoregulations. For example, the paramecium that lives in pond water that is hypotonic to its cytosol. Paramecium’s plasma membrane is less permeable to water and paramecium is equipped with a special apparatus called contractile vacuole. Contractile vacuole can force the excess water out of the cell as fast as the water enters the cell by osmosis. Active transport is the transport of solutes across plasma membrane against the concentration gradient with the aid of carrier proteins and the carrier proteins consume ATP in doing so. The main purpose of active transport is to maintain cytosolic concentration of certain molecules or ions. For example, the animal cells require the higher concentration of potassium ion, [K+] and lower concentration of sodium ion, [Na+] than the extracellular solution. Animal cells can do so by having the sodium - potassium pumps that always pump Na+ out and K+ into the cells: 1. The cytoplasmic Na+ ions (3 ions) bind to the pump. 2. Phosphorylation of the pump by ATP is stimulated. The ATP transfers its terminal phosphate group directly to the pump. 3. Phosphorylation causes the pump to change its conformation, expelling Na+ out of the cell. 4. Two K+ ions from extracellular solution bind to the phosphorylated pump, triggering the release of the phosphate group from the pump. 5. The pump restores its original conformation, translocates K+ ions into the cell. Another example of carrier protein that involves in active transport is the bacteriorhodopsin. Bacteriorhodopsin is the protein pump that found in plasma membrane of halophilic archaea-halobacterium. Bacteriorhodopsin utilizes the energy from the photons to drive active transport. When the oxygen is depleted, oxygendriven metabolic pathway is blocked and the bacteriorhodopsin is used as alternative. Voltage is the electrical potential energy due to the separation of the two opposite charges: positive charge and negative charge. Cells also have voltage across plasma membrane-membrane potential. The cytoplasm is negatively charged while the extracellular solution is positively charged. The separation of charges between the two sides of plasma membrane is due to the unequal distribution of anions and cations between the two sides of plasma membrane. The membrane potential always favors the passive transport of cations into the cell while the anions out of the cell. Two forces that drive the transport are chemical and electrical energy. The chemical energy is the concentration gradient of the ions and the electrical energy is the membrane potential. All together the two forces are called electrochemical gradient. Therefore,

the above passive transport is also the diffusion down the electrochemical gradient. One of the protein pumps that involves in this generation of electrochemical gradient is the sodium – potassium pump. The sodium – potassium pump generating the membrane potential by transporting 3 Na+ ions out of the cell for every 2 K + ions into the cell, actively. On doing so, there are net transfers of one positively charged ions out of the cell so that the outside of the cell remains positively charged. The protein pumps that generate membrane potential are called electrogenic pumps. The sodium – potassium pump is the major electrogenic pump in animal cells. The main electrogenic pumps in plants, fungi, and bacteria are the proton pumps that actively transporting the proton/H+ ions out of the cells. The proton pump involves in one more kind of active transport-indirect active transport. One of this indirect active transport is the cotransport or coupled transport. Cotransport occurs when the active transport of one type of solute indirectly drive the uphill diffusion of other solutes. In plant cells, proton pump actively generates the proton gradient and drives the active transport of amino acids, sugars, into the cells. In this case, for example, the proton pump is coupled by the sucrose-proton transporter that uses the diffusion of proton down its electrochemical gradient to drive the uptake of sucrose. The proton-sucrose cotransporter does not consume ATP but it use active transport of proton pump that generates the electrochemical gradient to drive the transport of sucrose against its concentration. Therefore, plants can load the sucrose produced by photosynthesis into the cells of veins and thus can distribute it from sucrose source to sucrose sinks. Proton pump H+ ATP

ADP + Pi

Cotransporter

Sucrose

Bulk Transports: Large molecules such as proteins, polysaccharides, and any other large particles across the plasma membrane by bulk transport that involving the vesicles. There are two types of bulk transports: the endocytosis and exocytosis. Exocytosis is the secretion of macromolecules out of the cells with the fusion of vesicle containing the macromolecules with the plasma membrane. The transport vesicles that are bud off from the Golgi apparatus travel along the microtubules by the motor proteins to the plasma membrane. On reaching the plasma membrane, the phospholipid bilayers of the vesicle membrane rearrange themselves so that it can be fused with the plasma membrane and the contents can be spill out of the cell. Pancreatic cells secrete the insulin into the blood stream by exocytosis. The neuron cells also secrete the neurotransmitter molecules into the synaptic cleft by exocytosis. The cell wall materials also secreted out of the plant cells by exocytosis to make cell wall. Plasma membrane

Substance to be secreted

Vesicle The arrangement of vesicle with plasma membrane, expelling the substance out of the cell. Cytosol

Cells take in macromolecules and particulate matters from outside of the cells by endocytosis. Endocytosis involves the formation of new vesicles from the plasma membrane. In endocytosis, the small areas of plasma membrane sink inwards into a pocket-like shape, and the pocket pinches off from the plasma membrane, forming the vesicles containing the materials. There are three types of endocytosis: the

phagocytosis, pinocytosis, and receptor-mediated endocytosis. Phagocytosis is the action of engulfing a particle by wrapping the pseudopodia around the particle and packaging it within a vesicle. The vesicle is then fuses with the lysosome where the particle is ingested. Pinocytosis is the action of gulping the droplets of extracellular fluid into the tiny vesicles. Plasma membrane

Pseudopodium

Food vacuole

Cytosol

Pinocytosis is rather the nonspecific endocytosis as it is not the fluid that needed by the cell, but the dissolved particles in the droplet.

Plasma membrane

Vesicle Cytosol

The most specific endocytosis is the receptor-mediated endocytosis as it involves the specific receptor sites on the membrane proteins exposed to the extracellular environments. The receptor proteins are usually clustered in an area called coated pits. The lining of the coated pit area on the cytoplasmic side is the fuzzy layer of coated proteins. The molecules that bind to the receptor proteins are called ligands. When the ligand binds to the receptors, the coated pits area forms a vesicle containing the ligand molecules. This type of endocytosis is used by the human cells to take in cholesterol. The cholesterol travels in the blood stream in the form of low-density lipoproteins (LDLs). The LDL is the ligand that will binds to the receptor proteins when it entering the cells. Familial Hypercholesterolemia is the inherited disease where there is a very high cholesterol level in the blood, due to the defective or missing of the LDL receptor proteins, causing the LDL molecules failed to enter the cells and accumulate in the blood stream, leading to the atherosclerosis disease.

Plasma membrane Coated pit

Receptor Ligand molecule

Chapter 5. Membranous Organelles Compartmentalization: If all enzymes and compounds for a particular process are localized within a specific region (usually within a membraneous organelle), then the high concentration of the substances are only needed in that region rather than throughout the cells. The specific region is called the compartment where the localized of the substances is called the compartmentalization. These organelles (the membrane-bounded compartments) are only present in eukaryotic cells. These organelles are highly specialized to compartmentalize activities. Eukaryotic cells require these compartments as they are usually larger than the prokaryotic cells. One example of the compartmentalization is the chloroplasts. Most enzymes, compounds, and pigments for photosynthesis are compartmentalized into the organelle to maintain their high concentration in the chloroplasts without having to maintain the concentration in other regions in the cells. One of the distinctive features of the eukaryotic cells is the presence of the endomembrane systems which regulate protein trafficking and perform metabolic functions. The endomembrane systems include: nuclear envelope, endoplasmic reticulum (ER), Golgi apparatus (GA), lysosomes, endosomes, vacuoles, and plasma membrane. Nuclear Envelope: Nuclear envelope is the double membranes that surround the content of the nucleus from the cytoplasmic environment. The outer membrane is continuous with the ER membrane, and the perinuclear space is continuous with the ER lumen. Perinuclear space is the region formed between the inner and outer membrane. Perinuclear space Outer membrane Inner membrane Nucleolus

Pore Endoplasmic Reticulum (ER): ER consists of extensive network of membranous sacs and tubules called cisternae, as well as associated vesicles that transporting the content of ER to GA. ER functions mainly in biosynthesis of proteins, lipids, etc. in eukaryotic cells, there are two types of ER: rough ER and smooth ER. Rough ER is so called as its cytoplasmic site of membrane has ribosomes bound to it, making it appears rough under microscope. However, not all membrane of rough ER has bound ribosomes, one part of rough ER called transitional element (TE) is lack bound ribosome. TE is very important in formation of transition vesicles/transport vesicles that shuttle the content of rough ER to GA. Rough ER functions mainly in protein synthesis. The proteins synthesized by the rough ER are membrane-bound proteins and soluble proteins to be secreted (secretory proteins). The bound ribosomes are the one that synthesizing the polypeptides through the process called translation. The polypeptide chain grows from the bound ribosome is threaded into the ER lumen through the pores formed by the ER’s membrane protein complex. Many of the polypeptides enter the ER co-translationally where the proteins are inserted into the ER lumen as polypeptide chains synthesized by ribosomes. Other proteins are inserted into the ER lumen post-translationally. If the polypeptide chains are destined to become membrane proteins, they would remain anchor to the lumen side of ER membrane. If the polypeptide chains are destined to become secretory proteins, they would be directly released into the ER lumen. Rough ER is the initial site for the folding of polypeptide chains, recognition and removal of misfolded proteins and the assembly of multimeric proteins. The process of recognition and removal of misfolded proteins is called degradation. In the degradation process, the misfolded proteins are exported to the cytosolic proteasomes before they have the opportunity to move on to GA. Beside the protein synthesis, rough ER also functions in membrane synthesis. ER membrane can expands and transferred its own membrane to the other endomembrane system through the formation of transport vesicles. Smooth ER is the ER that lack bound ribosome on the cytosolic side of it membrane. Smooth ER involves in several functions that are different from the rough ER. Smooth ER involves in biosynthesis of lipids, metabolisms of carbohydrates, drug detoxification, and calcium storage. Smooth ER contains many enzymes for synthesis of lipids such as oils, phospholipids, and steroids. Many sex hormones and steroid hormones are produced in smooth ER.

Smooth ER in liver cells contains enzymes such as glucose-6-phosphatase that functions in enzymatic breakdown of stored glycogen. Glucose-6-phosphatase is the membrane-bound enzyme (protein) that special to smooth ER in liver cells. It catalyzes the removal of phosphate group from glucose-6-phosphate into a free glucose (plasma membrane is impermeable to phosphorylated sugars). The liver glycogen also stored as granules associated to smooth ER. Smooth ER in liver cells also functions in detoxification of drugs and poisons. The enzymes in smooth ER can catalyze the hydroxylation (addition of hydroxyl group) of the drugs and poisons to make them more soluble and easier to flash from the body. One detoxification that is carried out by smooth ER is the elimination of barbiturate (sedative phenol barbital). Barbiturate, alcohol, and many other drugs induce the proliferation of smooth ER that can increase the rate of detoxification. This will lead to concomitant effect, which is the increase of tolerance of body to the drugs. Moreover, proliferation of smooth ER to one drug can also increase the tolerance of body to other drugs. In effect, higher and higher doses of drug are required to achieve the same effect or sedation from time to time. For example, barbiturate abuse decreases the effectiveness of certain antibodies and other useful drugs. Besides, smooth ER also functions in storage of calcium ions. For example, one special type of smooth ER is the sarcoplasmic reticulum in muscle cells. Calcium ATP synthase is the pump that actively transports calcium ions from the cytosol into the lumen of smooth ER, actively decreasing the concentration of calcium ions in cytosol. The muscle cells are in relaxed stage. When the neurotransmitters bind to the receptors on the surface of muscles, sodium ion influx triggers the efflux of calcium ions back into the cytosol. The increase of cytosolic concentration of calcium ions triggers the muscle contractions. Golgi Apparatus (GA): After leaving the ER, transport vesicles travel to the GA. Anterograde transport is the movement of materials (mostly by transport vesicles) from ER to GA and then other destinations. Retrogade transport is the movement of materials from GA back to ER, mostly to recycle the lipids and proteins that are no longer needed. GA is the flattened membrane-bounded cisternae, which are disk-shaped and they are stacked together into a Golgi stacks. Each Golgi stack consists of 3 to 8 cisternae. The GA lumen is the part of endomembrane system network of internal spaces. GA is a dynamic structure that is surrounded by numerous transport vesicles, which are engaged in the transfers of materials (such as lipids and proteins) between parts of the GA other structures.

A GA has two structural and functional polarities: the cis-face and the trans-face. Cisface is the receiving face of the GA and it is oriented towards the transitional ER. Cisface consists of the Cis-Golgi Network (CGN), the Golgi compartment that is nearest to the transitional ER. A transport vesicle travels from the ER, adds its membrane and contents to cis-face of GA by fusing with the CGN. Trans-face consists of TransGolgi-Network (TGN), the network of membrane-bound tubules that gives rise to coated transport vesicles that carrying processed proteins from GA to other destinations. Between the CGN and TGN is the mediate cisternae, the central sacs that where the products of ER are modified. Glycosylation: Glycosylation is the processes of production of glycoproteins in ER and GA by additions of carbohydrate side chains to specific amino acid residues in proteins. One most known example of glycosylation in the cells is the N-glycosylation, the addition of specific oligosaccharide side chains to N-terminus of certain asparagine residues. In ER, N-glycosylation occurs as two stages. The first stage is the core glycosylation. In ER, core oligosaccharide is built up by the sequential addition of monosaccharide units to an activated lipid carrier- the dolichol phosphate. The complete core oligosaccharide is then transferred from the dolichol phosphate to an Asn residue of N-terminus. For the second stage, the core oligosaccharide side chain that is already attaches on a protein is trimmed and modified specifically. In these modifications, three glucose units and one manose unit are removed by enzymes. Besides, the proteins in which the oligosaccharide side chains attach are also properly folded before the glycoproteins are free to travel to GA. In GA, the glycoproteins move from the CGN to TGN through the mediate cisternae where the terminal glycosylation takes place, giving rise to great diversity of glycoproteins. The terminal glycosylation involves the removal of few carbohydrate units from the modified core oligosaccharide side chains, followed by the further addition of carbohydrate units. Soluble lysosomal enzymes that are travelling from the ER to GA also undergo Nglycosylation. In GA, the manose residues on the carbohydrate side chain of the enzymes are phosphorylated and tagged. The tagged oligosaccharides bind with manose-6-phosphate receptor on the interior surface of TGN, forming a receptorligand complex. The complex is then packed into clathrin coated transport vesicles which will then conveyed into endosomes.

Lysosomes: Lysosomes are the double-membrane digestive compartments in animal cells. It contains diverse of hydrolases that can hydrolyze major macromolecules in acidic environment (pH 4 – 5). Examples of hydrolytic enzymes that contain in lysosomes are phosphatase, protease, peptidase, nuclease, lipase, etc. The double membrane protects the rest of the cells from the enzymes and acidic environment in lysosomal lumen. Even if the lysosomes break open, the neutral environment in cytosol makes the enzymes inactive. However, excessive leakage of lysosomal enzymes can leads to the autodigestion of the cells. The contents and structures of lysosomes and endosomes are similar but endosomes do not involve in digestive activity. Actually the lysosomes are derived from the late endosomes. The final stage in development of lysosomes is the activation of enzymes in late endosomes. The activation of lysosomal enzymes is by influx of hydrogen ions into the late endosomes. This is done by the proton pumps. The influx of hydrogen ions lowering the pH of lysosomal lumen.

Mature lysosomes

Autophagic lysosomes

Heterophagic lysosomes

Autophagic lysosomes are the lysosomes that function in autophagy. Autophagy is the process of recycling of the cell’s own organic materials. The materials can be macromolecules or a damaged organelle. The damaged organelle or the small amounts of macromolecules are surrounded by a layer of membrane, forming a vesicle. The vesicle is then fuses with the lysosome. The organic monomers can be returned to the cytosol for reused.

There are two types of autophagy:

Autophagy

Macrophagy: Autophagy of organelles, the organelle is surrounded by membrane, forming the autophagy vacuole that will fuses with the lysosomes.

Microphagy: Small amount of organic molecules are surrounded by membrane into a smaller vacuole that will fuses with the lysosomes.

Vesicle

Fusion

Lysosome