Competency | Learning Objectives |
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BI 1.1: Describe the molecular and functional organization of a cell and its subcellular components. |
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OVERVIEW
Biochemistry is the study of life on molecular level. Life is based on morphological units known as cell. Cells are often called the “building blocks of life”. Cells are the structural and functional units of all living organisms. Organisms can be classified as unicellular (consisting of a single cell such as bacteria) or multicellular (including plants and animals). Most unicellular organisms are classed as microorganisms.
In humans each cell type has unique structure; human cell types have certain architectural features in common, such as the plasma membrane, membrane around the nucleus and organelles, and a cytoskeleton. In this chapter, we review structure and function of organelles.
CELL TYPES AND SUBCELLULAR COMPONENTS
Cells are of two types: eukaryotic, which contain a nucleus, and prokaryotic, which do not. Prokaryotes are single-celled organisms, while eukaryotes can be either single-celled or multicellular.
Prokaryotic Cells (Greek: pro: before, karyon: nucleus)
Prokaryotes include bacteria and archaea. Prokaryotic cells are simpler and smaller than eukaryotic cells, and lack a nucleus, and other membrane-bound organelles. The DNA of a prokaryotic cell consists of a single circular chromosome. The nuclear region in the cytoplasm is called the nucleoid.
Archaea and Bacteria can be distinguished on genetic and biochemical grounds.
- Archaea lives in extreme environments: salt lakes hot springs, highly acidic bogs, and the ocean depths. Archaea may be the most primitive of the groups.
- Bacteria live in soils, surface waters, and the tissues of other living or decaying organisms.
Components of Prokaryotic Cell
Cell envelope: It generally consisting of a plasma membrane covered by a cell wall which, for some bacteria, may be further covered by a third layer called a capsule. The envelope gives rigidity to the cell and separates the interior of the cell from its environment, serving as a protective filter. Some eukaryotic cells (plant cells and fungal cells) also have a cell wall.
- Cytoplasm: It contains the genome (DNA) and ribosomes. The DNA is condensed in a nucleoid. The nucleoid is not separated from the cytoplasm by a membrane. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular. Plasmids encode additional genes, such as antibiotic resistance genes.
- Flagella and pili: Flagella and pili project from the cell's surface. These are structures (not present in all prokaryotes) made of proteins that facilitate movement and communication between cells (Figure 1.1).2
Eukaryotic Cells (eu: good, karyon: nucleus)
Eukaryote include single cell organism such as yeast, fungi and multicellular plants and animals. Their cell volume is 1,000 to 10,000 times larger than most prokaryotic cells. The main distinguishing feature of eukaryotes as compared to prokaryotes is:
- Eukaryotes have a defined nucleus with a well-defined membrane that contains the bulk of the cell's DNA.
- They also have intracellular organelles surrounded by membrane. These intracellular membrane systems establish distinct cellular compartments. By compartmentalization, different chemical reactions that require different environments can occur simultaneously.
There are other differences in chemical composition and biochemical activities between eukaryotes and prokaryotes. As an example:
- Prokaryotes do not contain histones, a highly conserved class of protein in all eukaryotes that complex with DNA.
- There are also differences in enzyme content and in ribosomes, involved in biosynthesis of proteins.
Table 1.1 and Figure 1.1 describe some of the major structural features of the prokaryote and eukaryote cells
Components of Eukaryotic Cell
All eukaryotic cells possess characteristic structure and organelles. A cell has three major components.
- Cytoplasm with its organelles.
- Endoplasmic reticulum
- Golgi apparatus
- Mitochondria
- Lysosomes
- Peroxisomes
- Nucleus
Table 1.2 shows major biochemical functions of subcellular organelles of the eukaryotic cell.
STRUCTURE AND FUNCTIONS OF CELL MEMBRANE
The cell membrane also called the plasma membrane, which envelops the cell. In animals, the plasma membrane is the outer boundary of the cell, while in plants and prokaryotes it is usually covered by a cell wall. This membrane serves to separate and protect a cell from its surrounding environment.
Structure of Cell Membrane
Most membranes composed primarily of lipids and proteins. The relative proportions of protein and lipid vary with the type of membrane, reflecting the diversity of biological roles. Plasma membrane consists of a double layer of phospholipids. Hence, the layer is called a lipid bilayer (Figure 1.2). The hydrophobic portions of the phospholipid molecules, are repelled by water but are mutually attracted to one another, and have natural tendency to attach to one another in the middle of the membrane, as shown in Figure 1.2.
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The cell membrane is sometimes referred to as a fluid mosaic membrane (Figure 1.3) because it consists of a variety (mosaic) of proteins and lipids. The lipids present in the membrane are in fluid form that allows the flexibility of the membrane without disturbing the structural integrity. The fluidity of the membrane is mainly dependent on the lipid composition of the membrane. The membrane proteins are loosely attached and float in fluid phospholipid bilayer. Most of the interactions among its components are noncovalent, leaving individual lipid and protein molecules free to move laterally in the plane of the membrane. The approximate composition of cell membrane is:
- Protein: 55%
- Phospholipids: 25%
- Cholesterol: 13%
- Other lipids: 4%
- Carbohydrate: 3%
The different types of membrane include cell membrane, nuclear membrane, membrane of the endoplasmic reticulum, and membrane of the mitochondria, lysosomes and Golgi apparatus.
Membrane Lipids
Membrane Phospholipids
The basic lipid bilayer is composed of phospholipid molecules which are amphipathic (partly hydrophobic 4and partly hydrophilic). One end of each phospholipid molecule (head group) is soluble in water; (hydrophilic). The other end (tail group) is soluble only in fats; (hydrophobic). The phosphate end of the phospholipid is hydrophilic and the free fatty acid portion is hydrophobic (Figures 1.4A and B).
Figures 1.4A and B: Structure of phospholipid: (A) Common glycerophospholipid; (B) Diagrammatic representation of phospholipid.
The phospholipid molecules spontaneously organize themselves in a bilayer (Figure 1.2); with the hydrophobic tails facing the interior of the bilayer forming a hydrophobic region held together by intermolecular forces between the tails. The hydrophilic heads form a hydrophilic region on either side of the bilayer that can interact with both the water-based cytoplasm and the exterior of the cell. The principle phospholipids in the membrane are:
- Glycerophospholipids: Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine
- Sphinogophospholipid: Sphingomyelin.
The lipid composition varies among different cell types, with phosphatidylcholine being the major plasma membrane phospholipid in most cell types. Each cell type and the organelles of each cell type have a characteristic set of membrane lipids. Plasma membrane for example, is enriched in cholesterol and contains no detectable cardiolipin; mitochondrial membrane is very low in cholesterol and sphingolipids but that contain cardiolipin.
Membrane Cholesterol
The cholesterol molecules in the membrane are also lipid in nature. Cholesterol, which is incorporated between the phospholipids, maintains membrane fluidity. The fluidity of a membrane depends on composition of lipids and the degree of unsaturation. The major determinant is its cholesterol-phospholipid ratio. In eukaryotes, the ratio is about 1:1. Higher cholesterol content reduces the fluidity of the membrane.
The changes in membrane fluidity may affect proteins that span the membrane (integral proteins), such as ion channels and receptors for neurotransmitters involved conducting the nerve impulse.
Functions of the Lipid Bilayer
- Cell membrane produces a permeability barrier between the interstitial fluid and the cytoplasm.
- The permeability of substance depends on whether it is lipid-soluble or water-soluble. Lipid-soluble substances such as oxygen, carbon dioxide, and alcohol can pass easily through the cell membrane, whereas water-soluble substances, such as ions, glucose and urea cannot pass easily.
Membrane Proteins
The protein composition of membrane from different sources varies even more widely than their lipid composition, reflecting functional specialization. Two types of membrane proteins differ in their association with the membrane. Most of the membrane proteins are glycoproteins.
- Peripheral membrane proteins: Peripheral proteins that are attached only to one surface of the membrane and do not penetrate all the way through. Peripheral protein molecules are often attached to the integral proteins. They are associated with the membrane through electrostatic interactions and hydrogen bonding with the hydrophilic domains of integral proteins and with polar head groups of membrane lipids.
Functions of Membrane Proteins
- Integral membrane proteins function primarily as channels (pores) through which water molecules and water-soluble substances, especially ions, can diffuse between extracellular and intracellular fluids. These protein channels have also selective properties that allow preferential diffusion of some substance over others.
- Other integral proteins act as carrier proteins transporting substances that otherwise could not penetrate the lipid bilayer.
- These proteins even transport substances in the direction opposite to their electrochemical gradients for diffusion, which is called “active transport”.
- They can also serve as receptors for hormones and neurotransmitter.
- Integral proteins spanning the cell membrane provide a means of conveying information about the environment to the cell interior.
- Peripheral proteins function almost entirely as enzymes or as controllers of transport of substances through the cell membrane “pores”.
- One of the main roles of peripheral proteins is to direct and maintain both the intracellular cytoskeleton and components of the extracellular matrix.
Membrane Carbohydrates
Membrane carbohydrates occur in combination with proteins or lipids in the form of glycoproteins or glycolipids. Some of the proteins and lipids on the external surface of the membrane contain short chains of carbohydrate (oligosaccharides) that extent into the aqueous medium. As well as many other carbohydrate compounds called proteoglycans are loosely attached to the outer surface of the cell. Thus the entire outside surface of the cell often has a loose carbohydrate coat called the glycocalyx. Carbohydrate constitutes 2–10% of the weight of cell membrane.
Functions of Membrane Carbohydrates
- Many of the carbohydrates have a negative electrical charge, which gives most cells an overall negative surface charge that repels other negative objects and restricts the uptake of hydrophobic compounds.
- The glycocalyx of some cell attaches to the glycocalyx of other cells, thus attaching cells to one another.
- Many of the carbohydrate act as hormone receptor such as insulin
- Some carbohydrate moieties involved into immune reactions.
Functions of Plasma Membrane
- The plasma membrane maintains the physical integrity of the cell by preventing the contents of the cell from leaking into the outside fluid environment and at the same time facilitating the entry of nutrients, inorganic ions and most other charged or polar compounds from the outside.
- The functions of the plasma membrane are coordinated by specialized adhesion receptors called integrins. Integrins are integral transmembrane proteins. Integrins represent important cell receptors that regulate fundamental cellular process; such as attachment, movement, growth and differentiation.
STRUCTURE AND FUNCTIONS OF SUBCELLULAR ORGANELLES
Cytoplasm is the internal volume bounded by the plasma membrane. The clear fluid portion of the cytoplasm in which 6the organelles are suspended is called cytosol. This contains mainly dissolved proteins, electrolytes and glucose. Five important organelles that are suspended in the cytosol are:
- Endoplasmic reticulum
- Golgi apparatus
- Mitochondria
- Lysosomes
- Peroxisomes
Endoplasmic Reticulum (ER)
The endoplasmic reticulum is the interconnected, folded network of tubular structures in the cytoplasm. A portion of the endoplasmic reticulum has ribosomes bound to it, which give it a rough appearance in contrast with smooth endoplasmic reticulum which is devoid of ribosomes (Figures 1.5A and B). Endoplasmic reticulum and Golgi apparatus are involved in formation of other cellular organelles such as lysosomes and peroxisomes.
Functions of Rough Endoplasmic Reticulum
- The rough endoplasmic reticulum is the site for synthesis of proteins that are destined to be exported from the cell. Virtually all integral membrane proteins of the cell, except those located in the membranes of mitochondria are formed by ribosomes bound to the endoplasmic reticulum.
- The endoplasmic reticulum also has mechanisms for maintaining the quality of the proteins synthesized. The endoplasmic reticulum has three different sensor molecules that monitor the amounts of improperly folded proteins that accumulate.
Functions of Smooth Endoplasmic Reticulum
- Smooth endoplasmic reticulum is involved in lipid synthesis and contains enzymes termed cytochromes P450 that catalyze hydroxylation of a variety of endogenous and exogenous compounds.
- These enzymes are important in biosynthesis of steroid hormones and removal of toxic substances
Figures 1.5A and B: Structure of endoplasmic reticulum. (A) Rough or granular endoplasmic reticulum; (B) Smooth or a granular endoplasmic reticulum.
Golgi Apparatus
The Golgi (named for its discoverer Camillo Golgi) apparatus is a flat, membranous sac. The Golgi apparatus is also referred to as Golgi complex. In Golgi apparatus proteins are processed, modified and prepared for export from the cell. It works in association with endoplasmic reticulum, where proteins for certain destinations are synthesized (Figure 1.6).
As shown in Figure 1.6, small transport vesicles (ER vesicles) continually pinch of from the ER and shortly thereafter fuse with the Golgi apparatus. In this way, substances entrapped in the ER vesicles are transported from the ER to Golgi apparatus. The transported substances are then processed in the Golgi apparatus to form lysosomes, peroxisomes, secretory vesicles and other cytoplasmic components.
Functions of Golgi Apparatus
The Golgi apparatus participates in post-translational modification of proteins: such as complex branched chain oligosaccharide addition, sulfation and phosphorylation.
7Proteins which are synthesized in the endoplasmic reticulum passed through layers of the Golgi apparatus where enzymes in Golgi membranes catalyze transfer of carbohydrate units to proteins to form glycoproteins or to lipids to make glycolipids, a process that is important in determining the proteins eventual destination. The modified proteins are then sorted, packaged and transported to destination inside or outside the cell. Golgi apparatus plays the role of post office mail sorting room, the mail in this case being newly synthesized proteins.
Mitochondrion (Power House of Cell)
Mitochondria are organelles in eukaryotic cells that supply energy for all cellular metabolic activities. The number of mitochondria in cells varies as do their energy needs. Muscle cells of the heart contain the largest number of mitochondria. Mitochondria are called Power plant of the cell, since they generate most of the cell's energy in the form of ATP. Erythrocytes are an exception which derive their ATP from glycolysis due to lack of mitochondria. Each mitochondrion is bounded by two membranes.
- The relatively porous smooth outer membrane is permeable to most molecules.
- The inner membrane, which is impermeable to ions and a variety of organic molecules. The inner membrane projects inwards into folds that are called cristae (Figure 1.7).
- Together, both membranes create two separate compartments: the intermembrane space (between the outer and the inner membranes) and the matrix which is bounded by the inner membrane. The matrix side and the cytoplasmic side also called the N and P sides respectively, because the membrane potential is negative on the matrix side and positive on the cytoplasmic side.
- The outer and inner membranes both contain mechanism for translocation of specific proteins. There is variety of transmembrane system in the inner membrane for translocation of various metabolites.
- The outer membrane is permeable to most small molecules and ions because it contains many mitochondrial porin (pore forming protein) also known as voltage-dependent anion channel (VDAC) that permit access to most molecules. In contrast inner membrane is impermeable to nearly all ions and polar molecules. Many transporters shuttles metabolites such as ATP, pyruvate, and citrate across the inner mitochondrial membrane.
Functions of Mitochondria
- The intermembrane space contains several enzymes involved in nucleotide metabolism.
- Whereas, the gel-like matrix (mitosol) consists of high concentration of enzymes required for the metabolic pathways of oxidation of pyruvate produced by glycolysis, fatty acids, and amino acids and some reactions in biosynthesis of urea and heme. The mitochondrial matrix is the site of most of the reactions of the citric acid cycle and fatty acid oxidation.
- Oxidative phosphorylation takes place in the inner mitochondrial membrane. Components of electron transport system and oxidative phosphorylation that are responsible for the synthesis of ATP are embedded in inner membrane. Also present are a series of proteins that are responsible for the transport of specific molecules and ions.
- Mitochondria also have a requisite machinery to catalyze protein synthesis. Mitochondria contain their own DNA, (mtDNA), which in human encodes 13 respiratory chain proteins, as well as small and large ribosomal RNAs and enough tRNAs to translate all codons
- In recent years, mitochondria have also been recognized as key regulators of apoptosis. Mitochondria have a key role in aging; cytochrome c, a component of the mitochondrial electron transport chain, is an initiator of apoptosis.
Genetic Diseases of Mitochondria
There are several hundred genetic diseases of mitochondrial function. Mutations in mtDNA are responsible for a number of diseases called mitochondriopathies that can be inherited. Mutations in mitochondrial DNA are transmitted from an affected mother to all her children but not from an affected father.
Many mitochondrial diseases involve skeletal muscle and central nervous systems. In some patients, exercise intolerance and muscle fatigue are due to mutations in mtDNA. Mitochondrial DNA damage may occur due to free radicals (superoxides) formed in the mitochondria.8
- The first disease to be identified as due to a mutation of mitochondrial DNA was Leber's Hereditary Optic Neuropathy, (LHON) which leads to sudden blindness in early adulthood caused by degeneration of the optic nerve. Mutation in patients with this disease is a single base substitution that replaces an arginine residue in one of the subunits of NADH-Q reductase with histidine. Mutation impairs electron flow through the respiratory chain and reduces ATP synthesis. They lead to blindness because the optic nerve has a high energy demand and depends almost entirely on oxidative phosphorylation for its ATP supply.
- It has been suggested that a single mutation for a mitochondrial tRNA leads to hypertension, high blood cholesterol and decreased level of plasma Mg2+.
- Mutation in mitochondrial rRNA results in antibiotic (such as streptomycin, paromomycin, and gentamycin) induced deafness.
- Organs that are highly dependent on oxidative phosphorylation, such as the nervous system and the heart, are most vulnerable to mutations in mtDNA.
Lysosomes
Lysosomes are organelles formed from Golgi apparatus and dispersed throughout the cytoplasm. The lysosomes are membrane bounded sacs containing hydrolytic enzymes. Lysosomes contain as many as forty different hydrolytic enzymes. The hydrolytic enzymes found in lysosomes include proteases, nucleases, glycosidases, lipases, phosphatases and sulfatases. All these enzymes function at acidic pH, so pH of lysosome matrix is maintain at about 5.
Among all organelles of the cytoplasm, the lysosomes have the thickest covering membrane to prevent the enclosed hydrolytic enzymes from coming in contact with other substances in the cell and therefore prevent their digestive actions. Disruption of the lysosomal membrane within cells leads to cellular digestion. Various pathological conditions such as arthritis, allergic responses, several muscular diseases, and drug-induced tissue destruction have been attributed to release of lysosomal enzymes.
Functions of Lysosomes
- Lysosomes are involved in digestion of intra- and extra-cellular substances that must be removed. Substances destined to be degraded are identified and taken up by lysosomes through endocytosis. Products of lysosomal digestion are released from lysosomes and are reutilized by the cell. Indigestible material called residual bodies are removed from the cell by exocytosis.
- During development, lysosomes play an important role in the formation of specialized tissues such as fingers and toes. For example, lysosomes digest the webbed tissues that join fingers and toes in the embryo.
Lysosomal Storage Disease
Genetic defects in lysosomal enzymes, or in proteins such as the mannose-6-phosphate receptors required for targeting the enzyme to the lysosome, lead to an abnormal accumulation of undigested material that may be converted to residual bodies particularly in neuronal cells. Genetic diseases such as the Tay-Sachs disease (an accumulation of partially digested gangliosides in lysosomes), and Pompe's disease (an accumulation of glycogen particles in lysosomes) are caused by the absence or deficiency of specific lysosomal enzymes. Such diseases, in which a lysosomal function is compromised, are known as lysosomal storage disease.
The lysosomal enzymes are synthesized at the rough ER and become glycosylated in the ER and Golgi apparatus. In the Golgi apparatus they finally acquire a mannose-6-phosphate residue on some of their oligosaccharides. Mannose-6-phosphate is a molecular tag that acts like a postal address to route the enzymes to the lysosomes. A partial list of lysosomal enzymes is given in Table 1.3. The enzyme content of lysosomes varies in different tissues and depends on specific tissue functions.
Peroxisomes
Peroxisomes (organelles having ability to produce or utilize hydrogen peroxide) are similar to lysosome in that they are membranous sacs containing enzymes. The enzyme content of cellular peroxisome varies according to the need of the tissue. Liver peroxisomes contain three important detoxification enzymes; catalase, peroxidase and D-amino acid oxidase.
Functions of Peroxisomes
- Peroxisomes contain enzymes that are used for detoxification rather than for hydrolysis.
- In peroxisomes, a number of molecules which are not metabolized elsewhere are oxidized by enzymes by using molecular oxygen directly and produce hydrogen peroxide (H2O2). Hydrogen peroxide is destroyed further by catalase and peroxidases. By having both peroxide producing and peroxide utilizing enzymes in one compartment, cells protect themselves from the toxicity of hydrogen peroxide
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Peroxisomal Diseases
Several diseases are associated with peroxisomes. Peroxisomal diseases are caused by mutations that affect either the synthesis of functional peroxisomal enzymes or their incorporation into peroxisomes. Defects in peroxisome lead to disorders such as:
Adrenoleukodystrophy: Mutation in the ATP-binding cassette (ABC) transport membrane protein of a fatty acid in the peroxisomal membrane
Zellweger's syndrome: Defective formation of peroxisomes, characterized by accumulation of long chain, saturated, unbranched fatty acids in liver and CNS, server neurological symptoms and early death.
Refsum's disease: Deficiency of phytanoyl-CoA hydroxylase required for α-oxidation of branched chain fatty acid; characterized by peripheral neuropathy, ataxia and retinitis pigmentosa.
Nucleus
Nucleus is the control center of the cell; it contains the DNA organized into chromosomes which carry genetic information. The nucleus is surrounded by a double membrane called nuclear envelope. The outer membrane is fused with the endoplasmic reticulum at multiple sites. Nuclear pores (multiprotein complexes) occur at points where the outer and inner membranes are connected (Figure 1.8). Nuclear pores permits controlled movement of particles and large molecules between the nuclear matrix and the cytoplasm.
It is now considered that the nuclear envelope plays important roles other than just as a barrier between the nuclear matrix and the cytoplasm. The space enclosed by the nuclear envelope is called nucleoplasm; within this the nucleolus is present. Nucleolus is an organized structure of DNA, RNA and protein. Nucleolus is a major site of RNA synthesis and the site of assembly of ribosome.
The remaining nuclear DNA is dispersed throughout the nucleoplasm in the form of chromatin fibers). Chromatins are complexes of DNA with specific proteins such as histones. In the nucleus, these chromatin fibers are associated with nuclear lamina, a fibrous network made of three proteins, A, B, and C; lying beneath the inner nuclear membranes. At mitosis chromatin is condensed into discrete structures called chromosomes. The organization of the nuclear envelope, nucleolus, and chromatin is shown in Figure 1.8.
Functions of Nucleus
- DNA, the repository of genetic information is located in the nucleus as a DNA-protein complex, chromatin, which is organized into chromosomes. The nucleus contains the proteins and enzymes of replication of DNA and for repair of DNA that has been damaged.
- The processing of RNA for assembly of ribosomes, required for protein synthesis in the cytosol, occurs in the nucleolus.
CYTOSKELETON
The cytoplasm of most eukaryotic cells contains network of several types of proteins filaments that interact extensively with each other and with the component of the plasma membrane forming three-dimensional meshwork. Such an extensive intracellular meshwork of protein has been called cytoskeleton. Cytoskeleton is not a rigid permanent framework of the cell but is a dynamic, changing structure.
Functions of Cytoskeleton
- The cytoskeleton gives cells their characteristic shape and form, provides attachment points for organelles, fixing their location in cells and also makes communication between parts of the cell possible.
- It is also responsible for the separation of chromosomes during cell division.
- The internal movement of the cell organelles as well as cell locomotion and muscle fiber contraction could not take place without the cytoskeleton. It acts as track on which cells can move organelles, chromosomes and other things.
Structure of Cytoskeleton
The cytoskeleton is an organized network of three protein filaments; Microfilaments, microtubules and intermediate filaments, differing in width, composition and specific function.
- Microfilaments consist of long thin strands of protein actin, which is also a main component of muscle. Actin filament form a meshwork just underlying the plasma membrane of many cells and are referred to stress fiber or cell cortex which is labile. They disappear as cell motility increases or upon malignant transformation of cells by chemical or oncogenic viruses.
- Microtubules are long, thin tubes composed of the protein tubulin. They rapidly assemble into tubular structures and disassemble depending on the needs of cells. Microtubules comprise the spindle fibers that separate chromosomes prior to cell division. Centrioles are composed of microtubules and function as the organizing center for the formation of spindle fibers.
- Intermediate filaments are so-called as their diameter is intermediate between that of microfilaments and of microtubules. These are formed from fibrous protein which cannot be easily disassembled as either the microtubules or the microfilaments can, except lamin. Protein structure of intermediate filaments varies with different tissue type. There are major seven classes of intermediate filaments as indicated in Table 1.4.
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CELL FRACTIONATION AND MARKER ENZYMES FOR DIFFERENT ORGANELLES
Investigation of the biochemical properties of organelles requires subcellular fractionation in which the cell is first mechanically homogenized using isotonic 0.25 M sucrose solution to break cells and disperse their contents in an aqueous buffer to maintain the pH at its optimum value for organelle stability. Sucrose solution is used because it is not metabolized in most tissues and it has an osmotic pressure similar to that in organelles, thus balancing diffusion of water 11into and out of the organelles, which would swell and burst in a solution of lower osmolarity.
- By gently homogenization in an isotonic sucrose solution, the cell membrane is ruptured keeping most of the internal organelles intact. However, large fragile structures such as the endoplasmic reticulum, is broken into pieces that spontaneously form vesicles called microsomes.
- Then homogenate is centrifuged at different speeds. Large particles sediment more rapidly than small particles and soluble material does not sediment. In differential centrifugation, the homogenate is subjected to a series of centrifugation steps of increasing time and gravitational force (Figure 1.9).
- The subcellular organelles, e.g., nuclei and mitochondria, which differ in size and specific gravity and thus sediment at different rates in a centrifugal field and can then, be isolated from homogenate by differential centrifugation. The dense nuclei are sediment first, followed by the mitochondria, and finally the microsomal fraction at the highest forces. After, all the particulate matter has been removed; the soluble remnant is the cytosol.
- Organelles of similar sedimentation coefficient obviously cannot be separated by differential centrifugation. For example, mitochondria isolated in his way are contaminated with lysosome and peroxisomes. These may be separated by isopycnic centrifugation technique.
Isopycnic Centrifugation Technique
In this technique, a density gradient is set up in a centrifuge tube; i.e., the density of the solution in the tube increases from the top to the bottom. Sucrose is often used as a medium. Colloidal materials such as Percoll, which form density gradients with a low osmotic pressure, are often preferred.
Particles are sediment to an equilibrium position at which their density equals that of the medium at that point in the tube (Figure 1.9). Different organelles are thus separated according to their density, their size and shape being immaterial.
After centrifugation to equilibrium, the gradient is fractionated and the separated organelles recovered as shown in Figure 1.10. Macromolecules, such as large proteins, nucleic acids and nucleoprotein complexes can also be separated by density gradient centrifugation technique.
Marker Enzymes for Different Organelles
The purity of isolated subcellular fraction is assessed by the analysis of marker enzymes. Marker enzymes are the enzymes that are located exclusively in a particular fraction, and thus become characteristic of that fraction.
Analysis of marker enzymes confirms the identity of the isolated fraction and indicates the degree of contamination with other organelles. For example, isolated mitochondria have a high specific activity of cytochrome oxidase but low catalase and acid phosphatase, the catalase and acid phosphatase activities being due to contamination with peroxisomes and lysosomes respectively. Some typical subcellular markers are given in Table 1.5.
MEMBRANE TRANSPORT
Fundamental Properties of Biological Membranes
Cell membranes are highly fluid, dynamic structures consisting of lipid bilayer and associated proteins. Cell membranes form closed compartments around the cytoplasm to define cell boundaries. The cell membrane has selective permeability. The lipid bilayer of biological membranes is basically impermeable to ions and polar molecules.12
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The lipid bilayer is not miscible with either the extracellular fluid (ECP) or the intracellular fluid (ICF). Therefore, it constitutes a barrier against movement of water molecules and water-soluble substances between extracellular and intracellular fluid compartments, thereby maintaining differences in composition between inside and outside of the cell (Table 1.6).
The membrane is sometimes referred to as a fluid mosaic (see Figure 1.3). Since, it consists of a mosaic (variety) proteins and lipid molecules that can move laterally in the plane of the membrane. The membrane mosaic is fluid because most of the interactions among its components are noncovalent, leaving individual lipid and protein molecules free to move laterally in the plane of the membrane.
- Selective membrane permeability is conferred by specific transporters and ion channels. Most of the membrane proteins can function as transport proteins. These proteins are highly selective for the types of molecules or ions that are allowed to cross the membrane. Different proteins function differently:
- Channel proteins: Some proteins have watery spaces all the way through the molecule and allow free movements of water as well as selected ions or molecules; these are called channel proteins.
- Carrier proteins: Others called carrier proteins bind with molecules or ions that are to be transported.
- The cells also transport certain macromolecules such as proteins, polysaccharides, and polynucleotides across the plasma membrane by independent mechanisms namely endocytosis and exocytosis.
- There are special areas of membrane structure—gap junction, through which adjacent cells may exchange material.
TRANSPORT MECHANISMS ACROSS CELL MEMBRANE
Transport mechanism through cell membrane can be broadly divided into three types (Figure 1.11):
- Passive transport
- Active transport
- Vesicular transport
Passive Transport
- In passive transports, the substances pass through the membrane from both sides. The direction of transport of molecule is always from a region of higher concentration to lower concentration. It does not require energy in the form of ATP.
- There are three types of passive transport as follows:
- Simple diffusion
- Facilitated diffusion
- Osmosis
Simple Diffusion
Diffusion is a process of passive transport in which molecules move from the area of higher concentration to the area of lower concentration. The energy that causes diffusion is derived by the kinetic energy generated due to random motion of molecules.
- The examples of substances that pass through cell membranes by simple diffusion are transport of O2, CO2, urea, ammonia and ions.
- Across a membrane, diffusion of a molecule exists on both sides of the membrane. The net movement of molecule ceases when the concentration of molecule on both sides becomes equal and a diffusional equilibrium is achieved. Simple diffusion can occur through the cell membrane by two pathways (Figure 1.12):13
- Through the interstices of lipid bilayer if the diffusing substance is lipid soluble and
- Through watery (aqueous) channels formed by transmembrane proteins.
Simple Diffusion of Lipid-soluble Molecules through the Lipid Bilayer
Simple diffusion of lipid-soluble molecules occurs rapidly through the interstices of the lipid bilayer (Figure 1.12). For example, oxygen, nitrogen, carbon dioxide and alcohols are lipid soluble, so all these can dissolve directly in lipid bilayer and diffuse through the cell membrane. The rate of diffusion of each of these substances through the membrane is directly proportional to its lipid solubility.
Simple Diffusion of Water and Other Lipid Insoluble Molecules through Protein Channels
Even though water is highly insoluble in the membrane lipids, it readily passes through protein channels that penetrate all the way through the membrane (Figure 1.12). Other lipid insoluble molecules can pass through the protein pore in the same way as water molecules if they are water-soluble and small enough like ions, glucose and urea.
- Protein pores and channels are tubular pathways all the way from the extracellular to the intracellular fluid. Therefore substances can move by simple diffusion directly along these pores and channels from one side of the membrane to the other.
- Pores are composed of integral cell membrane proteins that form open tubes through the membrane and are always open. However, the diameter of a pore and its electrical charges provide selectivity that permits only certain molecules to pass through. These protein pores are called aquaporins or water channels. They permit rapid passage of water through cell membranes but exclude other molecules. The pore is too narrow to permit passage of any hydrated ions. At least 13 different types of aquaporins have been found in various cells of the human body.
- Protein channels are highly selective for transport of one or more specific ions or molecules; as they have characteristic diameter, shape, and the nature of the electrical charges and chemical bonds along its inside surfaces. A polypeptide subunit forms a gate at one end of the channel that opens in response to a specific stimulus.Gating of protein channels provides a means of controlling ion permeability of the channels. The opening and closing of gates are controlled by the electrical potential across the cell membrane, e.g., Na+ and Ca+ channels and by the binding of a chemical substance ligand) either an ion or a specific molecule with the protein; this causes a conformational change 14in the protein molecule that opens or closes the gate, e.g., acetylcholine channel. Acetylcholine opens the gate of this channel.
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Facilitated Diffusion
This is also called carrier-mediated diffusion, as the process of diffusion is facilitated by a carrier protein in the membrane. There are many types of carrier proteins in membranes, each type having binding sites that are specific for a particular substance. Among the most important substances that cross cell membranes by facilitated diffusion are glucose and most of the amino acids. In case of glucose there are 12 glucose transporter molecules have been discovered in various tissues (Table 1.7). Like simple diffusion, facilitated diffusion is also a downhill transport and does not require energy (Figure 1.12).
- Facilitated diffusion is more rapid than simple diffusion. These diffusion processes are not coupled to the movement of other ions, they are known as uniport transport process.
- Sometimes, facilitated diffusion is regulated by hormones. For example, transport of glucose by GLUT-4 into muscle and adipose tissue is insulin dependent.
- In facilitated diffusion, the number of carrier proteins available determines the rate of diffusion. In simple diffusion, the rate of diffusion is proportional to the concentration of the substance.
Osmosis
Osmosis is the process of movement of water (solvent) from the solution (Solute + Solvent) with the lower concentration of solutes to the solution with higher concentration of solute, when both the solution are separated by a semipermeable (permeable to solvent but not the solute) membrane.
Active Transport
By passive transport processes, the composition of intracellular fluid tends to equalize with that of composition of extracellular fluid. However, this should never happen practically, as it threatens cell volume and intracellular solute concentrations that are not compatible with life. Therefore, nature maintains inequality of fluid composition of intracellular and extracellular compartments by providing special transport mechanisms to the cell membrane that oppose these equilibrating transport processes. These transport processes are called active transport mechanisms (Figure 1.13).
When a cell membrane transports molecules or ions uphill (towards high concentration) against a concentration gradient or uphill against an electrical or pressure gradient, the process is called active transport. There are two common characteristics of active transport mechanisms
- Uphill transport: The transport occurs against the electrochemical gradient of the substance transported.
There are two types of active transport according to the source of energy used to cause transport.
- Primary active transport
- Secondary active transport
Primary Active Transport
Primary active transport is the transport mechanism that directly utilizes energy derived from hydrolysis of ATP to ADP. The mechanism is operated by ion pumps.
- In this process, the solute is transported against its electrochemical gradients, which requires energy in the form of ATP.
- As the ion pumps hydrolyze ATP, these are also called ATPases.
- The examples of primary active transports are:
- Na+-K+ ATPase or Na+-K+ pump
- Calcium ATPase
- H+-K+ ATPase
- H+- ATPase.
- Active transport depends on carrier proteins. These carrier proteins are capable of transporting substance against the concentration gradient hence energy is required. There are four major classes of active transporters.
- P-type: P signifies phosphorylation. P-type transporters are phosphorylated and dephosphorylated during transport. Na+-K+ ATPase and Ca2+ ATPase are the examples of P-type ATPase.
- F-type transporters: F-signifies energy coupling factor type. The most important example of this class is the mitochondrial ATP synthase present in mitochondria.
- V-type transporter: V signifies vacuolar, V-type transporters pump protons into lysosomes, endosomes, Golgi vesicles and secretory vesicles.
- ABC transporters: ABC transporters transport a variety of compounds out of the cells those includes ions, steroids, cholesterol, peptides, bile acids, drugs and xenobiotics. The most important example of this class is:
- CFTR protein: Cystic fibrosis transmembrane conductance regulator (CFTR) protein is responsible for regulating the proper flow of chloride and sodium in and out of the cell membranes in the lungs and other organs. This protein functions as a channel across the membrane of cells that produce mucus, sweat, saliva, tears, and digestive enzymes. Cystic fibrosis occurs when the cystic fibrosis transmembrane conductance regulator (CFTR) protein is either not made correctly, or not made at all.
- MDR-1 protein (multidrug resistance-1 protein). P-glycoprotein 1 (permeability glycoprotein, Pgp) also known as multidrug resistance protein 1 (MDR1). This transporter pumps a variety of drugs including many anticancer agents out of the cells.
- BRCP (breast cancer resistance protein): BCRP physiologically functions as a part of a self-defense mechanism for the organism; it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract, as well as through the blood-brain, placental, and possibly blood-testis barriers. It was so named because it was initially cloned from a multidrug-resistant breast cancer cell line where it was found to confer resistance to chemotherapeutic agents in cancer cells.
Secondary Active Transport
Many cells have aided by other carrier mechanisms that transfer one solute against its concentration by using energy generated by gradient of other solute that was originally pumped by primary active transport (Figure 1.14). Since the transport depends on primary active transport of sodium by the Na+-K+ pump, it is known as a secondary active transport.
- Typical example of secondary active transport is reabsorption of glucose from intestine and kidney tubules across intestinal and renal epithelial cell.
- When sodium ions are transported out of the cells by primary active transport (Na+-K+ pump) large concentration gradient of sodium ions develops across the cell membranes, (high concentration outside the cell and low concentration inside). This gradient generates energy, as the excess sodium outside the cell membrane is always attempting to diffuse to the interior. This diffusion energy of sodium can pull glucose along with the sodium through cell membrane from luminal fluid into the cell.
Vesicular Transport
- Vesicular transport is special for macromolecules. Macromolecules cannot be transported by diffusion or active transport process. Therefore they are transferred across the cell membrane mainly by vesicular transport. Amino acids, sugars, waste products of metabolism, cellular secretions, hormones, neurotransmitters and organisms are transported by this mechanism.
- Transport process occurs by either fusion of vesicle or formation of vesicle is called vesicular transport.
- The process by which cells take up large molecules is called endocytosis and the process by which cells release large molecules from the cells to the outside is called exocytosis.
- Fusion of vesicle with the cell membrane occurs in exocytosis and formation of vesicle from cell membrane occurs in endocytosis.
- In vesicular transport, formation and transport of vesicles are facilitated by some vesicular transport proteins. These proteins are calthrin, coating proteins, dynamin and docking proteins.
Figure 1.14: Diagrammatic representation of secondary active transport. Gradient of ion has been established by primary active transport movement of solute (S1, often Na+) down its electrochemical gradient provides the energy to drive cotransport of a second solute (S2) against its electrochemical gradient.
Endocytosis
Endocytosis is the process of transport in which a substance is taken into the cell by means of vesicle formation. It is the only process by which most macromolecules, such as most proteins, polysaccharides and polynucleotides can enter cells.
Endocytosis occurs by two mechanisms: Constitutive and Clathrin-mediated.
Constitutive Endocytosis
Endocytosis by constitutive pathway occurs in almost all cells. It is called “constitutive”, as the process occurs continually and does not require any specific stimulus. The molecule or substance makes contact with the cell membrane that invaginates to form an endocytic vesicle. The non-cytoplasmic side of the membrane then fuses and the vesicle is pinched-off into the cytosol (Figure 1.15).
Clathrin-mediated Endocytosis
- Clathrin-mediated endocytosis occurs at the specific site of the cell membrane. Clathrin is fibrillar protein located in the cell membrane beneath the receptor protein. Clathrin-mediated endocytosis internalizes various organisms, growth factors and lipoproteins (Figure 1.16).
- These molecules first attach to specific receptors on the surface of the membrane.
- The receptors are generally concentrated in small pits on the outer surface of the cell membrane. These receptors are coated on the cytoplasmic side with a fibrillar protein called clathrin and contractile filaments of actin and myosin.
- Then immediately, the invaginated portion of the membrane breaks away from the surface of the cell forming endocyte vesicle inside the cytoplasm of the cell.
Digestion of Endocyte Vesicles
- Immediately after a endocytotic vesicle appears inside a cell, one or more lysosomes become attached to the vesicle and empty their acid hydrolases to the inside of the vesicles.
- The macromolecules present in vesicle are digested to yield amino acids, simple sugars or nucleotides that can diffuse through the membrane of the vesicle into the cytoplasm and reused by the cell.
- What is left of the digestive vesicle, called the residual body, represent indigestible substances. In most instances, this is finally excreted through the cell 18membrane by a process called exocytosis, which is opposite of endocytosis (Figure 1.17).
Exocytosis
Exocytosis is the release of macromolecules from cells to the exterior, which is reverse of endocytosis. By exocytosis, hormones, neurotransmitters, digestive enzymes and undigested foreign particles are released from cells.
- The undigestible substances produced within the cytoplasm may be enclosed in membranes to form vesicles called exocytic vesicles.
- These cytoplasmic exocytic vesicles fuse with the internal surface of the plasma membrane.
- The vesicle then ruptures releasing their contents into the extracellular space and their membranes are retrieved (left behind) and reused (Figure 1.17).
ASSESSMENT QUESTIONS
SHORT ESSAY QUESTIONS (SEQs)
- Draw the structure of eukaryotic cell and write functions of the subcellular organelles.
- Give structure and function of any two subcellular organelles.
- With the help of diagram, describe the fluid mosaic model of cell membranes.
- Enumerate transport processes across cell membrane with diagrams.
- Write mechanism and importance of endocytosis, and exocytosis.
SHORT ANSWER QUESTIONS (SAQs)
- Write types and functions of membrane proteins.
- What are marker enzymes? Name the marker enzymes for lysosomes and mitochondria.
- What are the functions of lysosomes?
- What are the functions of peroxisomes?
- What are the functions of membrane carbohydrates?
- Write difference between passive and active transport.
- Infusion of normal saline (0.9% NaCl) is preferred over isotonic 5% glucose solution; justify.
- What are aquaporins? Write importance of aquaporins.
- What are ABC transporters? Write most important example of ABC transporters
MULTIPLE CHOICE QUESTIONS (MCQs)
- The following is the metabolic function of ER:
- RNA processing
- Fatty acid oxidation
- Synthesis of plasma protein
- ATP-synthesis
- In biologic membranes, integral proteins and lipids interact mainly by:
- Covalent bond
- Both hydrophobic and covalent bond
- Hydrogen and electrostatic bond
- None of the above
- Plasma membrane is:
- Composed entirely of lipids
- Mainly made up of proteins
- Mainly made up of lipid and protein
- Composed of only carbohydrates and lipids
- Select the subcellular component involved in the formation of ATP:
- Nucleus
- Plasma membrane
- Mitochondria
- Golgi apparatus
- Mitochondrial DNA is:
- Maternal inherited
- Paternal inherited
- Maternal and paternal inherited
- None of the above
- All of the following statements about the nucleus are true, except:
- Outer nuclear membrane is connected to ER
- It is the site of storage of genetic material
- Nucleolus is surrounded by a bilayer membrane
- Outer and inner membranes of nucleus are connected at nuclear pores
- Golgi apparatus is produced from which organelle?
- Endoplasmic reticulum
- Plasma membrane
- Mitochondria
- Ribosomes
- Peroxisomes arise from:
- Golgi membrane
- Lysosomes
- Mitochondria
- Pre-existing peroxisomes and budding off from the smooth ER
- Na+ - K+ ATPase is the marker enzyme of:
- Nucleus
- Plasma membrane
- Golgi bodies
- Cytosol
- The rough endoplasmic reticulum in the cells is because of the presence of:
- In human which cell lacks nucleus:
- Lymphocyte
- Monocytes
- RBC
- Neutrophils
- Microtubules are made up of by which protein?
- Tubulin
- Myosin
- Actin
- None of these
- No membrane surrounds in this organelle:
- Lysosome
- Nucleolus
- Golgi body
- Nucleus
- The cytoskeleton includes all of the following, except:
- Microtubules
- Intermediate filaments
- Myosin filaments
- Actin filaments
- Ribosomes are found:
- Only in the nucleus
- In the cytoplasm
- Attached to the rough endoplasmic reticulum
- Both b and c
- The Golgi apparatus is involved in:
- Packaging proteins into vesicles
- Altering or modifying proteins
- Producing lysosomes
- All of the above
- Which of the following are involved with the movement or transport of materials or organelles throughout the cell?
- Rough endoplasmic reticulum
- Cytoskeleton
- Smooth endoplasmic reticulum
- All of the choices are true
- Lysosomes are produced by the:
- Nucleus
- Mitochondria
- Golgi apparatus
- Ribosomes
- Major site of RNA synthesis is:
- Nucleoplasm
- Nucleolus
- Nucleus
- All
- Mitochondria is an organelle of which process, except:
- Glycolysis
- Krebs' cycle
- Biosynthesis of urea
- Fatty acid oxidation
- Give name of organelle, which is surrounded by a double layered wall.
- Lysosome
- Plasma membrane
- Golgi apparatus
- Nucleus
- Assertion: A cell membrane shows fluid behavior.Reason: A membrane is a mosaic or composite of diverse lipids and proteins.
- Both Assertion and Reason are true and the Reason is the correct explanation of the Assertion.
- Both Assertion and Reason are true but the Reason is not the correct explanation of the Assertion.
- Assertion is true statement but Reason is false.
- Both Assertion and Reason are false statements
- Assertion: Eukaryotic cells have the ability to adopt a variety of shapes and carry out directed movements.Reason: There are three principal types of protein filaments; actin filament, microtubules and intermediate filaments, which constitute the cytoskeleton.
- Both assertion and reason are true and the reason is the correct explanation of the assertion.
- Both assertion and reason are true but the reason is not the correct explanation of the assertion.
- Assertion is true statement but reason is false.
- Both assertion and reason are false
- Gases such as oxygen and carbon dioxide cross the plasma membrane by:
- Secondary active transport
- Passive diffusion through the lipid bilayer
- Specific gas transport proteins
- Primary active transport
- A substance can only be accumulated against its electrochemical gradient by:
- Facilitated diffusion
- Passage through ion channels
- Diffusion through a uniport
- Active transport
- Which of the following is an example of primary active transport?
- Cl−- HCO3− exchange
- Na+ - H+ exchange
- Na+-Ca2+ exchange
- Na+, K+ ATPase
- The sodium pump:
- Exchanges extracellular Na+ for intracellular K+
- Is important for maintaining a constant cell volume
- Can only be inhibited by metabolic poisons
- Is an ion channel
- Which of the following statements regarding exocytosis is correct?
- Is always employed by cells for secretion
- Is used to deliver material into the extracellular space
- Takes up large molecules from the extracellular space
- Allows the retrieval of elements of the plasma membrane
- Endocytosis is used by cells to:
- Ingest bacteria and cell debris
- Retrieve elements of the plasma membrane after exocytosis
- Secrete large molecules into the extracellular space
- None of the above
- The sodium-potassium pump transports:
- More Na+ out than K+ in
- K+ out and Na+ in on a one-for-one basis
- Na+ out and K+ in on a one-for-one basis
- K+ and Na+ in the same direction
- Exocytosis is a process by which cells:
- Cystic fibrosis results from defective ion channels for:
- Na+
- Cl−
- Ca++
- H+
- Substances transported by facilitated diffusion:
- Move passively through specific channels from an area of greater concentration to one of lower concentration
- Must have movements coupled to those of other substances
- May flow to a region of higher concentration by the expenditure of energy
- Are restricted to only one direction through the membrane
- The methods of membrane transport that don't require protein channels or carriers are:
- Exocytosis
- Diffusion
- Phagocytosis
- All of the above
- In erythrocyte glucose transport is an example of:
- Simple diffusion
- Active transport
- Facilitated diffusion
- Ion driven active transport
- Which of the following is correct for active transport processes?
- Transport molecules or ions against concentration gradient.
- Transport molecules or ions against electrical gradient
- Are often referred to as pumps.
- All of the above
- Facilitated diffusion transport molecules:
- Against concentration gradient
- With the concentration gradient
- Always use energy
- Does not require carrier protein
- The exocytosis requires which ion:
- Ca2+
- Na+
- K+
- Fe+
ANSWERS FOR MCQs
1. c | 2. c | 3. c | 4. c | 5. a |
6. c | 7. a | 8. d | 9. b | 10. b |
11. c | 12. a | 13. c | 14. c | 15. d |
16. d | 17. d | 18. c | 19. b | 20. a |
21. d | 22. a | 23. a | 24. b | 25. d |
26. d | 27. b | 28. b | 29. a | 30. a |
31. a | 32. b | 33. a | 34. d | 35. c |
36. d | 37. b | 38. a |