Cell and Membrane Transport PDF

CHAPTER 1 Cell and Membrane Transport  Introduction  Cell Fractionation  Structure and Functions of a Cell and its...

CHAPTER 1 Cell and Membrane Transport  Introduction  Cell Fractionation  Structure and Functions of a Cell and its  Marker Enzymes Subcellular Components  Summary  Cytoskeleton  Exercise  Membrane Transport INTRODUCTION STRUCTURE AND FUNCTIONS OF A CELL AND ITS SUBCELLULAR COMPONENTS (FIGURE 1.1) ‘Cell’ means a small room or chamber, cells are the structural and functional units of all living organisms. The A cell has three major components: major parts of a cell are the nucleus and the cytoplasm. 1. Cell membrane (Plasma membrane) The electron microscope allowed classification of cells 2. Cytoplasm with its organelles into two major groups, prokaryotes and eukaryotes, 3. Nucleus. based on the presence and absence of the true nucleus. Cell Membrane (Plasma Membrane) Eukaryotes The cell is enveloped by a thin membrane called cell Eukaryotes have nucleus which is covered by membrane or plasma membrane. nuclear membrane. (Greek: Eue = true, karyon = nucleus). Animals, plants and fungi belong to the eukaryotes. Eukaryotic cells are much larger than prokaryotes. Unlike prokaryotes, eukaryotes have a variety of other membrane-bound organelles (subcellular elements) in their cytoplasm, including: – Mitochondria – Lysosomes – Endoplasmic reticulum and – Golgi complexes. The biochemical functions of subcellular organelles of the eukaryotic cell is given in Table 1.1. Prokaryotes Prokaryotes have no typical nucleus and subcellular components. (Greek: Pro = before). Bacteria and blue Figure 1.1: A diagrammatic representation of a typical green algae belong to the prokaryotes. eukaryotic cell 2 ESSENTIALS OF BIOCHEMISTRY Table 1.1: Biochemical functions of subcellular organelles of the eukaryotic cell Subcellular organelles Function Plasma membrane Transport of molecules in and out of cell, receptors for hormones and neurotransmitters Lysosome Intracellular digestion of macromolecules and hydrolysis of nucleic acid, protein, glycosaminoglycans, glycolipids, sphingolipids Golgi apparatus Post-transcriptional modification and sorting of proteins and export of proteins Rough endoplasmic reticulum Biosynthesis of protein and secretion Smooth endoplasmic reticulum Biosynthesis of steroid hormones and phospholipids, metabolism of foreign compounds Nucleus Storage of DNA, replication and repair of DNA, transcription and post-transcriptional processing Peroxisomes Metabolism of hydrogen peroxide and oxidation of long-chain fatty acids Nucleolus Synthesis of rRNA and formation of ribosomes Mitochondrion ATP synthesis, site for tricarboxylic acid cycle, fatty acid oxidation, oxidative phosphorylation, part of urea cycle and part of heme synthesis Cytosol Site for glycolysis, pentose phosphate pathway, part of gluconeogenesis, urea cycle and heme synthesis, purine and pyrimidine nucleotide synthesis Cell membranes mainly consist of lipids, proteins and smaller proportion of carbohydrates that are linked to lipids and proteins. The basic organization of biologic membranes is illustrated in Figure 1.2. The cell membrane is an organized structure consisting of a lipid bilayer primarily of phospho- lipids and penetrated protein molecules forming a mosaic-like pattern (Figure 1.3). Figure 1.2: The basic organization of biological membrane Figure 1.3: The fluid mosaic model of cell membrane  CELL AND MEMBRANE TRANSPORT 3 Membrane Lipids As a semipermeable membrane: The cell membrane permits only some substances to pass in either The major classes of membrane lipids are: direction, and it forms a barrier for other substances. – Phospholipids – Glycolipids The Fluid Mosaic Model of Cell Membrane – Cholesterol. They all are amphipathic molecules, i.e. they have In 1972, Singer and Nicolson postulated a theory of both hydrophobic and hydrophilic ends. membrane structure called the fluid mosaic model, Membrane lipids spontaneously form bilayer in which is now widely accepted. aqueous medium, burrying their hydrophobic tails and A mosaic is a structure made up of many different leaving their hydrophilic ends exposed to the water parts. Likewise, the plasma membrane is composed (Figure 1.2). of different kinds of macromolecules like phos- pholipid, integral proteins, peripheral proteins, Membrane Proteins glycoproteins, glycolipids and cholesterol. Proteins of the membrane are classified into two According to this model, the membrane structure is major categories: a lipid bilayer made of phospholipids. – Integral proteins or intrinsic proteins or trans- The bilayer is fluid because the hydrophobic tails of membrane proteins and phospholipids consist of an appropriate mixture of – Peripheral or extrinsic proteins. saturated and unsaturated fatty acids that is fluid at Integral proteins are either partially or totally normal temperature of the cell. immersed in the lipid bilayer. Many integral memb- Proteins are interspersed in the lipid bilayer, of the plasma rane proteins span the lipid bilayer from one side to membrane, producing a mosaic effect (Figure 1.3). the other and are called transmembrane protein The peripheral proteins literally float on the surface whereas others are partly embedded in either the of ‘sea’ of the phospholipid molecules, whereas the outer or inner leaflet of the lipid bilayer (Figure 1.2). integral proteins are like icebergs, almost completely Transmembrane proteins act as enzymes and submerged in the hydrophobic region. transport carriers for ions as well as water soluble There are no covalent bonds between lipid molecules substances, such as glucose. of the bilayer or between the protein components Peripheral proteins are attached to the surface of the and the lipids. lipid bilayer by electrostatic and hydrogen bonds. They Thus, there is a mosaic pattern of membrane proteins bound loosely to the polar head groups of the in the fluid lipid bilayer. membrane phospholipid bilayer (Figure 1.2). Fluid mosaic model allows the membrane proteins Peripheral proteins function almost entirely as to move around laterally in two dimensions and that enzymes and receptors. they are free to diffuse from place to place within the plane of the bilayer. Whereas they cannot tumble Membrane Carbohydrates from one side of the lipid bilayer to the other. The Singer-Nicolson model can explain many of Membrane carbohydrate is not free. It occurs in the physical, chemical and biological properties of combination with proteins or lipids in the form of membranes and has been widely accepted as the glycoproteins or glycolipids. Most of the integral most probable molecular arrangement of lipids and proteins are glycoproteins and about one-tenth of the proteins of membranes. membrane lipid molecules are glycolipids. The carbohydrate portion of these molecules protrude to Cytoplasm and its Organelles the outside of the cell, dangling outward from the cell surface (Figure 1.2). Cytoplasm is the internal volume bounded by the Many of the carbohydrates act as receptor for plasma membrane. The clear fluid portion of the hormones. Some carbohydrate moieties function in cytoplasm in which the particles are suspended is called antibody processing. cytosol. Six important organelles that are suspended in the Functions of Cell Membrane cytoplasm are: Protective function: The cell membrane protects the 1. Endoplasmic reticulum cytoplasm and the organelles of the cytoplasm. 2. Golgi apparatus Maintenance of shape and size of the cell. 3. Lysosomes 4 ESSENTIALS OF BIOCHEMISTRY Figures 1.4 A and B: Structure of endoplasmic reticulum 4. Peroxisomes Functions of golgi apparatus 5. Mitochondria The Golgi apparatus functions in association with the 6. Nucleus. endoplasmic reticulum. Proteins synthesized in the ER are transported to Endoplasmic Reticulum (ER) the Golgi apparatus where these are processed by addition of carbohydrate, lipid or sulfate moieties. Endoplasmic reticulum is the interconnected network These chemical modifications are necessary for the of tubular and flat vesicular structures in the transport of proteins across the plasma membrane. cytoplasm (Figures 1.4A and B). Golgi apparatus are also involved in the synthesis of Endoplasmic reticulum forms the link between intracellular organelles, e.g.lysosomes and peroxisomes. nucleus and cell membrane by connecting the cell membrane at one end and the outer membrane of Lysosomes the nucleus at the other end (see Figure 1.1). Lysosomes are vesicular organelles formed from A large number of minute granular particles called Golgi apparatus and dispersed throughout the ribosomes are attached to the outer surface of many cytoplasm. parts of the endoplasmic reticulum, this part of the Among the organelles of the cytoplasm, the lysosomes ER is known as rough or granular ER. have the thickest covering membrane to prevent the During the process of cell fractionation, rough ER is enclosed hydrolytic enzymes from coming in contact disrupted to form small vesicles known as with other substances in the cell and therefore, microsomes. It may be noted that microsomes as prevents their digestive actions. such do not occur in the cell. Many small granules are present in the lysosome. Part of the ER, which has no attached ribosomes, is The granules contain more than 40 different known as smooth endoplasmic reticulum. hydroxylases (hydrolytic enzymes). All the enzymes Functions of the ER are collectively called lysozymes. Rough ER functions in the biosynthesis of protein. Functions of lysosomes The smooth endoplasmic reticulum functions in the Lysozymes present in lysosomes digest proteins, carbo- synthesis of steroid hormones and cholesterol. hydrates, lipids and nucleic acids. Apart from the Smooth endoplasmic reticulum is the site of the digestive functions, the enzymes in the lysosomes are metabolism of certain drugs, toxic compounds and responsible for the following activities in the cell: carcinogens (cancer producing substances). Destruction of bacteria and other foreign bodies. Removal of excessive secretory products in the cells Golgi Apparatus of the glands. Golgi apparatus is present in all cells except in red blood Removal of unwanted cells in embryo. cells. It is situated near the nucleus and is closely related Peroxisomes to the endoplasmic reticulum. It consists of four or more membranous sacs. This apparatus is prominent in These organelles resemble the lysosomes in their secretory cells. appearance, but they differ both in function and in their synthesis.  CELL AND MEMBRANE TRANSPORT 5 They do not arise from Golgi membranes, but rather Nucleus from the division of pre-existing peroxisomes. Or perhaps through budding off from the smooth The cells with nucleus are called eukaryotes and those without nucleus are known as prokaryotes. Most of the endoplasmic reticulum. cells have only one nucleus but cells of skeletal muscles Functions of peroxisomes have many nuclei. The matured red blood cell contains Peroxisomes contain enzymes peroxidases and no nucleus. catalase which are concerned with the metabolism of peroxide. Thus, the peroxisomes are involved in Structure of Nucleus the detoxification of peroxide. The nucleus is spherical in shape and situated near Peroxisomes are also capable of carrying out the center of the cell. The nucleus is surrounded by β-oxidation of fatty acid. the nuclear envelope. The space enclosed by the nuclear envelope is called Mitochondria (Power House of Cell) nucleoplasm, within this the nucleolus is present. Mitochondria are called “Power Plant” of the cell since Nucleolus is an organized structure of DNA, RNA they convert energy to form ATP that can be used by and protein that is involved in the synthesis of cell. ribosomal RNA. The remaining nuclear DNA is A mitochondrion is a double-membrane organelle dispersed throughout the nucleoplasm in the form (Figure 1.5) that are fundamentally different in of chromatin fibers. At mitosis, chromatin is conden- composition and function. sed into discrete structures called chromosomes. — The outer membrane forms a smooth envelope. Functions of Nucleus It is freely permeable for most metabolites. — The inner membrane is folded to form cristae, The major functional role of the nucleus is that of: which give it a large surface area and are the site Replication: Synthesis of new DNA. of oxidative phosphorylation. The components Transcription: The synthesis of the three major types of RNA: of the electron transport chain are located on the inner membrane. 1. Ribosomal RNA (rRNA) The space within the inner membrane is called the 2. Messenger RNA (mRNA) mitochondrial matrix. It contains the enzymes of the: 3. Transfer RNA (tRNA). — Citric acid cycle — β-oxidation of fatty acid CYTOSKELETON — Some other degradative enzymes. The cytoplasm of most eukaryotic cells contains Mitochondria contain DNA (mtDNA) which network of protein filaments, that interact extensively encodes a few polypeptides involved in oxidative with each other and with the component of the plasma phosphorylation. membrane. Such an extensive intracellular network of protein has been called cytoskeleton. The plasma membrane is anchored to the cytoskeleton. The cytoskeleton is not a rigid permanent framework of the cell but is a dynamic, changing structure. The cytoskeleton consists of three primary protein filaments: 1. Microfilaments 2. Microtubules 3. Intermediate filaments. Figure 1.5: Structure of mitochondria 1. Microfilaments are about 5 nm in diameter. They are made up of protein actin. Actin filaments form a It is worth noting that sperms contribute no mito- meshwork just underlying the plasma membrane of cells chondria to the fertilized egg, so that mitochondrial and are referred to as cell cortex, which is labile. They DNA is inherited exclusively through the female line. disappear as cell motility increases or upon malignant Thus, mitochondria are maternally inherited. transformation of cells. The function of microfilaments is: — To help muscle contraction 6 ESSENTIALS OF BIOCHEMISTRY — To maintain the shape of the cell Biological membranes are semipermeable memb- — To help cellular movement. ranes through which certain molecules freely diffuse 2. Microtubules are cylindrical tubes, 20 to 25 nm in across membranes but the movement of the others diameter. They are made up of protein tubulin. is restricted because of size, charge or solubility. Microtubules are necessary for the formation and The two types of transport mechanisms are (Figure 1.6): function of mitotic spindle. They provide stability to 1. Passive transport or passive diffusion the cell. They prevent tubules of ER from collapsing. 2. Active transport. These are the major components of axons and dendrites. Passive Transport or Passive Diffusion 3. Intermediate filaments are so called as their diameter (10 nm) is intermediate between that of Passive transport is the process by which molecules microfilaments (5 nm) and of microtubules (25 nm). move across a membrane without energy (ATP). Intermediate filaments are formed from fibrous The direction of passive transport is always from a protein which varies with different tissue type. region of higher concentration to one of lower They play role in cell-to-cell attachment and help to concentration. stabilize the epithelium. They provide strength and There are two types of passive transport as follows: rigidity to axons. 1. Simple diffusion 2. Facilitated diffusion. Functions of Cytoskeleton Simple Diffusion The cytoskeleton gives cells their characteristic shape and form, provides attachment points for organelles, Lipid soluble, i.e. lipophilic molecules can pass through fixing their location in cells and also makes cell membrane, without any interaction with carrier communication between parts of the cell possible. proteins in the membrane. Such molecules will pass It is also responsible for the separation of chromo- through membrane along the concentration gradient, somes during cell division. i.e. from a region of higher concentration to one of The internal movement of the cell organelles as well lower concentration. This process is called simple as cell locomotion and muscle fiber contraction could diffusion. not take place without the cytoskeleton. It acts as “track” on which cells can move organelles, chromo- Facilitated Diffusion somes and other things. The movement of water soluble molecules and ions across the membrane requires specific trans- MEMBRANE TRANSPORT port system. They pass through specific carrier One of the functions of the plasma membrane is to proteins. A carrier protein binds to a specific regulate the passage of a variety of small molecules molecule on one side of the membrane and across it. releases it on the other side. This type of crossing Figure 1.6: Types of membrane transport mechanism  CELL AND MEMBRANE TRANSPORT 7 Figure 1.7: Uniport, symport and antiport transport of Figure 1.8: Mechanism of sodium-potassium pump substance across the cell membrane (primary active transport) the membrane is called facilitated diffusion or time pumps K + ions from outside to the inside carrier-mediated diffusion. generating an electrochemical gradient. An example of facilitated diffusion is the movement Carrier protein of Na+-K+ pump has three receptor of glucose and most of the amino acids across the sites for binding sodium ions on the inside of the cell plasma membrane. and two receptor sites for potassium ions on the These diffusion processes are not coupled to the outside. The inside portion of this protein has movement of other ions, they are known as uniport ATPase activity (Figure 1.8). transport processes (Figure 1.7). The pump is called Na+-K+ ATPase because the hydrolysis of ATP occurs only when three Na+ ions Active Transport bind on the inside and two K+ ions bind on the If a molecule moves against a concentration gradient, outside of the carrier proteins. The energy liberated an external energy source is required; this movement by the hydrolysis of ATP leads to conformational is referred to as active transport. change in the carrier protein molecule, extruding the Substances that are actively transported through cell three Na+ ions to the outside and the two K+ ions to membranes include, Na+, K+, Ca++, H+, CI–, several the inside. different sugars and most of the amino acids. Physiological importance of Na+-K+ pump Active transport is classified into two types The active transport of Na+ and K+ is of great physio- according to the source of energy used as follows : logical significance. The Na+-K+ gradient created by i. Primary active transport this pump in the cells, controls cell volume. ii. Secondary active transport. It carries the active transport of sugars and amino In both instances, transport depends on the carrier acids. proteins; like facilitated diffusion. However, in active transport, the carrier proteins function differently Secondary Active Transport from the carrier in facilitated diffusion. Carrier Secondary active transport uses an energy generated by protein for active transport is capable of transport- an electrochemical gradient. It is not directly coupled ing substance against the concentration gradient. with hydrolysis of ATP. Secondary active transport is classified into two types: Primary Active Transport 1. Co-transport or symport, in which both substances In primary active transport, the energy is derived move simultaneously across the membrane in the directly from hydrolysis of ATP. same direction (Figure 1.7), e.g. transport of Na+ and Sodium, potassium, calcium, hydrogen and chloride glucose to the intestinal mucosal cells from the gut. ions are transported by primary active transport. 2. Counter transport or antiport, in which both Primary active transport of Na+ and K+ substances move simultaneously in opposite (sodium-potassium pump) direction (Figure 1.7), e.g. transport of Na+ and H+ Na+-K+ Pump, a primary active transport process occurs in the renal proximal tubules and exchange that pumps Na+ ions out of the cell and at the same of Cl- and HCO3- in the erythrocytes. 8 ESSENTIALS OF BIOCHEMISTRY Transport of Macromolecules Across The receptors are generally concentrated in small pits the Plasma Membrane on the outer surface of the cell membrane. These receptors are coated on the cytoplasmic side with a The process by which cells take up large molecules fibrillar protein called calthrin and contractile is called endocytosis (Figure 1.9) and the process by filaments of actin and myosin. which cells release large molecules from the cells to Once the macromolecules (which is to be absorbed) have the outside is called exocytosis (Figure 1.10). bound with the receptors, the entire pit invaginates inward, and the fibrillar protein by surrounding the Endocytosis invaginating pit causes it to close over the attached There are two types of endocytosis: macromolecule along with a small amount of 1. Pinocytosis (cellular drinking) extracellular fluid. 2. Phagocytosis (cellular eating). Then immediately, the invaginated portion of the memb- rane breaks away from the surface of the cell forming Pinocytosis endocyte vesicle inside the cytoplasm of the cell. Pinocytosis is the cellular uptake of fluid and fluid contents and is a cellular drinking process. Phagocytosis Pinocytosis is the only process by which most Phagocytosis involves the ingestion of large particles such macromolecules, such as most proteins, polysaccha- as viruses, bacteria, cells, tissue debris or a dead cell. rides and polynucleotides can enter cells (Figure 1.9). It occurs only in specialized cells such as macro- These molecules first attach to specific receptors on phages and some of the white blood cells. the surface of the membrane. Phagocytosis occurs in much the same way as pinocytosis. Exocytosis Most of the endocytotic vesicles formed from pinocytosis fuse with lysosomes. Lysosomes empty Figure 1.9: Three stages of the absorption of macromolecules by endocytosis Figure 1.10: Stages in exocytosis  CELL AND MEMBRANE TRANSPORT 9 their acid hydrolases to the inside of the vesicle and begin hydrolyzing the proteins, carbohydrate, lipids and other substances in the vesicle. The macromolecular contents are digested to yield amino acids, simple sugars or nucleotides and they diffuse out of the vesicle and reused in the cytoplasm. Undigestible substances called residual body is finally excreted through the cell membrane by a process called exocytosis, opposite to endocytosis (Figure 1.10). 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. CELL FRACTIONATION To obtain purified preparations of organelles, the tissue is first carefully broken up in a homogenizing apparatus using isotonic 0.25 M sucrose solution. Sucrose solution is used because it is not metabolized in Figure 1.11: Subcellular fractionation of cell by differential centrifugation most tissues and it does not pass through membranes readily and thus, does not cause inter organelles to swell. Then homogenate is centrifuged at a series of contamination with other organelles. For example, increasing centrifugal force. (Figure 1.11). isolated mitochondria have a high specific activity The subcellular organelles, which differ in size and of cytochrome oxidase but low catalase and acid specific gravity, sediment at different rates and can phosphatase, the catalase and acid phosphatase be isolated from homogenate by differential activities being due to contamination with centrifugation. peroxisomes and lysosomes respectively. The dense nuclei are sedimented first, followed by Some typical subcellular markers are given in the mitochondria, and finally the microsomal Table 1.2. fraction at the highest forces. After all the particulate matter has been removed, the soluble remnant is the cytosol. Table 1.2: Marker enzymes of subcellular fractions Organelles of similar sedimentation coefficient Fraction Enzymes obviously cannot be separated by differential centri- Plasma membrane 5 Nucleotidase, Na+-K+-ATPase fugation. For example, mitochondria isolated in this way are contaminated with lysosome and peroxisomes. Nucleus DNA polymerase RNA polymerase These may be separated by isopyknic centrifugation technique. Endoplasmic reticulum Glucose-6-phosphatase Golgi bodies Galactosyl transferase MARKER ENZYMES Lysosomes Acid phosphatase β-glucuronidase The purity of isolated subcellular fraction is assessed Mitochondria Succinate dehydrogenase by the analysis of marker enzymes. Cytochrome C-oxidase Marker enzymes are the enzymes that are located Peroxisomes Catalase exclusively in a particular fraction and thus become Cytosol Lactate dehydrogenase characteristic of that fraction. Glucose-6-phosphate Analysis of marker enzymes confirms the identity dehydrogenase of the isolated fraction and indicates the degree of