The plasma membrane of all organisms is made of A, the phospholipid bilayer and C, the proteins. In mammals, including humans, there is also cholesterol (B) and, on the outside, carbohydrates (D). We will look at each of these components individually.
The phospholipid bilayer is made of two molecules of phospholipids, arranged so that the polar head groups are facing the watery environment outside and inside the cell. The hydrophobic fatty acids are inside forming an oily inner layer to the membrane. These fatty acids may be saturated and fairly stiff or unsaturated and oily. The consistency will be adjusted by the cell in order to keep the membranes fluid in different parts of the body and in different seasons of the year. Cholesterol is a lipid as are the phospholipids. Cholesterol is added to animal membranes to give it rigidity. (Plants and bacteria have cell walls for rigidity and do not have cholesterol.) Cholesterol is present up to 30 percent in the plasma membranes of animals.
All plasma membranes have proteins associated with them. The integral proteins span the membrane as shown in the illustration. And peripheral proteins are usually located on the inside of the cell. We will examine later how proteins can act as channels, transport, receptors or enzymes for the cell. Proteins also help to reinforce the cell shape. In mammals, there are carbohydrates on the outside of cells. Glycolipids are carbohydrates—”glyco” attached to lipids and glycoproteins are carbohydrates attached to proteins, usually integral proteins in the membrane. These glycolipids and glycoproteins are used in mammals as cell-cell identification by the immune system. One example is our ABO blood type.
Section II: Diffusion and Osmosis
Diffusion is the movement of molecules from an area of higher concentration to an area of lower concentration. In other words, diffusion goes down the concentration gradient. Diffusion is caused by the random movement of molecules that fill the available space. Because it is going down the concentration gradient and is caused by the random movement of molecules, diffusion does not require energy.
There are several factors affecting diffusion. Molecules that are cold diffuse more slowly than warm molecules. Larger molecules diffuse more slowly than smaller molecules. And molecules diffuse slowest in a solid, less slowly in a liquid and most quickly in a gas.
Osmosis is diffusion across a membrane. In cells, we usually look at the concentrations of water and salts inside and outside the plasma membrane when discussing osmosis. Isotonic, which means “same saltiness”, is when the salt concentrations are the same on both sides of the membrane. Hyp o tonic solution, which means “less salty” is when the solution outside the cell is less salty than inside. Hyp er tonic solution, which means “more salty”, is when the solution outside the cell is more salty than inside.
Let’s look at this in red blood cells (RBC). The center picture shows the RBC in an isotonic solution. Since the salt concentration is the same inside and outside the cells, there is no net movement of water. (Water will go into and out of the cell at the same rate) This is the normal situation for cells. The left shows the cells in a hypertonic solution that is much saltier than the RBC. In this situation water will leave the cells to try to balance the salt concentrations inside and out. The result is that the RBC become shriveled or crenated. On the right is the case of RBC in a hypotonic solution. The outside of the cells is less salty than the inside so water will enter the RBC to try to balance the salt concentrations inside and out. The RBC become swollen and may burst.
Section III: Transport Across Membranes
There are three types of transport across membranes, Passive transport, Facilitated diffusion and Active transport. We will look at each of these separately.
Passive transport is diffusion of molecules across a membrane down the concentration gradient and so does not require energy. Since these molecules will usually have to pass through the phospholipid bilayer, large molecules and polar or ionic molecules are excluded. Oxygen, carbon dioxide and small hydrocarbons which are also hydrophobic will be able to pass through the lipid bilayer. In the example of oxygen, it is a small uncharged molecule and is used by the cells as soon as it enters, so oxygen enters the cells by passive transport.
Facilitated Transport is passive transport down the concentration gradient that is assisted by integral membrane proteins. Facilitated transport is for those polar, ionic or large molecules that cannot pass through the lipid bilayer and includes water, ions, glucose and so forth. For example, osmosis with water occurs by facilitated transport using the integral membrane protein called aquaporin.
There are two types of integral membrane proteins that are involved with facilitated diffusion, one of which are the channel proteins. As the name implies, channel proteins, usually made of 3 or more polypeptides grouped together have a hydrophilic channel in the middle through which the molecules can cross the membrane. There are different channel proteins for different molecules: aquaporin is specific for water and the calcium channel is specific for calcium ions. Many channels are gated, meaning that they have a polypeptide covering the channel itself. A chemical or electric stimulus is needed to open the channel. Neurons have several types gated channels in their membranes.
The regular channel protein is on the left; it is always open. The channel on the right is gated with the polypeptide gate inside the cell. The gate is in the closed position so no molecules can stray into the cell.
Another type of proteins associated with facilitated transport are the carrier proteins. These change shape when a molecule enters the protein creating a rocking motion that allows the molecules to enter the cell one by one. Old ideas about carrier proteins were that they were like revolving doors, rotating through the membrane to bring molecules into the cell. However, the portion of these proteins that face the inside and outside of the cell contain hydrophilic amino acids. Rotating through the membrane would require bringing the hydrophilic amino acids in contact with the hydrophobic fatty acids, so cannot act like revolving doors.
The third type of transport across membranes is active transport. In active transport, proteins carry molecules across the membrane against the concentration gradient. This process is fighting the second law of thermodynamics and therefore requires energy, usually as ATP. An example of an active transport system are ion pumps which establish different concentrations of anions and cations across the membrane and so set up the membrane potential.
The sodium-potassium pump is a classic example of an active transport ion pump. There are more sodium ions outside the cell than inside, so the pump must use energy to bring the sodium out of the cell. Conversely, there are more potassium ions inside the cell than outside, so the pump must use energy to bring the potassium into the cell. In a. the pump has receptor sites that fit 3 sodium ions—yellow. In b. The channel changes shape bringing the sodium ions across the membrane to the outside. At the same time the potassium ions can now fit into the receptor sites. In c. The sodium ions are released to the outside and the potassium ions move the inside. In d. the potassium ions are released into the cell. Note shown is that ATP must be used to move the sodium and potassium ions up the concentration gradient.
Introduction to MolecularBiology
What is Molecular Biology?Study of biology at molecular levelBasic Molecular BiologyApplied Molecular Biology2
Which Molecular Level?• Nucleic AcidsDNARNA• Proteins3
What to know about nucleic acids?• StructureChemical propertiesPhysical propertiesTypes• Synthesis• FunctionBasic Molecular Biology4
What to know about proteins?StructureChemical propertiesPhysical propertiesSynthesisHow the sequence of a protein is determined?GeneticsFunction of the proteinsStructure/Function RelationshipBasic Molecular Biology6
Molecular biology seeks to explain therelationships between the structure andfunction of biological molecules and howthese relationships contribute to theoperation and control of biochemicalprocesses.9
• Medicine:BiochemistryParasitologyImmunologyBacteriologyVirologyPathologyLaboratory MedicineGeneticsetc.Applications of Molecular Biology(Molecular Medicine)11
Tools used in molecular studies• DNA cloningDNA cloning facilitates the isolation andmanipulation of fragments of an organism’sgenome by replicating them independentlyas part of an autonomous vector.Using Biological System12
• Polymerase Chain Reaction (PCR)PCR is used to amplify a sequence of DNA using a pair ofoligonucleotide primers each complementary to one endof the DNA target sequence.These are extended towards each other by a thermostableDNA polymerase in a reaction cycle of three steps:1. Denaturation2. Primer annealing3. PolymerizationUsing Chemical SystemTools used in molecular studies13
• DNA SequencingThe two main methods of DNA sequencing are theMaxam and Gilbert chemical method andSanger’s enzymic method.• RNA Sequencing• Protein SequencingDATA BASESGenBankTools used in molecular studies15
Some important events in thehistory of molecular biology1871 Discovery of the DNA.1943 DNA proves to be a genetic molecule capableof altering the heredity of bacteria :Avery, MacLeodand McCarty.1953 Postulation of a complementary double-helicalstructure for DNA.1960 Discovery of messenger RNA, and the demonstrationThat it carries the information that orders amino acidsin proteins. 17
DNA: The Transforming AgentIn 1943, Oswald Avery, C. M. MacLeod, and M. McCarty on bacteriumStreptococcus pneumoniae18
1961 Use of a synthetic messenger RNA moleculeto work out the first letters of the genetic code.Some important events in thehistory of molecular biology1966 Establishment of the complete genetic code.1970 Isolation of the first restriction enzyme, anenzyme that cuts DNA molecules at specific sites.1973 Beginning of DNA cloning in E. coli.1977 Formation of the first genetic engineering company(Genenetech) specifically found to use recombinant DNAmethods to make medically important drugs. 20
1977 Development of procedures for the rapidsequencing of DNA.Some important events in thehistory of molecular biology1978 The Nobel Prize in Medicine was awarded for thediscovery and use of restriction enzymes to HamiltonSmith and Daniel Nathans.1978 Somatotatin becomes the first human hormoneproduced by using recombinant DNA.1980 The Nobel Prize in Chemistry is awarded dually tofor the formation of the first recombinant DNA molecules(Paul Berg) and thedevelopment of powerful methods for sequencing DNAto Gilbert and Sanger.to Gilbert and Sanger.21
1981 Sickle-cell anemia becomes the first geneticdisease to be diagnosed antenatally directly atthe gene level by restriction enzyme analysis ofthe DNA.Some important events in thehistory of molecular biology1982 Human insulin produced by recombinant DNAmethods goes on the market under the trade nameHumulin.1985 Use of heat stable DNA polymerase in PCR.1990 First trial for gene therapy in humans22
24Cell TheoryCells were discovered in 1665 by Robert Hooke.Early studies of cells were conducted by- Mathias Schleiden (1838)- Theodor Schwann (1839)Schleiden and Schwann proposed the Cell Theory.
25Cell TheoryCell Theory1. All organisms are composed of cells.2. Cells are the smallest living things.3. Cells arise only from pre-existing cells.All cells today represent a continuous line ofdescent from the first living cells.
26Cell TheoryCell size is limited.-As cell size increases, it takes longer formaterial to diffuse from the cell membraneto the interior of the cell.Surface area-to-volume ratio: as a cellincreases in size, the volume increases10x faster than the surface area
35Prokaryotic CellsProkaryotic cell walls-protect the cell and maintain cell shapeBacterial cell walls-may be composed of peptidoglycan-may be Gram positive or Gram negativeArchaean cell walls lack peptidoglycan.
36Prokaryotic CellsFlagella-present in some prokaryotic cells-used for locomotion-rotary motion propels the cell
39Eukaryotic CellsEukaryotic cells-possess a membrane-bound nucleus-are more complex than prokaryotic cells-compartmentalize many cellular functionswithin organelles and theendomembrane system-possess a cytoskeleton for support andto maintain cellular structure
The Plasma MembraneA. Phospholipid BilayerTwo layers of phospholipidsHydrophilic head groups facethe waterHydrophobic fatty acids face insideFatty acids may be saturated or unsaturated.Fatty acid structure is adjusted to keepmembranes fluid like oil, not solid like butter.A. CholesterolAdds rigidity to the membrane.Up to 30% of animal plasma membranes. 41
The Plasma MembraneC. ProteinsIntegral proteins span the membranePeripheral proteins are mostly on inside.Act as channels, transport, receptors, enzymesHelps to reinforce cell shapeD. Carbohydrates on the outsideGlycolipids—attached to phospholipidsGlycoproteins—attached to proteinsThe sugar chains form a sticky, sugar layer on theoutside of the plasma membrane called theglycocalyx.Used in mammals for cell-cell identification as partof immune system, such as blood types.42
45Eukaryotic CellsNucleus-stores the genetic material of the cell inthe form of multiple, linear chromosomes-surrounded by a nuclear envelopecomposed of 2 phospholipid bilayers-in chromosomes – DNA is organized withproteins to form chromatin
Chromosomes• DNA of eukaryotes is divided into linearchromosomes.– exist as strands of chromatin, except duringcell division– associated with packaging histones,packaging proteins• nucleosomes47
48Eukaryotic CellsRibosomes-the site of protein synthesis in the cell-composed of ribosomal RNA andproteins-found within the cytosol of the cytoplasmand attached to internal membranes
49Endomembrane SystemEndomembrane system-a series of membranes throughout thecytoplasm-divides cell into compartments wheredifferent cellular functions occur1. endoplasmic reticulum2. Golgi apparatus3. lysosomes
50Rough endoplasmic reticulum (RER)-membranes that create a network ofchannels throughout the cytoplasm-attachment of ribosomes to themembrane gives a rough appearance-synthesis of proteins to be secreted, sentto lysosomes or plasma membrane
51Smooth endoplasmic reticulum (SER)-relatively few ribosomes attached-functions:-synthesis of membrane lipids-calcium storage-detoxification of foreign substances
57Microbodies-membrane bound vesicles-contain enzymes-not part of the endomembrane system-glyoxysomes in plants contain enzymesfor converting fats to carbohydrates-peroxisomes contain oxidative enzymesand catalase
58Vacuoles-membrane-bound structures with variousfunctions depending on the cell typeThere are different types of vacuoles:-central vacuole in plant cells-contractile vacuole of some protists-vacuoles for storage
59MitochondriaMitochondrion-organelles present in all types ofeukaryotic cells-contain oxidative metabolism enzymes fortransferring the energy withinmacromolecules to ATP-found in all types of eukaryotic cells
60Mitochondria-surrounded by 2 membranes-smooth outer membrane-folded inner membrane with layerscalled cristae-matrix is within the inner membrane-intermembrane space is located betweenthe two membranes-contain their own DNA
62Cytoskeleton-network of protein fibers found in alleukaryotic cells-supports the shape of the cell-keeps organelles in fixed locations-helps move materials within the cell
63CytoskeletonCytoskeleton fibers include-actin filaments – responsible for cellularcontractions, crawling,-microtubules – provide organization tothe cell and move materials within the cell-intermediate filaments – provide structuralstability
65Cell MovementCell movement takes different forms.-Crawling is accomplished via actinfilaments and the protein myosin.-Flagella undulate to move a cell.-Cilia can be arranged in rows on thesurface of a eukaryotic cell to propel a cellforward.
67Cell walls-present surrounding the cells of plants,fungi, and some protists-the carbohydrates present in the cell wallvary depending on the cell type:-plant and protist cell walls - cellulose-fungal cell walls - chitin
68Extracellular matrix (ECM)-surrounds animal cells-composed of glycoproteins and fibrousproteins such as collagen-may be connected to the cytoplasm viaintegrin proteins present in the plasmamembrane
Diffusion• Diffusion is the movement of moleculesfrom an area of higher concentration to anarea of lower concentration (which we call“down the concentration gradient”)• Random movement of molecules fills theavailable space.• Does not require cellular energy in theform of ATP.72
Osmosis and TonicityOsmosis is diffusion across a semi-permeablemembrane.In cells, we usually compare water and saltconcentrations inside & outside the cell.Isotonic is salt concentrations same on bothsides of the membraneHypotonic (“less salty”): one side is less saltythan the other.Hypertonic (“very salty”): one side is more saltythan the other.74
Examples Using Red Blood CellsSaltier outsidethan inside: waterleaves cells,shriveling them.Saltier inside thanoutside: water enterscells, swelling them,sometimes tobursting.Salt conc. sameinside and out.Normal for cells.No net movementof water.75
Types of Transport Across Membranes1. Passive Transport substances movedown their concentration gradient2. Facilitated Diffusion— protein-assisteddiffusion3. Active Transport— substances moveagainst their concentration gradient;requires energy77
Passive Transport• Diffusion across a membrane down theconcentration gradient• Does not require cellular energy (ATP)• Usually pass through the lipid bilayer– Oxygen– Carbon dioxide– Small hydrocarbons (also hydrophobic)78
Facilitated TransportPassive transported assisted by proteinsGoes down concentration gradient and sodoes not require energy.For molecules that cannot easily passthrough lipid bilayer:◦ H2O◦ ions◦ glucose, etc.Osmosis with water occurs by facilitatedtransport using the protein aquaporin.79
Channel ProteinsChannel proteins are 1 of 2 types ofintegral membrane proteins in facilitateddiffusion.◦ Often made of 3 or more subunits (polypeptides)grouped together.◦ Have hydrophilic channel in the middle formolecules to pass through.◦ Specific for type of molecule: H2O, Ca++, etc.◦ Many are gated and require a chemical orelectrical stimulus to open the channel as in nervecells (neurons). 80
Carrier Proteins• Carrier proteins are facilitated transportproteins that bring molecules across themembrane by changing shape in a rockingmotion.82
Active Transport• Carries molecules against theconcentration gradient• Requires energy usually as ATP.Ion pumps are active transport pumpsEstablish different concentrations of anionsand cations across membrane so set up acharge across the membrane or membranepotential.83