The CellProkaryotes and eukaryotes

CellsCells :structural and functional units of all living organismsunicellular, consisting of a single cell and multicellular, (an estimated 1013 cells in humans!) Each cell can take in nutrients, convert these nutrients into energy, carry out specialized functions, and reproduce as necessary. Each cell stores its own set of instructions for carrying out each of these activities.There are two general categories of cells: prokaryotes and eukaryotes

Life on Earth.

Cellular components.

Eukaryote and prokaryotes.

The cell.

The cell.

Where do viruses fit?.

The Nucleus and Nucleolus .

The Nucleus.

Nucleoproteins.

Nucleosome and beads on a string conformation.

Chromosomes.

The Nucleus and NucleolusThe nucleus is the most obvious organelle in any eukaryotic cell. It is a membrane-bound organelle and is surrounded by a double membrane. It communicates with the surrounding cytosol via numerous nuclear pores. Within the nucleus is the DNA responsible for providing the cell with its unique characteristics. The DNA is similar in every cell of the body, but depending on the specific cell type, some genes may be turned on or off - that's why a hepatocyte is different from a epithelial cell .When a cell is dividing, the DNA and surrounding protein condense into chromosomes that are visible by microscopy.The prominent structure in the nucleus is the nucleolus. The nucleolus produces ribosomes, which move out of the nucleus to positions on the rough endoplasmic reticulum where they are critical in protein synthesis.

Endoplasmic reticulum(ER).

RE.

Endoplasmic reticulum(ER)Is a vast membrane structure. The ER membrane is a continuation of the outer nuclear membraneWhen viewed by electron microscopy, some areas of the endoplasmic reticulum look "smooth" (smooth ER) and some appear "rough" (rough ER). The rough ER appears rough due to the presence of ribosomes on the membrane surface. Smooth ER is important in the synthesis of lipids and membrane proteins. Rough ER is important in the synthesis of other proteins.Information coded in DNA sequences in the nucleus is transcribed as messenger RNA. Messenger RNA exits the nucleus through small pores to enter the cytoplasm. At the ribosomes on the rough ER, the messenger RNA is translated into proteins. These proteins are then transferred to the Golgi in "transport vesicles" where they are further processed and packaged into lysosomes, peroxisomes, or secretory vesicles.

The Golgi.

Golgi complex regulates the insertion of plasma membrane proteins .

Golgi ApparatusThe Golgi apparatus is a membrane-bound structure with a single membrane. It is actually a stack of membrane-bound vesicles that are important in packaging macromolecules for transport elsewhere in the cell. The stack of larger vesicles is surrounded by numerous smaller vesicles containing those packaged macromolecules. The enzymatic or hormonal contents of lysosomes, peroxisomes and secretory vesicles are packaged in membrane-bound vesicles at the periphery of the Golgi apparatus.

Mitochondria

An eukaryotic cell contains many mitochondria, occupying up to a quarter of the cytoplasmic volume. The size of a mitochondrion is about 1.5-2 mm in length, 0.5-1 mm in diameter.It has two membranes: outer membrane and inner membrane. Mitochondria also have their own DNA ( mtDNA), which encodes some of the proteins and RNAs in mitochondria. However, most proteins operating in mitochondria still originate from nuclear DNA.The major role of mitochondria is to produce ATP In animal cells, the major sources for the synthesis of ATP are fatty acids and glucose. The generation of ATP involves a series of electron transport in the mitochondria

Mitochondrion.

Mitochondrial compartments.

ATP synthesis in the cristae.

Electron Transport on the inner mitochondrial membrane.

The respiratory chain is located on the inner mitochondrial membrane.

Protein import by mitochondria through protein "porin". .

Mitochondria ( apoptosis and aging)Release of cytochrome c from mitochondria into the cytoplasm, in concert with Apaf1, can activate caspase 9, which then activate caspase 3 to execute the death program. Cytochrome c and other small molecules may pass through the mitochondrial permeability transition pore in the outer membrane, the adenine nucleotide translocator (ANT) in the inner membrane, and several auxiliary proteins that include the Bcl-2 family involved in apoptosis. The Bcl-2 family of proteins may be divided into three groups:Anti-apoptotic. They share sequence homology at BH1, BH2, BH3, and BH4 domains (BH = Bcl-2 homology). Examples: Bcl-2 itself and Bcl-xL. Pro-apoptotic. They share sequence homology at BH1, BH2 and BH3 domains. Examples: Bax and Bak BH3-only proteins. They are pro-apoptotic, but share homology only at the BH3 domain. Examples: Bid, Bik, and Bim.Electrons may leak from the electron transport chain, producing free radicals. This has been suggested to be the major mechanism involved in the aging process.

MitochondriaMitochondria provides energy to the cell. They are the power centers of the cell. They are about the size of bacteria but may have different shapes depending on the cell type.Mitochondria are membrane-bound organelles, and like the nucleus have a double membrane. The outer membrane is fairly smooth. But the inner membrane is highly convoluted, forming folds called cristae. The cristae greatly increase the inner membrane's surface area. It is on these cristae that NADH and FADH2 from food oxidation is combined with oxygen to produce ATP

Prokaryotic Ribosome(70S).

80S and 70S Ribosomes.

Composition of Ribosomes.

Lysosomes, Peroxisomes, Secretory Vesicles

Lysosomes: Lysosomes (common in animal cells but rare in plant cells) contain hydrolytic enzymes necessary for intracellular digestion. In white blood cells that engulf bacteria, lysosome contents are carefully released into the vacuole around the bacteria and serve to kill and digest those bacteria. Uncontrolled release of lysosome contents into the cytoplasm can also cause cell death . Peroxisomes: This organelle is responsible for protecting the cell from its own production of toxic hydrogen peroxide. As an example, white blood cells produce hydrogen peroxide to kill bacteria. The oxidative enzymes in peroxisomes break down the hydrogen peroxide into water and oxygen. Secretory Vesicles: Cell secretions - e.g. hormones, neurotransmitters - are packaged in secretory vesicles at the Golgi apparatus. The secretory vesicles are then transported to the cell surface for release.

Membrane and Cytosol

Cell Membrane: Every cell is enclosed in a membrane. The membrane is a double layer of lipids (lipid bilayer) but is made quite complex by the presence of numerous proteins that are important to cell activity. These proteins include receptors, pores, and enzymes. The membrane is responsible for the controlled entry and exit of ions like sodium (Na) potassium (K), calcium (Ca++). Cytosol: The cytosol also called cytoplasm is an ammorphous viscous fluid within which all the other cell organelles reside and where most of the cellular metabolism occurs. It is full of proteins that control cell metabolism including signal transduction pathways, glycolysis, intracellular receptors, and transcription factors etc.

FUNCTIONS OF BIOLOGICAL MEMBRANES

Compartmentation Cells use plasma membrane as an envelope. Subcellular organelles nuclei, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, etc.Control of the passage of materials Control the flow of nutrients, waste products, ions, etc. Contain pumps and gates.Biochemical processes Oxidative phosphorylation. electron transport

Processing of information Sensory stimuli Intercellular communication(gap junctions Nerve impulses Hormonal actionsCell-cell recognition . immunological

The chemical components of membranes

General composition. The components Lipid -- cholesterol, phospholipid and sphingolipid Proteins Carbohydrate -- as glycoproteinDifferences in composition among membranes (e.g. myelin vs. inner mitochondrial membrane) Illustrate the variability of membrane structure. This is due to the differences in function. Example: Mitochondrial inner membrane has high amounts of functional electron transport system proteins. Plasma membrane, with fewer functions (mainly ion transport), has less protein.Membranes with similar function (i.e. from the same organelle) are similar across species lines, but membranes with different function (i.e. from different organelles) may differ strikingly within a species.

Common membrane phospholipids.

Phosphatidyl choline

Phosphatidylcholine.

Cholesterol is another common membrane lipid.

Amphipathic molecules. .

Structure-based classification of membrane lipids .

Lipid BilayerAmphipathic lipids in association with water form complexes in which their polar regions are in contact with water and their hydrophobic regions are away from water. Various micelle structures. E.g., the spherical micelle is a stable configuration for amphipathic lipids that have a conical shape, such as fatty acids. A bilayer. This is the most stable configuration for amphipathic lipids with a cylindrical shape, such as phospholipids.

Phosphatidylcholine.

Membrane fluidityThe interior of a lipid bilayer ishighly fluid In the liquid crystal state, hydrocarbon chains of phospholipids are disordered and in constant motion. At lower temperature, a membrane containing a single phospholipid type undergoes transition to a crystalline state in which fatty acid tails are fully extended, packing is highly ordered, and van der Waals interactions between adjacent chains are maximal. Kinks, due to cis double bonds in fatty acids, interfere with packing of lipids in the crystalline state, and lower the phase transition temperature.

Membrane fluidityPresence of cholesterol in a phospholipid membrane inhibits transition to the crystalline state. However interaction with the relatively rigid cholesterol decreases the mobility of hydrocarbon tails of phospholipids. Phospholipid membranes that include cholesterol have a fluidity intermediate between the liquid crystal and crystal states. Two strategies by which phase changes of membrane lipids are avoided: Cholesterol is abundant in membranes, such as plasma membranes, that include many lipids with long-chain saturated fatty acids. In the absence of cholesterol, such membranes would crystallize at physiological temperatures. The inner mitochondrial membrane lacks cholesterol, but includes many phospholipids whose fatty acids have one or more double bonds, which lower the melting point to below physiological temperature.

Membrane FluidityCholesterol is abundant in membranes, such as plasma membranes, that include many lipids with long-chain saturated fatty acids. In the absence of cholesterol, such membranes would crystallize at physiological temperatures. The inner mitochondrial membrane lacks cholesterol, but includes many phospholipids whose fatty acids have one or more double bonds, which lower the melting point to below physiological temperature.

Bilayer and micelle.

Lipid bilayer.

Lipid Bilayer.

Lateral mobility and Flip Flop.

Lipid bilayer.Color scheme: PO4 = green, N(CH3)3 = violet, water = blue, terminal CH3 = yellow, O = red, glycol C = brown, chain C = grey

Schematic diagram of membrane structure.

Schematic diagram of membrane proteins.

A membrane-spanning a-helix is the most common structural motif found in integral proteins

Membrane structure.

Plasma membrane.

A cartoon of EM view of membranes via freeze fracture/freeze technique

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Blood-group antigensThe antigens which determine blood types belong to glycoproteins and glycolipids. There are three types of ABO blood-group antigens: O, A, and B. They differ only slightly in the composition of carbohydrates.All humans contain enzymes which catalyze the synthesis of the O antigen. Humans with A-type blood also contain an additional enzyme (called A-type enzyme here) which adds N-Acetylgalactosamine to the O antigen. Humans with B-type blood contain another enzyme (called B-type enzyme ) which adds Galactose to the O antigen. Humans with AB-type blood contain both A-type and B-type enzymes while humans with O-type blood lack both types of enzymes.

Blood group antigens.

Distribution of lipids in membranes

There can be large membrane to membrane differences in lipids (compare phosphoglycerides and cholesterol in plasma membrane vs. inner mitochondrial membrane). There can be large differences within classes of lipids (compare the cardiolipin of the inner mitochondrial membrane to other membranes). There are also patterns of differences among the fatty acyl groups of the lipids of various membranes

The proteins of membranesClassificationPeripheral: Are loosely associated with the membrane surface. In integral: Are deeply imbedded in the membrane. Roles of membrane proteins. Catalytic: enzymes ReceptorsTransport Structural Carbohydrates of membranes are present attached to protein or lipid as glycoprotein or glycolipid. Typical sugars in glycoproteins and glycolipids include glucose, galactose, mannose, fucose and the N-acetylated sugars like N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid (sialic acid). Membrane sugars seem to be involved in identification and recognition.

Transmembrane proteins(1) single-pass (2) multiple-passTrans-membrane proteins have membrane spanning portions containing alpha helically arranged sequences of 20-25 hydrophobic amino acids. Short strings of hydrophilic amino acids separate the hydrophobic sequences from each other:These hydrophilic stretches tend to be found exposed to the more aqueous environments associated with the cytoplasm or the extra cellular space.

Aliphatic side-chains.Residues with aliphatic side-chains (leucine, isoleucine, alanine, valine) predominate in the middle of the bilayer.

Polar amino acids in membranes.Tyrosine and tryptophan are common near the membrane surface. Lysine and arginine are often at the lipid/water interface, with the positively charged groups at the ends of their aliphatic side chains extending toward the polar membrane surface.

Lipid anchorLipid anchor: Some proteins bind to membranes via a covalently attached lipid anchor, that inserts into the bilayer The attached lipid may be a fatty acid such as palmitate or myristate. Palmitate is usually attached via an ester linkage to the thiol of a cysteine residue. A protein may be released from the plasma membrane to the cytosol via depalmitoylation, hydrolysis of the ester linkage. An isoprenoid, such as a farnesyl residue, is attached to some proteins via a thioether linkage to a cysteine thiol. Fatty acid or isoprenoid chains link proteins to the cytosolic surface of the plasma membrane

Lipid anchors.

Membrane structure

The amphipathic properties of the phosphoglycerides and sphingolipids These charges are responsible for the hydrophilicity. The long hydrocarbon chains of the acyl groups are hydrophobicPhospholipids in an aqueous medium spontaneously aggregate into orderly arrays. MicellesLipid bilayersLiposomes are structures related to micelles, but they are bilayers,.The properties of phospholipids determine the kinds of movement they can undergo in a bilayer. Rotation ,Lateral diffusion ,flexing of the acyl chains,Transverse movement from side to side of the bilayer (flip-flop).

The fluid mosaic model. A lipid bilayer composed of phospholipid and cholesterol Proteins. Integral proteinss.Peripheral proteins are loosely attached to the surface.Membrane surfaces have asymmetry There are differences in lipid/protein composition between the sides of a membrane. Different catalytic proteins (enzymes) appear on the two sides of membranes. Carbohydrate is mostly on the outer surface.egRBCs Some proteins (ankyrin, spectrin) are associated with the inner surface of the membrane. Other proteins transfix the membrane (glycophorin), or loop back and forth from side to side

Passive (simple) diffusion.- Water and small lipophilic organic compounds can cross. Large molecules (e.g. proteins) and charged compounds do not cross.Movement is DOWN the concentration gradient ONLY Rate of diffusion depends on charge on the molecule -- electric charge prevents movement. size -- smaller molecules move faster than larger molecules. lipid solubility -- more highly lipid-soluble molecules move faster. the concentration gradient -- the greater the concentration difference across the membrane, the faster the diffusion.Molecules may cross the membrane in either direction, depending only on the direction of the gradient.

Passive (simple) diffusion..

Protein channelsProtein channels transport specific ions. Ion channels exist for Na+, K+ and Ca++ movement. These channels are specific for a given ionic species. Channels consist of protein, which forms a gate that opens and closes under the control of ligand or membrane potential. Ion movement is always down the concentration gradient.

Membranes carriers (mediated transport).

A carrier must be able to : Recognition its ligand Translocation Release -- on the other side of the membrane Recovery -- return of the carrier to its original condition. Carriers resemble enzymes in some of their properties. They are NOT enzymes, They are enzyme-like in the following ways: They are specific. Their dissociation constants are analogous to Km of enzymes. Inhibited by specific inhibitors. They exhibit saturation. Nomenclature: Uniport:,Symport,: Antiport.

Passive mediated transport( facilitated diffusion).The characteristics:. Faster than simple diffusion Movement is down the concentration gradient only No energy input is required. The carrier exhibits specificity, saturation kinetics ,specific inhibitabilityExamples : Glucose transport in many cells. A uniport system Adding a substance analogous to glucose can inhibit glucose transport specifically. It is specific for glucose. The Km for glucose is 6.2 mM Involves attachment of glucose outside the cell. Conformational change of the carrier protein. Release of the glucose inside the cell. Chloride-bicarbonate transport in the erythrocyte membrane. An antiport system: both ions must move in opposite directions simultaneously. The system is reversible, and can work in either direction. Movement is driven by the concentration gradient

Passive mediated transport(facilitated diffusion)..

Active mediated transportIs against a concentration gradient, and requires energy. There are two sources of energy . ATP hydrolysis may be used directly. The energy of the Na+ gradient may be used in a symport mechanism. The energy of the Na+ going down its gradient drives the movement of the other substance. But since the Na+ gradient is maintained by ATP hydrolysis, ATP is the indirect source of energy for this process.A carrier operating by active transport. Can move substances against a concentration gradient. Requires energy. Is unidirectional Exhibits :specificity, saturation kinetics and specific inhibitability.Release from the carrier into a higher concentration is explained by:The affinity of the translocase for the substance must decrease. (conformational change?). Require energy in the form of ATP.

Active mediated transport.

Ca++ transport is a uniport system, using ATP. There are two Ca++ translocases of importance. In the sarcoplasmic reticulum, important in muscle contraction. A different enzyme with similar activity in the plasma membrane.The Na+-K+ pump (or Na+-K+ ATPase). An antiport system. Importance: To maintain the Na+ and K+ gradients. Stoichiometry: 3 Na+and 2 K+ in/per one ATP hydrolyzed. Specificity: Specific for Na+, but it can substitute for the K+. The structure of the Na+-K+ pump is a tetramer of two types of subunits, 22. The Na+-K+ ATPase is specifically inhibited by the ouabain.

Proposed mechanism of the Na+/K+ ATPase Na+ attaches on the inside of the cell membrane. The protein conformation changes due to phosphorylation of the protein by ATP, and the affinity of the protein for Na+ decreases. Na+ leaves. K+ from the outside binds. K+ dephosphorylates the enzyme. The conformation now returns to the original state. K+ now dissociates.

Na+ linked glucose transportNa+ linked glucose transport is found in intestinal mucosal cells. It is a symport system; glucose is transported against its gradient by Na+ flowing down its gradient. Both are transported into the cell from the intestinal lumen. Na+ is required; one Na+ is carried with each glucose. The Na+ gradient is essential; it is maintained by the Na+-K+ ATPase.

Membrane receptors

Cell-cell communication is by chemical messenger. There are four types of signals. Nerve transmission Hormone release Muscle contraction Growth stimulationThere are four types of messenger molecules:i)steroids ii)small organic molecules iii)peptides iv) proteinsThe messenger interact with the cell by either: By diffusion through the cell membrane (e.g.. steroid hormones). Binding to a receptor on the plasma membrane.

Signaling.

Some receptors involve second messengersFormation of an intracellular molecule called a second messenger. Second messenger formation is a means of amplifying the original signal. The formation and removal of the second messenger can be controlled and modulated.Cyclic AMP (cAMP) is a second messenger that mediates many cellular responses. The mechanism of action of cAMP is to activate an inactive protein kinase A. This process is an amplification of the original signal. Since the protein kinase is activated by cAMP it is called protein kinase A.cAMP is synthesized by the enzyme, adenyl cyclase.

Inositol triphosphate (IP3) and diacylglycerol (DG)

IP3 and DG are synthesized by , phospholipase C, which has phosphatidylinositol 4,5-bisphosphate (PIP2) phosphodiesterase activity.. The phosphodiesterase is controlled by a G-protein in the membrane.Mechanism: IP3 and DG have separate effects. IP3 releases Ca++ from the endoplasmic reticulum. The Ca++ then activates certain intracellular protein kinases. DG activates protein kinase c, Note that both IP3 and DG activate protein kinases, which in turn phosphorylate and affect the activities of other proteins.Termination of the signal occurs at several levels. IP3 is hydrolyzed. Ca++ is returned to the endoplasmic reticulum or pumped out of the cell. The GTPase activity of the G-protein hydrolyses the GTP, terminating the activity of the phospholipase C.

Insulin (IR) and growth factor receptors.

IR a tetramer with two kinds of subunits, alpha and beta. Many of the cellular responses are well known, e.g. Glucose transport and protein phosphorylation Insulin and many growth factors activate a protein kinase which phosphorylates a tyrosyl residue in the target proteins, including the receptor itself. The phosphorylation of tyrosyl residues is unusual; usually seryl or threonyl residues become phosphorylated.

Termination of the insulin signals involves internalization and degradation .The receptor-insulin complex migrates to a region of the plasma membrane with the protein clathrin coating its inner surface. This region forms a "coated pit," a region that invaginates and pinches off, forming an intracellular "coated vesicle."

Second messengers.

Adenyl cyclase is controlled by Gs and Gi. The action of the G-proteins. Structure: G-proteins are complexes of three different subunits, alpha, beta and gamma. Mechanism: Receptor-messenger interaction stimulates binding of GTP to the alpha-subunits. The alpha-subunit with its bound GTP then dissociates from the beta-gamma complex. The alpha-subunit with its bound GTP then acts on adenyl cyclase. alphas-GTP stimulates adenyl cyclase. alphai-GTP inhibits adenyl cyclase.Termination of the signal occurs at several levels. The alpha-subunit of the G-protein has GTPase activity. cAMP already formed is cleaved by cAMP phosphodiesterase. The hormone gradually and spontaneously dissociates from the receptor.cAMP is degraded by cAMP phosphodiesterase. cAMP + H2O -> AMP

Disease process and membrane receptorsHIV and CD4 receptors/CCRX co receptorsCholera toxin and GM1 receptor.The adenosine diphosphate (ADP)-ribosyltransferases bind nicotinamide adenine dinucleotide (NAD), and catalyze the transfer of the ADP-rebose moiety to an acceptor nucleophile.

Cholera toxinCholera toxin catalyzes covalent modification of Gs. ADP-ribose is transferred from NAD+ to an arginine residue at the GTPase active site of Gs. This ADP-ribosylation prevents Gs from hydrolyzing GTP. Thus Gs becomes permanently activated. Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of Gi, making the inhibitory G incapable of exchanging GDP for GTP. Thus the inhibitory pathway is blocked. ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.

Mechanisms of ADP Ribosylation

Cholera toxin (CT)ADP ribosylated G protein loses GTPase activity and perpetually activate adenylate cyclaseCholera toxin has ADP ribosylating activityCT has A and B fragmentsThe B fragment is responsible for GM1receptor recognition and translocation of the A-fragmentA is the catalytic domain.The A1 fragment of cholera toxin (CTA), catalyze the transfer of ADP-ribose to arginine-201 of the -subunit of the adenylate cyclase regulatory protein Gs.GTPase action of G protein is lostc-AMP accumulates, triggering a signaling cascade which eventually culminates in massive diarrhea

Glucose 6-Phosphate Dehydrogenase Deficiency

It is the most common genetic alteration in the world (>400 million people). There are varying degrees of this deficiency (depending on the mutation that occurs in the gene for G6P DH) The most severe are eliminated early in development, however, the majority are phenotypically silent. Even the latter can develop a phenotype under conditions that stress the NADPH levels (i.e. oxidative stress); these include:

Glucose 6-Phosphate Dehydrogenase DeficiencyMetabolic stress can be triggered by: Sulfur DrugsMalaria DrugsInfection with some viruses and bacteriaFava BeansHemolytic Anemia develops if oxidative species, like H2O2, cannot be properly detoxified by RBCs

G6PD.

G6PD.

Oxidation and reduction of Glutathione H2O2 2 H2O Glutathione Peroxidase 2 GSH GS-SG Glutathione Reductase NADP+ NADPH