BIO 201 with Todd Hennessey Sixth Week of Notes
BIO 201 with Todd Hennessey Sixth Week of Notes BIO 201
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This 32 page Class Notes was uploaded by ChiWai Fan on Saturday March 5, 2016. The Class Notes belongs to BIO 201 at University at Buffalo taught by TODD HENNESSEY in Spring2015. Since its upload, it has received 101 views. For similar materials see CELL BIOLOGY in Biology at University at Buffalo.
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Date Created: 03/05/16
Cell Biology Feb 29 , Mar 2, 4 (All images taken from Todd Hennessey’s slides) Clarification: At 25 C: T = 273 + C = 298K R = 8.314 472 J K−1 mol−1 F = 96,485 coulombs/mol Plasma membrane receptors 1. A signal arrives at a target cell (neural transmitter, hormone, taste, smell) 2. The signal molecule binds to a receptor protein in the cell surface or inside the cytoplasm 3. Signal binding changes the 3D shape (conformation) of the receptor and exposes its active site 4. The activated receptor activates a signal transduction pathway to bring about cellular changes Short term changes: enzyme activation, cell movement Long term changes: altered DNA transcription Every DNA cells have the entire DNA needed to do anything that other cells do. (Making proper protein if ordered to do so) You can turn liver cell into brain cell by regulating gene transcription, they have the same DNA but it is different DNA that is being transcribed into liver or brain cell. General drug effects of antagonists (inhibitors) (left image) A. The substrate could be a ligand (anything that binds to receptor) B. The enzyme could be a receptor C. Inhibitors can be drugs that affect signal transduction D. Competitive inhibitor fits the right size but does not activate it, blocking it so another inhibitor can bind somewhere else (shown in red [D]) E. Inhibitors are antagonists (inhibitor-for slowing down) F. An agonist is another compound that can also activate a receptor An example of an antagonist (right image) The adenosine receptor in the brain inhibits arousal (inhibiting arousal=awake) Adenosine is the natural ligand Caffeine (competitive inhibitor) binds to the receptor but it doesn’t activate it because only its STRUCTURE fits, doesn’t mean it can sufficiently activate it Caffeine is an antagonist of the adenosine receptor GPCRs—proteins that bind GTP G protein-coupled receptors (GPCRs) are involved in many diseases, and are also the target of approximately 40% of all modern medicinal drugs. The 2012 Nobel Prize in Chemistry was awarded to Brian Kobilka and Robert Lefkowitz for their work that was "crucial for understanding how G-protein–coupled receptors function." G-protein coupled receptors (GPCRs) (3 phosphates is GTP, 2 phosphates is GDP) A. Ligand binds, causing a conformational change in the receptor B. GTP binds to activate a G-protein (can’t do this until ligand binds to receptor) C. The activated G-protein stimulates a response and then it is terminated by GTP hydrolysis 1. Hormone binding to the receptor activates the G protein. GTP replaces GDP 2. Part of the activated G protein activates an effector protein that causes changes in cell function 3. The GTP on the G protein is hydrolyzed to GDP. Signal transduction by GPCRs o Plasma membrane o Cytoplasm o Nucleus There’s up to thousands of different GPCRs in an organism doing different things Allow communication from the outside of cell to the inside of cell General GPCR pathways Metabotropic GPCRs communicates with others to function cAMP can cause many cellular responses depending on the cell If you have that receptor, then you can regulate that specific thing, if not, then it is probably not necessary for you. GPCR generally: short term changes in the cellular metabolism/ regulating gene transcription/ effecting membrane potential (there’s many different ways of doing it) Several important enzymes affected by GPCRs A. Phosphorylation and DE phosphorylation: using these to regulate activity of protein (more active, less active). You need kinase for phosphorylation (put phosphate on) for active enzyme. Protein phosphatase takes a phosphate off B. G-protein activation and inactivation: to activate it with receptor (ligand) binding, to inactivate it use GTPase. This is like a signal depending on you. C. cAMP production and breakdown: ATP can be turned into cAMP with Adenylyl cyclase then to AMP by Phosphodiesterase. (cells want real control; they want everything perfect cAMP—The enzyme that makes cAMP is called adenylyl cyclase The steady state swimming pool of life Keeping the level the same is steady state, not equilibrium Even with a little change, it makes everything dramatic Two ways to raise the water level: 1. Increase influx 2. Decrease efflux The amounts of things in cells are dependent upon the relative rates of synthesis and breakdown Rate of water-in equals rate of water-out. If you want less water inside, you either drain out water or stop adding in water. cAMP regulation: Two ways to increase cAMP: 1. Increase adenylate cyclase activity 2. Decrease phosphodiesterase activity Caffeine is also an inhibitor of phosphodiesterase. What does it do to [cAMP]? Excessive cAMP is bad! Some cells can’t tolerate it and it will die. Different cells want different amount of things. What can cAMP do? Effect ion channel activities, gene transcriptions and many many many more How do you smell? A. This is an example of signal transduction. B. Odorants cause electrophysiological changes in receptor cells in the nose C. GPCR coupled to an ion channel 1. Binding of an odorant to its receptor activates a G protein 2. The G protein activates the synthesis of cAMP 3. cAMP causes ion channels to open 4. changes in ion concentration inside the cell initiate a signal to a specific area of the brain One effect of changing Ca++ concentrations In this specific cell, you want perfect amount of Calcium 1. The receptor binds the hormone 2. The activated G protein subunit dissociates and activates phospholipase C. 3. The activated enzyme produces the second messenger and activates other protein and enzymes What is the effect? Activate PKC to phosphorylate other proteins Main point: communication from outside of cell to inside of cell What can internal Ca++ do if you’re changing it? 1. Ion channels 2. Gene transcription 3. And tons of others as long as the calcium amount is just right Acetylcholine receptor: a ligand-gated ion channel The change in permeability leads to depolarization of the muscle cell Receptor kinases Receptor tyrosine kinase (RTK) Ligand binding causes receptor dimerization because of conformational change. Soluble, intracellular receptors An example of a soluble receptor (regulated gene transcription if a protein got in) Summary so far: Extracellular signals can be transduced by receptors A. Plasma membrane receptors 1. GPCRs a. Metabotropic b. Ionotropic 2. Ligand-gated ion channels 3. Others (RTKs, etc.) B. Intracellular receptors C. Soluble cytoplasmic receptors What can they affect? A. Intracellular responses through cAMP, Ca++, phosphorylation, etc. B. Membrane potentials C. Gene transcription D. Other things we haven’t mentioned Mar 2, 2016 Receptors and Cancer Growth factors Not all mutations are “down” mutations. Some are “up” mutations Growth factors stimulate cell division Mutated oncogenes can cause unrestricted growth in the absence of growth factors (cancer) Signal transduction pathway of passing down messages Increase cell growth by decreasing cell breakdown We cannot generalize mutation: some are good, bad, increases, decreases A mutation is a change in the DNA sequence of a gene This is just an example. There are many kinds of genetic mutations Could a mutation cause a non-functional protein to become functional? YES. A mutation can keep on a inactivated protein A mutated receptor can be “on” in the absence of ligands A modified receptor Under normal circumstances membrane-bound receptors require the binding of their ligand to be in an activated state. In contrast, receptors encoded by some oncogenes do not require the regulatory step of ligand binding to be active. How could this happen? 1. Same conformational change by mutation and ligand binding to receptor 2. Ligand binds to receptor, it can activate then deactivate. Now it is a receptor that is ALWAYS ON. Now cell keeps divided and never stops. When A growth factor Receptor Protein Kinase This is a growth factor response Could a mutation cause activation of this pathway without a receptor? How? If theres many proteins in the pathway, and it is mutated, it can work without needing any of the required factors Signal Transduction and Cancer Normal cell: Stimulation of pathway (like a door bell) it goes on then off Cancer cell: it cannot be turned off Tumor suppressors Genes act like a Brake Pedal to suppress excessive cell growth Different tumor suppressors can turn of different regions neccessary Some receptors involved in cancers Role of ECM and Cell Adhesion in Cancer Cancer is the result of abnormal cell proliferation. The spread of a tumor to other parts of the body is called metastasis. Metastatic cells have special cell adhesion properties. They are: o Are less adhesive. o Are able to penetrate several barriers (vascular barriers). o Are able to invade normal tissues. During growth and development of a tumor there is loss of E-cadherin leading to less adhesion between tumor cells. Changes in the numbers and types of cell-adhesion molecules can promote metastasis. Cancer cells lose their adhesiveness To other tumor cells and pass across the basement membrane leading into a blood or lymph vessel where they can spread throughout the body. Various factors implicated in cancers Chapter 8: Energy don’t thnk of ATP for this class Three types of “energy”: 1. Potential energy. Stored energy 2. Kinetic energy. Energy used to do work 3. Entropy. Energy unavailable to do useful work due to loss of energy First law of thermodynamics: Energy cannot be created or destroyed but it can be converted from one form to another <know these Back to thermodynamics Change in energy can be measured in calories or joules. Terminology The total amount of energy added to the system is +ΔH The total amount of energy released by the system is –ΔH The free, usable energy released from the system is –ΔG The free energy which is required (consumed) is +ΔG Increase in disorder is +TΔS Decrease in disorder (increase in order) is -TΔS It takes energy to impose order on a system; we’re fighting against disorder Some more terms Exergonic: Reactions that release free energy due to –ΔG Ex.: Catabolism; lighting a match Endergonic: Reactions that require energy due to +ΔG Ex.: Anabolism have to be coupled by exergonic At equilibrium, ΔG = 0 dead cell and can’t be coupled However, –ΔG does not tell us anything about the rates of such reactions. Rate and delta G are different things Exergonic and Endergonic Reactions Transformation of energy Two characteristics of ATP account for the free energy released: Phosphate groups have a negative charge and repel each other—the energy needed to get them close enough to bond is stored in the P~O bond. The free energy of the P~O bond is much higher than the energy of the O—H bond that forms after hydrolysis. Overcoming the Activation Energy Initiates Reactions This needs two main things to occur: 1. -ΔG to make it thermodynamically possible 2. An enzyme to overcome the activation energy Enzymes Lower the Energy Barrier An enzyme is a catalyst that lowers the activation energy Ex. Mom is catalyst when she pulls you off from being a couch potato and she was not consumed...and she lowered the activation energy The final equilibrium does not change, and ΔG does not change. An uncatalyzed reaction has greater activation energy than does a catalyzed reaction There is no difference in free energy between catalyzed and uncatalyzed reactions What Are Enzymes? Catalysts speed up the rate of a reaction. The catalyst is not altered by the reactions. Some reactions are slow because of an energy barrier—the amount of energy required to start the reaction, called activation energy (Ea). Enzymes can lower the energy barrier by bringing the reactants together. Activation energy ____________________________________________________________________________________ Mar 4 Enzymes can lower the energy barrier by bringing the reactants together. The reaction must have negative delta G though Reactants are called substrates. Substrate molecules bind to the active site of the enzyme. The three-dimensional shape of the enzyme determines the specificity Taking a substrate apart Putting substrates together An example of an enzymatic activity Products are broken down and it became clear after adding lysozyme. In catalyzing a reaction, an enzyme may use one or more mechanisms: A. Orienting substrates B. Inducing strain in substrates C. Adding chemical groups—chemical alteration Enzymes can orient substrate molecules, bringing together the atoms that will bond. Two substrates are bound next to each other in the active site This proximity catalyzes the reaction Bring them together into the right orientation so they bind together which lowers activation energy Enzymes can induce strain in substrate molecules, making them unstable. Binding to the active site produces a strain on the substrate to help to break it down and we’re not using any ATP here with no energy added Induced fit: enzyme changes shape when it binds the substrate, which alters the shape of the active site. Ionic bonds, vanderwaals, hydrogen bonds between the substrate and enzyme can change the shape of active site. Enzymes can temporarily add chemical groups to substrates Electric charge transferred to the substrate The enzyme is changed during this reaction The enzyme modifies the substrate by adding charges Reaction rates Above represents one concentration of a substrate Will the slope vary with concentration of the substrate? Slope: initial velocity (initial rate) Rate of reaction: product formed over time Effects of Substrate Concentration on Reaction Rates More product formed over time Less product formed over time Higher Substrate Concentration Lower Substrate Concentration Higher rate Lower rate Reaction rate is dependent upon concentration Higher substrate concentration= higher rate How many pearl necklaces can be made in a factory per minute? If plenty of pearl, you can make products faster Workers (enzymes) are not used up in the process Effects of substrate concentration on reaction rates When you maxed out rate with max substrate concentrationsaturation Saturate: keep on adding but nothing more can happen The rate of a catalyzed reaction depends on substrate concentration. FALSE!!! Because it will eventually hit its maximum concentration causing saturation Correction: The rate of a catalyzed reaction depends on substrate concentration UNTIL it reaches the maximum rate At saturation, all enzymes are bound to substrate. It is working at maximum rate. Km and Vmax Since the reaction rate can saturate at high concentrations, saturation is Vmax (the maximum velocity or maximum reaction rate) Km is the concentration of substrate where the rate is ½ Vmax. So cell can decrease or increase it as it wishes In most cells, the substrate concentration in vivo is about at the Km. Why? Cells want to regulate its enzymatic activity by reversible inhibition Note: Reaction rates (initial velocity) tell you about the enzyme’s activity. It is product formed over time Example: umoles hydrolyzed/sec. Km is a concentration. They tell you about the effects of substrate concentrations on Reaction Rates Reversible and irreversible enzyme inhibition An example of Irreversible Inhibition Covalent bond formed Acetylcholine (ACh) is a neurotransmitter that triggers muscle contraction Acetylcholinesterase (AChE) breaks down Ach to regulate contraction Nerve gas inhibits AChE This causes loss of muscular control Death is caused by asphyxiation due to loss of control over respiratory muscles If you have irreversible inhibitor, it’s really hard to get rid of it Reversible Inhibition Usually non-covalent bonds (easy to reverse) The competitive inhibitor effect is concentration dependent. Competing for the same substrate depending on concentration. Allosteric Regulation of Enzymes (other site) An allosteric inhibitor binds to another site instead of the active site Other kinds of allosteric effectors can also be activators pH Affects Enzyme Activity How could pH affect enzymatic activity? Changing the pH could affect enzymes conformation by changing its charges Example: at low pH —COO– may react with H+ to form —COOH which is no longer charged. This could affect the conformation and enzyme function. Temperature Affects Enzyme Activity Different cells have different enzyme optimum. Think of fish in water and see urchin eggs. How to get to know an enzyme (protein-based), using what you know so far in BIO201B 1. What size is it? SDS-PAGE 2. Is it soluble or membrane bound? Centrifugation 3. Where is it in the cell? Fluorescent antibody 4. What does it do? What are its substrates, products and reaction rates? Use an appropriate enzyme assay (ADP over time) Chapter 9 (Friday ended here) Metabolism Anabolism: Synthesis Catabolism: Breakdown Energy derived from catabolism (like the breakdown of sugars) can be used to drive anabolism (like the synthesis of sugars) The Circle of Life From another book: “Solar energy is the ultimate source of all biological energy.” Chloroplasts use energy from light to make carbohydrates and generate O 2 Mitochondria produce water and CO from c2rbohydrates and O 2 Regulation of cellular biochemical pathways
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