Cell Molecular Bil
Cell Molecular Bil BIL 255
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This 27 page Class Notes was uploaded by Quinten Beatty on Thursday September 17, 2015. The Class Notes belongs to BIL 255 at University of Miami taught by C. Mallery in Fall. Since its upload, it has received 8 views. For similar materials see /class/205741/bil-255-university-of-miami in Biology at University of Miami.
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Date Created: 09/17/15
Bil 255 Spring Semester electron transport chain electron transport chain 255 Spring Mallery Mitochondrial Membrane Transport membrane impermeant to most everything esp to H wig outer membrane porins molecules 5000 10000d m b ATP synthase 3 1 I c carrier proteins phosphate translocases 39 z I ADPATP translocases pyruvatelH symporter 1316 d glycerolP amp malate shuttles inner membrane 70 protein amp 30 lipid holds a redox proteins of ETC mtDNA 16500 np39s code for 20 mito proteins including cytooxidase subunits I II III ATP synthase units 6 amp 8 5 subunits of NADH dehydrogenase and 22 tRNA39s and 2 rRNA39s electron transport chain 255 Spring Mallery 2 mitoDNA 16500 np39s codes for 20 of mitochondrial proteins 13 cytooxidase subunits I II 111 ATP synthase units 6 amp 8 5 subunits of NADH dehydrogenase and 22 tRNA39s and 2rRNA39s including 100039s copies per cell maternally inherited frequent point mutations sequence analysis mtDNA amp Human Evolution genetic variation among peoples forensic uses of Mito DNA mitochondrial diseases electron transport chain 255 Spring Mallery How Electron Transfer Works REDOX POTENTIAL how measured panel 131 empirical measure of tendency to gain e39s strong reducing agent has negative AEo39 strong oxidizing agent has positive A Eo AGo39 anEo NADH ltgt NAD H 2e39 O32V H20 ltgt 02 2H 2e 082V AGo39 10023 114 262 Kcal Electron Transfer Chain39s Order gt Increasing Redox Potential from to see fig 1321 p426 electron transport chain 255 Spring Mallery 4 Components of the ETC Pyridine nucleotides NAD 328 enzyme bound hydrogen carriers accepts 2e39s andor protons shows spectral shift 340nm Flavoproteins FMN amp FAD 412b protein bound hydrogen carriers spectral shift 340 370 amp 460 nm Iron sulfur proteins FeS 1322 p426 nonheme iron electron carriers Ubiquinone CoQ 1320 p425 semiquinone amp hydroquinone mobile membrane bound nonprotein hydrogen carriers Cytochromes a a3 b562 b566 c1 c 1323 p427 amp above colored proteins with bound Fe atoms ferrric vs ferrous iron porphyrin heme bound protein carriers How Oxidative Phosphorylotion Works fig 1313 p417 electron transport chain 255 Spring Mallery 5 Respiratory Assemblies Mitochondrial Components Respiratory Assemblies lNTERMENBEANE Culnchmmz c O NADHQ red uctase 5 1 o Succinate dehydrogenase m a o CytochromeCReductase o Cytochrome Oxidase ATR K mun SPACE 2H HI202 ATP Synthase creates a hydrophilic channel for H flow makes 100 ATP per 300 H per sec m ADP Pi gt ATP Fo membrane piece amp stalk F1 soluble piece 5 proteins rotational models electron transport chain 255 Spring Mallery 6 Oxidative Phosphogxlation Making of ATP Synthesis of ATP made via a proton motive gradient generated by transfer of e39s to O2 to make H20 through series of redox proteins Mechanism Chemiosmotic Coupling Mitchell 1961 fundamental mechanism arose early in evolution was retained 3 steps fig 139 v ETC passage of 6 thru membrane carrier proteins electron flow hydride ion H gt H 2e v generates a proton motive force g adient pH difference pH 10 units 80 matrix vs 0 perimito space amp a membrane potential charge 140mV in out v ATP Synthase which links ADP amp P making ATP uncouplers as DNP destroy H gradient no ATP electron transport chain 255 Spring Mallery 7 ATP Synthase Structure 39mushroom39 shaped complex composed of 2 membrane subunits F1 extrinsic amp F0 intrinsic Humbeto Fernandez 6039s sees lollipops on inner mito membranes Efraim Racker 1966 isolates lollipop Coupling Factor 1 F1 ATP synthase of liver mitochondria about 15000 present F1 5 polypeptides nuclear DNA 3a331y16amp1 arranged like sections of grapefruit 3 catalytic sites for ATP synthesis one on each 3 subunit F0 3 polypeptides in ratio of 1a 2b and 12c Cring electron transport chain 255 Spring Mallery 8 Binding Charge Mechanism of ATP Synthesis A Rotary Motor Paul Boyer 1979 1 H movement changes binding affinity of synthases39s active site thus when ADP amp P bind to active site they readily condense into ATP removed from aqueous solution Keq 1 and AG close to zero thus ATP forms easily 2 active site 3 subunits changes conformation through 3 successive shapes LTO L loose ADP amp P loosely bound to site T tight ADP amp P tightly bound favoring condensation without water 0 open site has low affinity to bind ATP thus releases it 3 conformational changes result in rotation of subunits relative to central stalk y d amp 3 subunits of F1 form hexagonal ring that rotates around central axis y stalk extends from F0 amp interacts with 3 339s differently as it rotates 3600 electron transport chain 255 Spring Mallery 9 Pathway of the Protons through Fo rotational model of Cring amp y stalk 12 Cproteins reside in lipid bilayer Cring Cring is attached to v stalk of F1 H diffuse through Fo rotating the 12c39s of F0 ring each C protein has a halfchannel space with a charged ASP C39s bind H amp via shape changes Crotates 300 CCW next C in ring picks up H amp thus the ring cycles thru 3600 release of H into matrix happens at end of cycle Karp 529 4 H moves ring 1200 v stalk shifts 1200 gt 339s change 4 H result in ATP being made rotation of Cring drives v stalk through 3600 amp 3 conformations of F1 LTO to make ATP electron transport chain 255 Spring Mallery 10 design of metabolism Bil 255 The Design of Metabolism Biological Order and Cell Energy Transformations 39 CELLS Do OBEY LAWS of CHEMISTRY amp PHYSICS B ells possess Potential Energy by having different bonds 2 kinds oftraditional energy 1 Potential Energy stored energy due to mass in position 2 Kinetic Energy energy of movemen ex heat thermal energy which flows from higher heat the design Of metabolism orgreater molecular motionto lower heat content radiant energy kinetic energy of photons light hen molecules absorb light radiant gt thermal chlorophyll lightgt ATP in photosynthesis mechanical energy pushpull of cytoskeletal filaments electrical energy energy of moving electrons deslgn ur metabullsm Mallery l deslgn ur metabullsm Mallery ENERGY in cells is housed THERMODYNAMICS SCIENCE of ENERGY TRANSFORMATIONS n a mOIeCUIes CHEMICAL BONDS 1st Law of Thermodynamics Energy can neither be created nor destroyed but is converti cells possess Chemical Potential Energy nuclear blast mass ofU235 gt heatlight all forms of energy are interconvertible amp hus all are expressed in same units of measure Joule but biologists use more common calorie calorie is heat A1gm1oC 1 Kcal 1000 cal 4184 Joule 1 cal 4184 J occurs in such forms as chemical concentrations gradients across membranes dl use from hlgher to lower 2nd Law of Thermodynamics ENTROPY referred to as a measure of degree of order of the Universe e electrical gradients potential differences is commonly s membranes amp thus its randomness Entropy disorder can only increa Ent is m ximum disorder quotheatquot a separatlon Of Charge Events in the Universe have a direction gt max entropy as much as 200000 volts per cm Mallery 4 deslgn ur metabullsm Mallery deslgn ur metabullsm Mallery Bil 255 Mallery design of metabolism The Rules of the Universe are simple Cities crumble Stars go Supernova ampwe are all equlibrium ing dying Yet WOW Cells are highly ordered wings ofa bird human eye spider s web and all cells feed grow and differentiate HOW in light of the 2nd law of thermodynamics FOOD light energy amp covalent bond energy Cell reactions mmmu may mmm cell wan mm ollvnl a HEAT overall increased entropy Entropy must increase heat yet disorder within one part of Universe can decrease at the greater expense of the Total Surroundings design afmetabalism Mallery 5 ENERGY IN gt CELL STRUCTURE gt ENERGY OUT What we need to be able to do is measure Energy in systems esp energy able to do wor Willard Gibbs ease190 applied the principles of Thermodynamics to chemical systems to determine the energy content and changes within a chemical reaction and derived the FREE ENERGY EQUATIONS A6 AH A5 free energy enthalpy entropy AG is a numa ical measure of how far a reaction is from equilibrium AG is measure amount energy in system able to do work stay away from equilibrium Disorder increases thus entropy increases when useful energy that which could be used to do work is dissipated as heat biological systems are are ISOTHERMAL eg held at constant temppressure 4 to a 45 and thus AH 0 design at metabolism Mallery a What Gibbs showed was that quotcell chemical systems change so that Free Energy is minimized thus DG can PREDICT the Direction of Cellular Reactions TOWARD EQUILIBRIUM and to Maximum ENTROPY Any natural process occurs spontaneously if and only if the associated change in G for the system is negative AG lt 0 when DG is negative a reaction is spontaneous R gt P amp there is a decrease in entropy Likewise a system reaches equilibrium when the associated change in G r the system is zero AG zero amp no spontaneous process will occur ifthe change in G is positive As gt 0 design at metabolism Mallery CHEMICAL REACTION A ltgt B Which Way J Willard Gibbs 18391903 AG AGO RTlnpr change in 39ee energy content ofa reactiondepends upon 1 energy is stored in molecule39s covalent bonds 2 remember temperature is negligible cells are isothermal ie AG actual free energy AGo39 standard free energy change under std conditions R gas onstant 2 x 103 Kdmol T absolute temp 2730K In natural log conversion log 2303 at equilibrium AG 0 and plr Keq ifwe solve above equation for AGo39 we can see relationship of Keq to AGO design at metabolism Mallery a Bil 255 design of metabolism Free Energy Equation AB AGO RT In P The difference between AG and AGO R equilibrium AB 0 N30 i5 aflxedvaiuefor a given reaction amp indicates i h which direction that reaction Wiii proceed under stah dard con dltlorl Y standard condltl 0 he i Within a c ll thusAe cab be quot S 39earrmg39w AGO 39 39 RT 39quot le il used to predict the direction of a reaction at a given equilibrium JEL Keq R1 determined by the concentrations present at that time amp AG i5 is a m asure of howfar a reaction is from equii brium then 2500 RT In Keq 20 293 2303 g10 Keq thus he of reactantsamp roducts to favorthe r0 ress ofurlfavored reactions AGO 1364 lg10 Keq p p g desigmmaaaaim Mailerv design Mmetahailw Maiierv an a mquot ivatm 39 t Z Z to ca which way this reaction goes aso39 cell equilibrium 135039 1364 Igm224 is dependent upon existing concentrations R gt Pi Kgq DHAPGJP 224 1354 lgn 135 142 colmole mow RT nPR but when DHAP 0001M a 63 01M AB 1a42cni 1354lgn 001 1a4213532 was cni Thus under standard condition the from 53 toward DHAP but under specific cellular condition where the ratio of reactant pro uc s is cla the reaction isn39t favored reaction is favored A P s R nse 61 goes in other direction from DHAP to 63 This is what happens in glxcolEisi39 but the pathway shirts ratios and pulls it to eat it design at metahailw design Mmetahailw Maiierv MaiieN 2 Mallery Bil 255 design of metabolism CHEMICAL REACTIONS A ltgt B which way amp Why EXERGONIC REAC110N is one which releases free e Product B ltltlt ex 39 rgy Reactant A burning wood cellulose glucose monomers potential energy breaks bonds relea se heat amp light gt C02 amp H20 nergy stored in covalent bonds How does Metabolism create more order in chemical reactions COUPLED REACTION via ATP hydrolysis if AG for the reaction cell respiration h slower m l C gt D is but less than the DG of ATP hydr sis eterotrophy cellular burning of glucose u tistep process to capture amp release e oly then the reaction can be driven to completion by coupling ATP hydrolysis energy can be coupled to conformational changes in enzyme ENDERGONIC REACTION requires input of energy for A gt B as kinasesi which PhDSPhD Product B gtgtgte Reactant A 39 photosynthesis au glucose made 39 rylat roteins converting then from i totrophy om coZ HZO lightgt cano6 energy poor energy rich design er metabolism e nactive to active amp vice versa energy gained in the stressed conformation is released when he protein relaxes Mallery design er metabolism Mallery M Design of Metabolism Design of Metabolism 2 Categories of metabolic reactions Catabolic cell respiration in heterotrophs nzme catal ed metabolic pathways g 32 Anabolic biosynthesis in autotrophs oxidation removal of e s from foodstuffs 3 steps 1 Digestion of polymers foods into monomers 2 GLYCOLYSIS gt AcoA splits sugar monomers coupling reactions that are energetically unfavorable 3 Oxidation of AcoA gt co 2 NADH gt H 20 with reactions that are energetically favo D P gt ATP done by linking hydrol is of ATP favored to reactions linking atoms together not favored creating new biological order FREE ENERGY EQUAT39ONS AG Aquot 39 TAS a numerical measure of how far a reaction is from equilibrium design er metabolism Mallery is design er metabolism Mallery Mallery Bil 255 Mallery Design Of Metabolism or how biological order comes about Organisms are classi ed by the nutritional habits Autotroghs light energy is convened into covalent chemical bond energy e COZ oxidized form H20 4 V NADPH ATP A H V CHZO reduced form Heterotroghs food stuffs more energetically stable CHZOn NAD 4 gtC02 HZO ATP NADH Key Cell energy intermediates NADH amp NADPH FAD amp ATPquot design of metabolism Malay 17 design of metabolism Design of metabolism OXIDATION I REDUCTION Redox Reactions e amplor H transferred between oxidized amp reduced forms AH A e39 H oxidation removal of e from substrate reduction gaining of e amp o en a proton H fig 312 NAD respiration NADH so2 c6H1zo6 4 sco2 GHZO NADP photosynthesis NADPH g 310 design a metabolism Malay i8 KEY METABOLIC REACTIONS 6 major categories of biochemical reactivity Biochemical reactivityis bond breaking amp reforming these are 39 39 39 39 cells 1functional group transfers guATP ltgt GGP ADP 2 redox reaction oxidlreduction PGAId NAD ltgt 13diPGA NADH 3 rearrangement isomerizations glucose6P ltgt fructose6P 4 CC breaking or reformation fruc16bP ltgt DHAP 3PGAId 5 Condensations proteinnaa1 ltgt proteinn1 H20 6 Hydrolysis gluglun HZO ltgt quglun1 design ufmetabuiism Malay iB BIL 255 CMB Methods Protocols amp Instrumentation in Cell amp Molecular Biology Mallery Methodologies in CMB Methodologies Techniques amp Procedures used in Cell amp Molecular Biology Cell Biology Dictionarii A Table of Glossaries Glossgrv of Techniques Etiongl Humgn Genome GlossajL Genergl Procedures amp Protocols Cell Bio Genergl Procedures amp Protocols Moleculgr Bioloqv mcb5e pages 184193 amp 165173 Mallery Methodologies in CMB 2 Early Approaches in CMB 1910 to 1960 Equipment advances of last 50 years are epitome of modern scientificage Light Microscopy 02 um My type 5 mcb 542 1876 Abbe optimizes microscope designs lens amp condensers 18862eiss lens resolution near limits of light 190039s embedding amp sectioning microtome 1 to 10 um thin tissue sections selective staining stains attach to specific molecules picture 1924Lacassagne autoradiography preparation tracking images definitions amp procedures 1941 Coons fluorescence microscopv fluorescent tagged antibodies amp Green Fluorescent Protein gene from jellyfish protein allows dynamic tagging Mallery Methodologies in CMB 3 Electron Microscopy 20nm mcb 550 1931 Ruska 1st Transmission Electron Microscope TEM 1952 Palade Porter EM stains for ultrastructure fig microtome 1957 Robertson unit membrane hypothesis 1964 Muhlethaler develops freeze fracture EM preparation amp coating 1965 Charles Oatley 1st Scanning EM Stereoscan 1974 Nobel Prize to G Palade C deDuve A Claude for their quotinner workings of cellsquot Mallery Methodologies in CMB Investigations of Cells the Results of Microscopy major EUKARYOTIC ORGANELLES microscopy has used fixed sectioned cells which are static mcb522a divide organelles by presence or absence of membranes Single Membrane Bound Organelles membrane bound vesicles of ectracellular milieu internalized by ENDOCYTOSIS a endocytosis cathrin protein quotcoatedquot membrane pits pinch of endosome vesicles 1 endosomes b phagocytosis whole cells engulfed amp passed to lysosomes for digestion c autophagy wornout organelles fuse with lysosome mcb520a amp endosomes amp lysosomes Mallery Methodologies in CMB 2 lysosomes several hundred single membrane bound vesicles exclusive to animals plants use vacuoles have acid pH environment to help denature proteins HATPases amp Cl transporters gt HCI contains hydrolytic enzymes nucleases proteases phosphatases glycosylases cytosolic amp nuclear proteins are not digested within lysosomes but rather proteasome TaySachs tt defective lysosomal enzyme degrades ganglosides glysolipids buildup in neurons z dementia blindness and death 3 plant vacuole membrane limited interior space up to 80 cell volume containing membrane transporters that accumulate ions nutrients amp wastes mcb524 lumen holds digestive enzymes acid pH optima tonoplast membrane permeable to water influx helps establish turgor pressure 520 ATM Mallery Methodologies in CMB 6 4 peroxisomes spherical 0210 um organelle containing oxidases catalase that use 02 to oxidize removes e39s from molecules as H202 amp other toxins degrade FA39s to acetyl groups used to make cholesterols esp impt in liverkidney cells Xlinked adrenoleukpdystrophy ADL no FA digestion occurs leads to several neurolinked defects and death mcb521 plants contain glyoxysomes which oxidize lipids very similar to peroxisomes 5 endoplasmic reticulum network of closedflattened membrane sacks called cisternae found in al nucleated cells involved in proteinlipid biosynthesis 2 types SER smooth lacks ribosomes mcb522 makes FA amp lipds esp in hepatocytes detoxifies hydrophobic chemical including carcinogens amp pesticides RER rough membranes bound w ribosomes mcb521 makes plasma membrane proteins amp exportable proteins of ECM abundant in cells making antibody protein plasma cells pancreas digestive enzymes amp hormones Mallery Methodologies in CMB 7 6 Golgi Complex series of flattened membrane sacks cisternae that take up ER transport vesicles and process contents via glycosylation adding carbohydrate residues 3 divisions cis where ER vesicles enter mcb522b medial where modifications glycosylations occur trans vesicle packages amp budded off here for secretion mcb523 Mallery Methodologies in CMB Double Membrane Bound Organelles 7 nucleus synthesizes DNA rRNA tRNA primary transcript mRNA preccursor largest double membrane bound outer membrane contiguous with ER perinuclear space 2 5nm is contiguous with lumen of ER mcb519 contains pores of protein complexes mcb 1218 regulates nucleoplasmcytoplasm exchange via NLS of 7 aa sequence Cterminus prolyslyslysargysva nucleolus regions of rDNA that makes rRNA nucleoplasm 39cytoplasm39 of the nucleus heterochromatin condensed dark EM color inactive DNA mcb525 euchromatin noncondensed light EM color active DNA lamins fibrous proteins adjacent to inner nuclear membrane form frame for nuclear shape Mallery Methodologies in CMB 8 mitochondria conducts ATP production of cell via oxidative metabolism of glucose amp fatty acids outer membrane 5050 lipidprotein contains porin mcb514 transports most ligands lt 10K inner membrane 2080 lipidprotein strictly regulates most transport into mitoplasm cristae infoldings of inner membrane mcb526 pi 1 amp pi 2 chloroplast largest green plant cell organelle 0520 pm by 10 um double membranes with extensive inner membranelimited sacks called thylakoids mcb527 absorbs light energy via chlorophyllous pigments converts light energy into ATP amp NADPH chemiosmosis reduces CO2 into CHZO Mallery Methodologies in CMB Similarities of Mitochondria amp chloroplasts 1 make ATPNADPH via same mechanism chemiosmosis oxidative creation of H gradient coupled to ATP synthase 2 show mobility throughout cell 3 divide by fission independent of cell39s division 4 autonomously replicate their own DNA mito 16569 nucleotide pairs about 37 genes chlp 10fg or 120 genes highly supercoiled amp repetitiveup to 6 copies 5 both contain 70s bacterial size ribosomes 6 synthesize their own proteins on own protein synthesizing machinery Mallery Methodologies in CMB 11
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