Bài giảng Biochemistry 2/e - Chapter 10: Membrane Transport

Outline 10.1 Passive Diffusion 10.2 Facilitated Diffusion 10.3 Active Transport 10.4 - 10.6 Transport Driven by ATP, light, etc. 10.7 Group Translocation 10.8 Specialized Membrane Pores 10.9 Ionophore Antibiotics

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Chapter 10Membrane Transportto accompanyBiochemistry, 2/ebyReginald Garrett and Charles GrishamAll rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 Outline10.1 Passive Diffusion 10.2 Facilitated Diffusion 10.3 Active Transport 10.4 - 10.6 Transport Driven by ATP, light, etc. 10.7 Group Translocation 10.8 Specialized Membrane Pores 10.9 Ionophore Antibiotics Passive DiffusionNo special proteins needed Transported species simply moves down its concentration gradient - from high [c] to low [c] Be able to use Eq. 10.1 and 10.2 High permeability coefficients usually mean that passive diffusion is not the whole story Facilitated DiffusionG negative, but proteins assist Solutes only move in the thermodynamically favored direction But proteins may "facilitate" transport, increasing the rates of transport Understand plots in Figure 10.3 Two important distinguising features: solute flows only in the favored direction transport displays saturation kinetics Active Transport SystemsEnergy input drives transport Some transport must occur such that solutes flow against thermodynamic potential Energy input drives transport Energy source and transport machinery are "coupled" Energy source may be ATP, light or a concentration gradient The Sodium Pumpaka Na,K-ATPase Large protein - 120 kD and 35 kD subunits Maintains intracellular Na low and K high Crucial for all organs, but especially for neural tissue and the brain ATP hydrolysis drives Na out and K in Alpha subunit has ten transmembrane helices with large cytoplasmic domain Na,K TransportATP hydrolysis occurs via an E-P intermediate Mechanism involves two enzyme conformations known as E1 and E2 Cardiac glycosides inhibit by binding to outside Na,K TransportHypertension involves apparent inhibition of sodium pump. Inhibition in cells lining blood Vessel walls results in Na,Ca accumulation Studies show this inhibitor to be ouabain! Calcium Transport in MuscleA process akin to Na,K transport Calcium levels in resting muscle cytoplasm are maintained low by Ca-ATPase - a Ca pump Calcium is pumped into the sarcoplasmic reticulum (SR) by a 110 kD protein that is very similar to the alpha subunit of Na,K-ATPase Aspartyl phosphate E-P intermediate is at Asp-351 and Ca-pump also fits the E1-E2 modelThe Gastric H,K-ATPaseThe enzyme that keeps the stomach at pH 0.8 The parietal cells of the gastric mucosa (lining of the stomach) have an internal pH of 7.4 H,K-ATPase pumps protons from these cells into the stomach to maintain a pH difference across a single plasma membrane of 6.6!The Gastric H,K-ATPaseThis is the largest concentration gradient across a membrane in eukaryotic organisms! H,K-ATPase is similar in many respects to Na,K-ATPase and Ca-ATPase Osteoclast Proton PumpsHow your body takes your bones apart! Bone material undergoes ongoing remodeling osteoclasts tear down bone tissue osteoblasts build it back up Osteoclasts function by secreting acid into the space between the osteoclast membrane and the bone surface - acid dissolves the Ca-phosphate matrix of the bone An ATP-driven proton pump in the membrane does this! The MDR ATPaseaka the P-glycoprotein Animal cells have a transport system that is designed to recognize foreign organic molecules This organic molecule pump recognizes a broad variety of molecules and transports them out of the cell using the hydrolytic energy of ATP The MDR ATPaseMDR ATPase is a member of a "superfamily" of genes/proteins that appear to have arisen as a "tandem repeat" MDR ATPase defeats efforts of chemotherapy Light-Driven H + TransportThe Bacteriorhodopsin story Halobacterium halobium, the salt-loving bacterium, carries out normal respiration if O2 and substrates are plentiful But when substrates are lacking, it can survive by using bacteriorhodopsin and halorhodopsin to capture light energy Purple patches of H. halobium are 75% bR and 25% lipid - a "2D crystal" of bR - ideal for structural studies BacteriorhodopsinProtein opsin and retinal chromophore Retinal is bound to opsin via a Schiff base link The Schiff base (at Lys-216) can be protonated, and this site is one of the sites that participate in H+ transport BacteriorhodopsinLys-216 is buried in the middle of the 7-TMS structure of bR, and retinal lies mostly parallel to the membrane and between the helices Light absorption converts all-trans retinal to 13-cis configuration - see Figure 10.22 BacteriorhodopsinThe protons visit the aspartates.... Asp-85 and Asp-96 lie on opposite sides of a membrane-spanning helix These remarkable aspartates have pKa values around 11! (Why?) Protons are driven from Asp-96 to the Schiff base at Lys-216 to Asp-85 and out of the cell HalorhodopsinHalorhodopsin transports Cl - instead of H + Halorhodopsin has Lys-242 Schiff base but no aspartates and no deprotonation of Schiff base during the transport cycle Secondary Active TransportTransport processes driven by ion gradients Many amino acids and sugars are accumulated by cells in transport processes driven by ion gradients Secondary Active TransportSymport - ion and the amino acid or sugar are transported in the same direction across the membrane Antiport - ion and transported species move in opposite directions Several examples are described in Table 10.2Group TranslocationThe phosphotransferase system (PTS) Discovered by Saul Roseman in 1964 Sugars are phosphorylated from PEP during transport into E. coli cells Four proteins required: EI, HPr, EII, and EIII Group TranslocationEI and HPr are universal and work for all sugars EII and EIII are specific for each sugar Mechanism involves transfer of P from PEP to EI and then to HPr and then to 2 sites on EIII and then finally phosphorylation of sugar PorinsFound both in Gram-negative bacteria and in mitochondrial outer membrane Porins are pore-forming proteins - 30-50 kD General or specific - exclusion limits 600-6000 Most arrange in membrane as trimers High homology between various porins Porin from Rhodobacter capsulatus has 16-stranded beta barrel that traverses the membrane to form the pore (with eyelet!)Why Beta Sheets?for membrane proteins?? Genetic economy Alpha helix requires 21-25 residues per transmembrane strand Beta-strand requires only 9-11 residues per transmembrane strand Thus, with beta strands , a given amount of genetic material can make a larger number of trans-membrane segmentsThe Pore-Forming ToxinsLethal molecules produced by many organismsThey insert themselves into the host cell plasma membraneThey kill by collapsing ion gradients, facilitating entry by toxic agents, or introducing a harmful catalytic activityColicinsProduced by E. coliInhibit growth of other bacteria (even other strains of E. coli)Single colicin molecule can kill a host!Three domains: translocation (T), receptor-binding (R), and channel-forming (C)Clues to Channel FormationC-domain: 10-helix bundle, with H8 and H9 forming a hydrophobic hairpinOther helices amphipathic (Fig. 10.30)H8 and H9 insert, with others splayed on the membrane surfaceA transmembrane potential causes the amphipathic helices to insert!Other Pore-Forming ToxinsDelta endotoxin also possesses a helix-bundle and may work the same wayThere are other mechanisms at work in other toxinsHemolysin from Staphylococcus aureus forms a symmetrical poreAerolysin may form a heptameric pore - with each monomer providing 3 beta strands to a membrane-spanning barrelAmphiphilic Helices form Transmembrane Ion ChannelsMany natural peptides form oligomeric transmembrane channelsThe peptides form amphiphilic -helicesAggregates of these helices form channels that have a hydrophobic surface and a polar centerMelittin (bee venom), magainins (frogs) and cecropin (from cecropia moths) are examplesAmphipathic HelicesMelittin - bee venom toxin - 26 residues Cecropin A - cecropia moths - 37 residuesMagainin 2 amide - frogs - 23 residues See Figure 10.35 to appreciate helical wheel presentation of the amphipathic helixThe Magainin PeptidesDiscovered by Michael ZasloffHe noticed that incisions on Xenopus laevis (African clawed frog) healed without infection, even in bacteria-filled aquarium waterHe deduced that the frogs produced a substance that protected them from infection!The CecropinsProduced by Hyalophora cecropia (the cecropia moth - see Figure 10.36)Induced when the moth is challenged by bacterial infectionsThese peptides are thought to form -helical aggregates in membranes, creating an ion channel in the center of the aggregateGap JunctionsVital connections for animal cells Provide metabolic connections Provide a means of chemical transfer Provide a means of communication Permit large number of cells to act in synchrony Gap JunctionsHexameric arrays of a single 32 kD protein Subunits are tilted with respect to central axis Pore in center can be opened or closed by the tilting of the subunits, e.g. as response to stress Ionophore AntibioticsMobile carrier or pore (channel) How to distinguish? Temperature! Pores will not be greatly affected by temperature, so transport rates are approximately constant over large temperature ranges Carriers depend on the fluidity of the membrane, so transport rates are highly sensitive to temperature, especially near the phase transition of the membrane lipids ValinomycinA classic mobile carrier A depsipeptide - a molecule with both peptide and ester bonds Valinomycin is a dodecadepsipeptide The structure places several carbonyl oxygens in the center of the ring structure Potassium and other ions coordinate the oxygens Valinomycin-potassium complex diffuses freely and rapid across membranesSelectivity of ValinomycinWhy? K + and Rb + bind tightly, but affinities for Na + and Li + are about a thousand-fold lower Radius of the ions is one consideration Hydration is another - see page 324 for solvation energies It "costs more" energetically to desolvate Na+ and Li+ than K+ GramicidinA classic channel ionophore Linear 15-residue peptide - alternating D & L Structure in organic solvents is double helical Structure in water is end-to-end helical dimer Unusual helix - 6.3 residues per turn with a central hole - 0.4 nm or 4 A diameter Ions migrate through the central pore