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how can we test for the mobility of membrane proteins?
put antibodies on beads which will attach to protein of interest
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how can we test for the mobility of protein?
laser trap, immobile proteins will not be displaced b/c of force
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what is the movement of protein dependent on?
temperature
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how do we know that membrane proteins are mobile and can be fused?
using rhodamine (mouse) and fluorescein (human) labeled membrane protein
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what is used to measure lateral diffusion rates in membranes?
FRAP (flourescence recovery after photobleaching)
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order the permeability of molecules
small hydrophobic, small uncharged polar, larger uncharged polar, ions
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what does it mean that carriers have enzymatic qualities?
carrier proteins bind specific solutes, resulting in conformational changes
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what is electrochemical gradient?
- sum of electrical potential difference and solute concentration gradient
- sometimes used in transport
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what are the cellular processes driven by energy stored in ion gradients?
- chemiosmotic (uptake/efflux of nutrients, metabolites, salts)
- osmotic volume regulation (H2O follows ions)
- chemical (H+Na+ driven ATP synthesis
- cell homeostasis (pH regulation, efflux or sequestration of toxic solutes
- signal transduction (Ca entry, action potentials)
- mechanical (H+ driven flagellar rotation)
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what are the characteristics of transporters and active transport?
- like enzymes without modifying substrate--binds to itself (kinase use ATP and put phosphate on substrate)
- conformational change is essential
- three ways to transport (ATP driven, light driven, coupled transporters)
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what does the bacteriorhodopsin use as energy, substrate and distribution?
use light, H+ is the substrate, distributed in halobacteria
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what does the photoredox use?
- light as energy
- H+ substrate
- photosynethic organisms
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what does the electron transport chain NADH oxidase use?
H+, use redox potential, H+ substrate and distributed in mito and bacteria
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ion trasnporting decarboxylases use?
use decarboxylation, Na is the substrate found in bacteria
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H+ pyrophosphatases used how where ?
energy comes from pyrophosphate, H* in plants, fungi and bacteria
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ATPases use what?
universal distribution, various ions and solutes, uses ATP
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how is energy conserved in pumps?
in the form of transmembrane electrical or chemical gradient of the transported ion or solute. PE of these ion gradients drive energy requiring processes
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what is another name for pumps b/c of its ability to transduce electromagnetic or chemical energy directly into transmembrane concentration gradients?
primary active transporters?
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how does secondary transporters play a role in pumps?
use ion concentration created by pumps to drive other reactions
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what does bacteriorhodopsin do?
it is a 7 pass transmembrane proton which change in retinal shape. use light to pump protons out of cell which creates proton gradient for ATP synthase to make ATP
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describe the process of bacteriorhodopsin
- retinal in middle covalently attached to multipass protein
- when light passes through, retinal changes shape from trans to cis (isomerization), causes conformational change in protein
- Asp85 has low pKa (serves as proton acceptor) Asp96 provides proton (proton donor)
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what are the three types of transport ATPases?
- F-type (V-type)
- P-type
- ABC
- F and P type generate electrical or chemical gradients across membranes
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F-type distribution, substrate and function
- ATP synthesis or ATP driven H+ pumping
- in bacteria, chloroplast, inner mitochondria membrane, H+ substrate
- F1 is water soluble catalyzes ATP hydrolysis or synthesis
- F0 is embedded in membrane/passively conducts protons across bilayer
- stalk connects F1 to F0 to couple proton translocation to ATPsynthesis
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V-type distribution and functions
- ATP driven H+ pumping, H+ substrate, found in archaea, eukaryotic membranes, pumps protons into organelles
- lysosomes, maintain low pH in cell compartments
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where does ABC work, function substrate?
- ABC in plasma membrane, various solutes, solute transport, pump small molecules instead of ions
- Cl transport
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what is the role of P-type?
- cation pumping, Na K, Ca, in plasma membrane and ER
- related multi-pass transmembrane pumps, phosphorylate themselves during cycle
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what is unique about ATP powered pumps?
- move ions against gradient
- transmembrane proteins with ATP binding site on cytosolic side
- coupling ATP hydrolysis and transport (one proton is absorbed with light and one proton is transported into cell)
- ATP powered pumps generate ionic gradiens across cell membrane (cytosolic K is high and Na is low; Cl is low and Ca is very low)
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what are some examples of p type pumps?
- Ca in SR in muscle relaxation
- Na/K ATPase maintains intracellular Ka and K in cells
- H ATPases maintain acidity of lyosomes
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how do we determine the rotation of the subunits of ATPase?
- attach bead on isolated domain of ATpase, in presence of ATP, bead will rotate
- rotation of the gamma subunit insdie the alpha/beta subunits
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describe the process of the ATPase
- H+ energy is stored in the bond energy of ATP
- H+ move into rotor, protonate forms can attach and rotate motor exit (proton energy into mechanical rotational energy)
- rotational energy is transmitted to shaft attached to motor, goes into ATPase which catalyzes the formation of ATPase
- position of shaft influences the surrounding subunits (these changes drive synthesize of ATP from ADP)
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what drives the chemioosmotic cycle?
proton gradient which drives rotation
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what do P Type Na K ATPase uses to catalyze electrogenic antiport of K and Na?
ATP hydrolysis accounts for 30% of ATP hydrolysis in most cells (70% in brain)
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describe the process of Na/K pump
3Na binds, ATP binds, hydrolyzes, phosphorylation, Na released 2 K binds, which triggers release of phosphate
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what is the function of Na-K ATPase?
maintain gradient that encompases PE for work; ATP binding leads to release of K on cytoplasm and regeneration of E1 conformation
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what is the calcium P-Type intracellular pump composed of?
10 transmembrane alpha helices that regulates osmolarity and membrane potential
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what is the occluded state of Ca?
when Ca not accessible on either side of membrane to prevent protein leak
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