Ch 13 Text 2

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  1. ·         Cells must control their __ to facilitate specific processes, such as __ and __, and prevent __

    ·         These ionic gradients are generated by a specific transport system, an enzyme that is called the __
    • intracellular salt concentrations
    • signal transduction and action potential propagation
    • unfavorable interactions with high concentrations of ions such as Ca2+
    • Na+-K+ pump (ATPase)
  2. o   The __ provides the energy needed for the active transport of __ out of the cell and__ into the cell, generating the gradients. The reason it is an ATPase is because what? This ATPase requires__
    • hydrolysis of ATP by the pump
    • Na+
    •  K+
    • the hydrolysis of ATP takes place only when Na+ and K+ are present

  3. ·         The __ accompanying the transport of Na+ and K+ can be calculated.
    o   Under typical cellular conditions, the hydrolysis of a single ATP molecule per transport cycle provides __ to __.
    §  The active transport of Na+ and K+ is of great physiological significance
    ·         More than __ by a resting animal is used to pump these ions. 
    • change in free energy
    • sufficient free energy
    • drive the uphill transport of these ions
    • one-third of the ATP consumed
  4. ·         The Na+-K+ gradient in animal cells controls __, __, and drives the __.
    ·         There are other families of ion pumps, such as the __, which does what, and the __, the enzyme responsible for __
    • cell volume
    • renders neurons and muscle cells electrically excitable
    • active transport of sugars and amino acids

    • sarcoplasmic reticulum Ca2+ ATPase (or SERCA)
    • transports Ca2+ out of the cytoplasm and into the sarcoplasmic reticulum of muscle cells

    • gastric H+-K+ ATPase
    • pumping sufficient protons in the stomach to lower the pH to 1.0
  5. o   These enzymes are referred to as __ because they form a __; and, in this formation, a __ is linked to the __ in the ATPase to form __. 
    • P-type ATPases
    • key phosphorylated intermediate
    • phosphoryl group from ATP
    • side chain of a specific conserved aspartate residue
    • phosphorylaspartate
  6. ·         P-type ATPases couple __ and __ to do what?
    o   Each pump can exist in two conformational states, one with __and the other with __
    §  To pump ions in a single direction, the free energy of ATP hydrolysis must be coupled to the __
    • phosphorylation and conformational changes
    • pump calcium ions across membranes

    • ion-binding sites open to one side of the membrane 
    • ion-binding sites open to the other side
    • interconversion between these conformational states
  7. ·         Example using SERCA, which is 80% of the __ and plays role in __
    o   Muscle contraction is caused by __. To relax it means doing what?
    • sarcoplasmic reticulum membrane
    • relaxation of contracted muscle
    • rise in cytoplasmic calcium ion level
    • removing Ca2+ from the cytoplasm into the SR by SERCA
  8. Structure of SERCA
    - single 110 kd polypeptide with transmembrne domain of 10 alpha helices, which has binding sites for two calcium ions, which can corrdiate to seven oxygen atoms coming from a combo of side chain glutmate, aspartte, threonine, and asparagine residues, backbone carbonyl groups and water
  9. o   It also has a large cytoplasmic headpiece that has three domains. Explain them
    One (N) binds the ATP nucleotide

    another (P) accepts the phosphoryl group on a conserved aspartate residue

    the third (A) serves as an actuator, linking changes in the N and P domains to the transmembrane part of the enzyme
  10. o   When __ is not bound and a __ is present in the __, the __ and __ are __, and the __ __ relative to its position in SERCA with Ca2+ bound and without the phosphoryl analog

    • Ca2+
    • phosphorylaspartate analog
    • P domain
    • N and A domains
    • closed around the phosphorylaspartate analog
    • A domain
    • rotates substantially
  11. o   Also, the transmembrane part of the enzyme __ and the well-organized Ca2+-binding sites are __, making the sties now accessible from the __
    • rearranges substantially
    • disrupted
    • side of the membrane opposite the N, P, and A domains
  12. ·         Mechanism for Ca2+ pumping by SERCA
    Step 1
    o   The catalytic cycle begins with the enzyme in its unphosphorylated state with two calcium ions bound (E1). IT can exchange calcium ions only with calcium ions from the cytoplasmic side of the membrane
  13. ·         Mechanism for Ca2+ pumping by SERCA
    Step 2
    o   In the E1 conformation, the enzyme can bind ATP. The N, P, and A domains undergo substantial rearrangement as they close around the bound ATP, but there is no substantial conformational change in the transmembrane domain. The calcium ions are now trapped inside the enzyme
  14. ·         Mechanism for Ca2+ pumping by SERCA
    Step 3
    o The phosphoryl group is then transferred from ATP to Asp351
  15. ·         Mechanism for Ca2+ pumping by SERCA
    Step 4
    o   Upon ADP release, the enzyme again changes its overall conformation, including the membrane domain. This new conformation is referred to as E2 or E2-P (if phosphorylated). The process of interconverting the E1and E2 conformations is called eversion. 

    • §  In the E2-P conformation, the Ca2+-binding sites become disrupted and the calcium ions are released to the side of the membrane opposite that which they entered; ion transport has been achieved
  16. ·         Mechanism for Ca2+ pumping by SERCA
    Step 5
    o   The phosphorylaspartate residue is hydrolyzed to release inorganic phosphate
  17. ·         Mechanism for Ca2+ pumping by SERCA
    Step 6
    o   With the release of phosphate, the interactions stabilizing the E2 conformation are lost, and the enzyme reverts to the E1 conformation
  18. ·         Mechanism for Ca2+ pumping by SERCA
    Step 7
    o   The binding of two calcium ions form the cytoplasmic side of the membrane completes the cycle. 
  19. ·         This mechanism can apply to other P-type ATPases
    o   __ is an __, with an A subunit homologous to SERCA and an aspartate residue analogous to Asp 351. The B subunit doesn’t __
    • Na+-K+ ATPase
    • a2B2 tetramer
    • directly take part in ion transport
  20. ·         __ and __ are potent inhibitors of the __ derived from plant steroids; they are cardiotonic steroids and inhibit the __ when applied on the extracellular face of the membrane
    ·         __inhibiting the Na+-K+ pump leads to __, diminishing the gradient and resulting in __
    o   The increase in intracellular levels of Ca2+ enhances the ability of cardiac muscle to contract
    • Digitoxigenin and ouabain
    • Na+-K+ pump
    • dephosphorylation of the E2-P form of the ATPase 

    • Digitalis 
    • higher Na+ level in side cell
    • slower extrusion of Ca2+ by the sodium-calcium exchanger
  21. ·         The __ has 16 proteins—2 transport H+ ions, 2 transport Ca2+, 3 transport Na+, and 2 transport metals
    o   Five members participate in the __ and help maintain __ by __; they are “__”
    • P-type ATPase family
    • transport of phospholipids with amino acid head groups
    • membrane asymmetry
    • transporting lipids such as phosphatidylserine from the inner to the outer leaflet of bilayer membrane
    • flippases
  22. o   All members of this family use __ to drive __ by means of __, which are induced by the __ at an analogous __site in each protein
    • free energy of ATP hydrolysis
    • membrane transport
    • conformational changes
    • addition and removal of a phosphoryl group
    • aspartate
  23. ·         Another family of active-transport proteins are pumps from tumor cells, which have a membrane protein that acts as an ATP-dependent pump that extrudes a wide range of small molecules from cells that express it.
    o   The protein is called the __; and, it will do what?
    “multidrug-resistance (MDR) protein or P-glycoprotein

    pump the drug out of the cell before the drug can exert its effects
  24. ·         Structure of MDR protein: __
    o   ATP binding domains are __
    §  The ABC proteins are members of the __
    four domains—two membrane-spanning domains and two ATP-binding domains

    ATP binding cassettes (transporters with this domain are ABC transporters)

    P-loop NTPase superfamily
  25. ·         Structure of ABC protein (in Vibrio cholera) [MsbA]: __of 62-kd chains—the amino terminal half of each protein contains the __, and the carboxyl-terminal half contains the __
    o   Prokaryotic ABC proteins are made up of __ or as a __of __ and __.
    • dimer 
    • membrane-spanning domain
    • ATP-binding cassette
    • multiple subunits
    • heterotetramer 
    • two membrane-spanning domain subunits and two ATP binding-cassette subunits
  26. ·         A mechanism of active transport by these ABC transporters has been developed

    Step One
    o   The catalytic cycle begins with the transporter free of both ATP and substrate. The transporter can interconvert between closed and open forms
  27. ·         A mechanism of active transport by these ABC transporters has been developed

    Step Two
    o   Substrate enters the central cavity of the open form of the transporter from inside the cell--> binds--? conformational change in ATP binding cassettes--> increased ATP affinity
  28. ·         A mechanism of active transport by these ABC transporters has been developed

    Step Three
    o   ATP binds to the ATP-binding cassettes, changing their conformations so that the two domains interact strongly with one another
  29. ·         A mechanism of active transport by these ABC transporters has been developed

    Step Four
    o   The strong interaction between the ATP-binding cassettes induces a change in the relation between the two membrane-spanning domains, releasing the substrate to the outside of the cell
  30. ·         A mechanism of active transport by these ABC transporters has been developed

    Step Five
    o   The hydrolysis of ATP and the release of ADP and inorganic phosphate reset the transporter for another cycle
  31. ·         Eukaryotic ABC transporters do what?; prokaryotic ABC transporters do what?
    o   A specific binding protein acts in concert with the bacterial ABC transporter, doing what and what?
    o   These binding proteins are present in the __, the compartment between the two membranes that surround some bacterial cells
    • export molecules from inside the cell
    • import specific molecules from outside the cell
    • delivering the substrate to the transporter and stimulating ATP hydrolysis inside the cell
    • periplasm
  32. ·         ABC transporters use a different mechanism from the P-type ATPases to couple the ATP hydrolysis reaction to conformational changes
    o   Nonetheless, the net result is the same: the transporters are converted from one conformation capable of __ to another that __
    • binding substrate from one side of the membrane
    • releases the substrate on the other side
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Ch 13 Text 2
2014-11-08 18:43:11
Test Three
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