Calcium Homeostasis

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ATPases

• toni scarpa
• physiology biophysics
• c.w.r.u.
• east 541
• axs15_at_po.cwru.edu

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General Resources

• Nicholls, D. and Ferguson, S. Bioenergetics, 2nd
  Edition, Academic Press, 1992
• Scarpa, A., Carafoli, E. and Papa, S., Ion Motive
  ATPases structure, function and regulation,
  Annals N.Y. Academy Sciences, Vol 671, 1992.

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Oxidation of Biological Molecules

• Body Content
• Aside for mineral and water our body is
  constituted by carbon molecules, not in stable
  form
• The energetically stable form of proteins,
  carbohydrates and fat is CO2 and H2O
• The process is not explosive and it is usually
  coupled to a reaction called oxidation
• Oxidation seldom requires movement of oxygen and
  it usually involves the removal of electrons (and
  vice versa in case of reduction)

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Biological Oxidation are Coupled to ATP Synthesis

• ATP common currency in the cell
• ATP is not transported in or out of the cell
• ATP is used for many cellular functions

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ATPase The Biochemical Reaction
ATPase

• The equilibrium constant is shifted to the right
  ADP and Pi 5 orders of magnitude gtATP
• The reaction is in principle reversible, but not
  physiologically because the conc of ATP
• Mg, cations are cofactors
• Cations, charge inside or outside could also be a
  substrate and a product of the reaction

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Why ATP

• Molecule with the right stability in water
• The inappropriate use of high energy bonds
• Energy is not confined to a single element within
  ATP molecule, but it is function of the
  displacement from equilibrium of the observed
  mass action ratio
• ADP, through an hypothetical ADPase, could
  provide the same energetics
• It is not the absolute concentration of ATP which
  is important for energetics but the ratio between
  ATP and ADP and Pi

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Standard Free Energy

• ?Go or Standard Gibbs Energy are a restatement of
  equilibrium constant and immutable for any
  coupled reaction
• ?Go -3.2 RT log10 K
• It reflects the displacement from equilibrium
  when each components of the reaction under
  standard conditions are kept at 1 M
• It gives no information on the energetics within
  the cell which depends on effective concentration
  ratios

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ATP Displacement from Equilibrium
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Displacement from Equilibrium and Gibson Energy
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Standard Free Energy of Phosphometabolites

• ATP ADP Pi - 7.3
  Kcal/mol
• ADP AMP Pi -7.3
• ATP AMP PPi -8.0
• AMP H2O Adenosine Pi -3.4
• PC Creatine Pi -10.3
• PA Arginine Pi -7.7

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Measurements of ATP

• NADH absorbance/fluorescence measurement of
  coupled reactions
• Luciferin-luciferase
• HPLC
• 31 P NMR

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Measurement of ATP and Posphometabolites
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HPLC of Phosphometabolites
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Metabolic NMR
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Labelled ATP
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ATP Buffers

• ATP conc within cells if fixed at 5 mM and
  that of ADP at 20 uM an remain unchanged
• Several ATP buffers in organs with high metabolic
  domands
• Those are trasiently active when metabolic demand
  for ATP exceeds max ATP synthesis within that
  cell
• PhosphoCreatine ADP Creatine ATP
• ADP ADP ATP AMP

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CellularP-metabolites
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Mitochondria as Amplifiers
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ATPases Significance

• As cells are impermeable to ATP, ATP is recycled
  within each cell.
• ATP synthesis and ATP hydrolysis are more coupled
  in some cells than others.
• A human body recycles at rest 50-100 pounds of
  ATP/day and hundred during work
• Over 50 of all food ingested and oxygen inspired
  fuels ion motive ATPases

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Classes of ATPases

• P class
• Are autophosphorylated during catalysis on an
  aspartate within the ATPase
• Examples Na-K ATPase, Ca ATPases
• Located on plasma membranes or organelles
• V class
• Located in secretory vesicles
• Transport H inside, multimeric
• F class
• Located in mitochondria and chloroplasts
• Not facing cytosol and high ATP
• Multimeric, physiologically ATP is synthesised

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ATPaseshere,there,everywhere
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ATPase Inhibitors

• P Class
• all, vanadate, non-specific
• Na-K, ouabain (digitalis)
• Ca, thapsigargin
• H-K, omoprazole
• V class
• H, DCCD, non-specific
• F class
• H ,oligomycin

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Common Characteristics

• Structure
• Catalysis
• Direction
• Reversibility
• Stoichiometry
• Regulation
• Isoforms

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Possible Domains of P class ATPase
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ATP Synthase
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Reversibility of ATPase reactions
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Types of ATPases
Plasma Membrane
Secretory Vesicles
Mitochondria
SR ER
H K
Na K