1. preparative ultracentrifugation
    rotates extracts of broken cells at high speeds to separate them
  2. velocity sedimentation
    sucrose gradient, separated by size and shape
  3. equilibrium centrifugation
    cesium chloride or sucrose gradient, separated based on buoyancy
  4. ion-exchange chromatography
    slows movement of molecules with opposite charge found on the ions, separated by charge
  5. gel-filtration chromatography
    porous matrix, molecules that can fit in the pores will be slowed down, separated by size
  6. affinity chromatography
    uses substrates on the matrix to bind specifically targetted protein, separated based on property
  7. SDS-PAGE electrophoresis
    separate proteins by size, detergent (SDS) and reducing agent (ß mercaptoethanol) unfold protein and separate polypeptide subunits
  8. isoelectric focusing
    proteins will separate according to native charge
  9. unsaturated fatty acids
    double bond present
  10. saturated fatty acid
    no double bonds present
  11. fatty acid composition
    carboxylic acids; long hydrocarbon tails
  12. phospholipid composition
    hydrophilic head group; two hydrophobic fatty acid tails; three carboxylic acids; phosphate group
  13. amphiphilic
    containing both a hydrophobic and hydrophilic region
  14. three main types of phospholipids
    (phosphatidyl) -ethanolamine, -serine, -choline
  15. which of the three phospholipids is negatively charged
  16. types of lipid movement (4)
    flexion, rotation, lateral diffusion, flip-flop (rarely)
  17. negative charges appear on what side of the membrane
  18. transmembrane proteins
    amphiphilic; hydrophobic regions pass through the membrane; hydrophilic regions interact with the water on either side
  19. kinds of membrane proteins (7)
    single pass, multi pass, ß barrel, amphiphilic α on surface, covalently attached lipid chain, GPI-anchored, noncovalent interactions with other membrane proteins
  20. protease
    cleaves single-chain multipass membrane protein into two-chain multipass membrane
  21. ß barrel locations
    outer membrane of mitochondria, chloroplasts, and many bacteria
  22. S-S bond formation
    noncytosolic side
  23. detergent composition
    hydrophobic tails, hydrophilic head
  24. detergent function
    creating barriers around hydrophobic portions of molecules as they are transfered; create bubbles around them
  25. membrane transport proteins
    transfer solutes across cell membranes; multipass transmembrane proteins
  26. ways of restricting lateral mobility of proteins
    aggregation, intracellular scaffold, extracellular scaffold, cell-cell interaction
  27. symporter
    movement of two different molecules in the same direction
  28. symporter example(s)
    Na+/glucose co-transporter
  29. three types of transporter
    coupled, atp-driven, light-drive
  30. antiporter
    transfer of two different molecules in the opposite direction
  31. antiporter example(s)
    • Na+(--)H+ exchanger
    • Na+(--)driven Cl-(--)HCO3- exchanger
    • Na+(--)independent Cl-(--)HCO3-exchanger
  32. Na+(--)H+ exchanger
    H+ is directly transported out of the cell and Na+ into the cell
  33. Na+(--)driven Cl-(--)HCO3- exchanger
    influx of NaHCO3 to an efflux of HCl
  34. four types of ATP-driven pumps
    • P-type
    • F-type
    • V-type
    • ABC-type
  35. P-type pump 
    ions, uses atp, phosphorylates itself
  36. F-type pump
    called ATP synthases; bacteria, mitochondria, chloroplasts; 
  37. V-type pump
    only pump H+; found in lysosomes, vesicles, and vacuoles
  38. ABC pump
    small molecules only, not ions
  39. concentration of Ca+ in cells
    low in cytosol, high in extracellular space
  40. concentration of K+ in cells
    high in cytosol, low in extracellular space
  41. concentration of Na+ in cells
    low in cytosol, high in extracellular space
  42. P-type pump examples
    • P-type Ca2+ ATPase
    • P-type Na+-K+ ATPase
  43. three mechanisms for intracellular trafficking of proteins
    • gated
    • transmembrane
    • vesicular
  44. gated transport
    cytosol to nucleus
  45. transmembrane transport
    • cytosol to:
    • mitochondria
    • ER
    • plastids
    • peroxisomes
  46. vesicular transport
    ER to golgi mainly
  47. GTPase-activating protein (GAP)
    found in cytosol; RanGTP->RanGDP
  48. Guanine exchange factor (GEF)
    found in nucleus; RanGDP->RanGTP
  49. GTPase Ran
    required for nucleur import and export
  50. ER roles
    • lipid and protein biosynthesis
    • intracellular Ca2+ store for signaling responses
  51. microsomes
    when the ER breaks and forms small, enclosed, authentic versions of ERs still capable of regular ER functions
  52. two types of proteins captured by ER
    • transmembrane proteins partly translocated across ER and embedded in it
    • water-soluble proteins fully translocated across membrane and released in lumen
  53. protein disulfide isomerase (PDI)
    caralyzes the oxidation o ree SH groups to form disulfide bonds
  54. S-S bond location
    extracellular spaces, rarely in cytosol
  55. BiP
    • pulls proteins post-translationally into ER
    • recognizes incorrectly folded proteins
  56. precursor oligosaccharide composition
    • asparagine side chain (N-linked)
    • 2 N-acetylglucosamine
    • 9 mannose
    • 3 glucose
  57. dolichol
    holds precursor oligosaccharide in ER membrane; transfers chain to target asparagine
  58. oligosaccharyl transferase
    drives the reaction of precursor oligosaccharide to the asparagine; found with every translocator
  59. oligosaccharide assembly
    2 N-acetylglucosamines and 3 mannoses on cytosolic side, flips to lumen, then rest of the sugars added
  60. calnexin and calreticulin
    binds to incompletely folded proteins containing one terminal glucose
  61. glucosyl transferase
    detects if the protein is folded properly, if not adds a glucose so it can rebind to calnexin
  62. pathways of unfolded protein response
    • IRE1
    • PERK
    • ATF6
  63. pathway 1 of unfolded protein response
    • signal need for more er chaperones by activating a kinase
    • kinase turns into endoribonuclease
    • endoribonuclease removes RNA intron
    • two exons ligated to form active mRNA
    • mRNA translated to make gene regulatory protein
    • gene regulatory protein enters nucleus and activated gene encoding ER chaperones
    • chaperones made in ER, help fold proteins
  64. phosphatidylcholine
    lipid made primarily in the ER; occurs exclusively in the cytosolic leaflet
  65. scramblase
    equilibriates prospholipids between the two lealets of the lipid bilayer
  66. two common chaperones
    Hsp70 and Hsp60
  67. Hsp70
    acts early; bind to hydrophobic patches; hydrolyzes ATP
  68. Hsp60
    barrel-shaped; acts after protein has been fully synthesized; 
  69. proteasome
    degrades proteins; recognizes ubiquitin chains; 
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