BIO 371 E2 LAB 7

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shockwave
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BIO 371 E2 LAB 7
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2012-04-20 17:56:37
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BIO 371 E2 LAB
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BIO 371 CELL LAB 7 GSU 2012
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  1. WHEN WAS CELL FRACTIONATION DEVELOPED?
    • 1930s Claude, Bensley & Hoerr....MITOCHONDRIA
    • & GRANICK ISOLATED CHLOROPLASTS.
  2. the purity of a cell fractionation can be confirmed how?
    microscope or biochm markers
  3. IN CELLULAR FRACTIONATION AND APPROPRIATE MEDIUM CONSISTS OF WHAT?
    OFTEN A BUFFERED ISOTONIC SUGAR OR SALT SOLUTION
  4. to maintain integrity of the organelles and enzymes during the fractionation procedure, all solutions and containers must be ________
    ice cold
  5. DEFINE HOMOGENIZATION?
    A procedure that disrupts the cell boundaries and releases the cell contents, undamaged, into the suspension medium.
  6. THE SUSPENSION OF CELLS IN A MEDIUM IS TERMED WHAT?
    Homogenate.
  7. THE BEST MEDIUM FOR LIVER CELLS IS WHAT?
    Isotonic sucrose (0.25 mol)
  8. WHAT IS RELATIVE CENTRIFUGAL FORCE?
    WHAT IS IT EXPRESSED IN?
    • THE FORCE GENERATED BY THE CENTRIFUGE
    • MEASURED IN G UNITS.
    • EACH G UNIT BEING THE FORCE OF THE EARTH'S GRAVITY EXERTED UPON A MASS.
  9. the RCF is a function of of the_______centerifugation in _____ ______ ________.
    • SPEED
    • REVOLUITIONS PER MINUTE (RPM) AND THE DISTANCE OF THE PARTICLE FROM THE AXIS OF ROTATION.
  10. HIGH-SPEED CENTRIFUGE CAN REACH SPEEDS OF WHAT?
    25,000.
  11. ultra centrifuge may have a maximum speed in excess of what?
    75,000
  12. two common types of rotors used in centrifuges are what?
    swinging bucket and fixed angle
  13. OF THE TWO TYPES OF ROTORS IN CENTRIFUGES WHICH IS MORE EFFICIENT FOR SEDIMENT PARTICLES?
    FIXED ANGLE ROTOR
  14. WHAT TYPE OF ROTOR WOULD YOU USE FOR DENSITY GRADIENTS EXTRICATION?
    SWING BUCKET
  15. IN DIFFERENTIAL CENTRIGUGATION HOMOGENATE IS CENTIGUGED REPEATLY.
    NAME THE LEVELS.
    • 600g/10 MNS..SEDIMENTS NUCLEAR FRACTION (PELLET HAD NUCLEI, INTACT CELLS AND TISSUE DEBRIS)
    • 10,000g/ 30MNS REFRIDIATED. MITO FRACTION( PELLETS LYSOMES, MITO AND MICROBODIES)
    • 105,000g MICROSOMAL FRACTION. (PELLETS RIBSOMES & ENDOPLASMIC RECTCULUM)
  16. UTILIING DIFFERENTIAL CENTRIFUGATION WHAT STEP WOULD ONE PRODUCE/FIND THE CYTOSOL OF THE CELL.
    MICROSOMAL SUPERNATANT.
  17. NAME THE FRACTIONS PRODUCED BY DIFFERNETIAL CENTRIFUGATION
    • NUCLEAR
    • MITOCHONDRIAL
    • MICROSOMAL
  18. WHAT WOULD SEDIMENT FASTER FROM A RAT'S LIVER CELL AND WHY?
    NECLUES OR MITOCHONDRIA?
    • NECLEUS.
    • SIZE 8-10 µm

    MITOCHONDRIA 1µm
  19. THE METHOD USED TO ISOLATE THE CHLOROPLAST FROM SPINACH WAS DEVELOPED BY WHO AND WHEN?
    • WHATLEY & ARNON 1962.
    • STRAIN.
    • 200g/1 MN
    • 1300g/5MNS..SEDIMENTING MOST OF THE CHLOROPLASTS.
  20. NAME THE TWO TYPES OF CENTRIFUGATION.
    DIFFERENTIAL AND DENSITY
  21. OF THE TWO TYPES OF CENTRIFUGATRION, WHICH PRODUCES BETTER RESOULTION?
    DENSITY GRADIENT
  22. WHAT IS THE DENSITY LEVELS OF SURCOSE IN A DENSITY GRADIENT RUN?
    MOST DENSE IS AT THE BOOTTOM, WHERE LEAST DENSE IS NEAR THE TOP OF THE TUBE.
  23. DENSITY GRADIENT...SPERATION BY WHAT?
    WHAT IS THE FIRST TO SEDIMENT 1ST?
    • SIZE
    • LARGEST.
    • IN THE EXPERIEMNT THE NUCLEI WAS 1ST, CLOSEST TO THE BOTTOM.
  24. DENSITY GRADIENT EXPERIEMNT..NAME THE LEVELS FROM TOP DOWN.. WHAT STOPED WHERE?
    • AT 10,000g/20MNS
    • CYTOSOL WAS THE TOPMOST LAYER
    • MICROSOMAL LAYER
    • MITOCHONDRIAL FRACTION
    • NUCLEI NEAR BOTTOM
  25. WHAT IS THE SEDIMENTATION COFFICIENT?
    NAME THE FORMULA AND ITS UNITS.
    SMALL S.

    • 210 log (X2/X1) X= DIST IN CM
    • s = -------------------
    • (rpm)^2 (T2 - T1) T= TIME IN SEC.
  26. THE s VALUES OF MOST CELLULAR MACROMOLECULES LIES BETWEEN _____ AND ______.

    ALSO NAME THE UNIT.
    1 X 10 ^ -13 & 200 X10^-13 SECONDS.

    • SVEDBERG UNIT "10^-13"
    • EX: 14 X 10^-13 = 14S
  27. THE FACTORS THAT DETERMINE HOW FAST A PARTICLE SEDIMENTS ARE WHAT?
    • RADUIS AND EFFECTIVE DENSITY.
    • RADIUS IS MORE IMPORTANT.
  28. KNOWN THE SEDIMENTATION COEFFICENT OF A MARCOMOLECULE, ONE CAN DETERMINE WHAT?
    MOLECULAR WEIGHT
  29. A SERIES OF _______ CHM RXNS, COLLECTIVE KNOWN AS ____ ____, ENABLES THE LIVING CELL TO RELEASE THE ENERGY ______ IN THE CHM BONDS OF MACROMOLECULES. WHAT MACROMOLECULE IS USED?
    • EXERGONIC
    • CELLULAR RESPIRATION
    • "STORED"
    • GLUCOSE.
  30. CELLULAR RESPIRATION OCCURS IN 2 WAYS. NAME THEM.
    ANAEROBIC AND AEROBIC RESPIRATION.
  31. NAME 2 PRODUCTS YEAST PRODUCE VIA FERMENTATION.

    WHAT IS PRODUCED FROM FREMENTATION IN MUSCLES?
    ETHYL ALCOHOL AND CO2

    LACTICE ACID
  32. REMEMBER TO REVIEW THE CHLOROPLAST STRUCTURES!!!
  33. WHAT IS SUBSTRAITE LEVEL PHOSPHORLATION?
    WHEN ATP IS FROM FROM P
  34. YEAST CONTAIN LACTICE ACID DEHYDROGENASE, PYRUVIC ACID DECARBOXLASE & ALCOHOL DEHYDROGENASE.
    WHERE ARE THEY USED?
    DO AMINAL CELLS HAVE THE SAME THINGS?
    • LACTICE ACID DEHYDROGENASE ..GLYCOLSIS
    • PYRUVIC ACID DECARBOXLASE & ALCOHOL DEHYDROGENASE...FERMENTATION.

    AMINAL CELLS ONLY HAVE LACTIC DEHYDROGENASE.
  35. THE CONVERSION OF PYRUVIC ACID TO LACTIC ACID IS A _____ PROCESS.

    THE CONVERSION OF LACTIC ACID TO PYRUVIC ACID IS A _____ PRCESS.
    • REDUCTION.
    • OXIDATION
  36. PYRUVATE + ____ + ____ -->
    LACTIC ACID & ______.
    • 2 NADH + 2H
    • 2 NAD +
  37. METHYLENE BLUE WAS UTILIZED AS WHAT ?
    SUBSTUTE FOR NAD+, WHICH WAS THE NATURAL e- ACCEPTOR (THE COENZYME). IT WAS WORKING ON THE LACTATE IONS.
  38. WHAT IS THE FORMULA WHEN METHYLENE BLUE WAS USED IN ANAEROBIC RESPIRATION?

    WHN IS MB OXIDIZED/REDUCED?
    • MB + 2 [H+] + 2e- -----> MB-H2
    • OXIDIZED (BLUE) REDUCED (COLORLESS)
  39. FERMENTATION AND GLYCOLSIS YIELD 4 ATP MOLECULES VIA WHAT?
    SUBSTRATE-LEVEL PHOSPHORYLATION.

    BUT REMEMBER THAT YOU USE 2 FOR A NET GAIN OF 2 ATP.
  40. WHAT IS THE SUMMARY FORMULA FOR ANAEROBIC RESPIRATION?
    • GLUCOSE + 2 ADP + 2 P -----> 2 ATP AND EITHER
    • ETHYL ALCOHOL & CO2 OR LACTIC ACID.
  41. WHAT TYPE OF RESIPRATION OCCURS IN THE KREBS CYCLE?
    AEROBIC RESPIRATION
  42. IN AEROBIC RESP. WHAT IS REMOVED FROM THE PYRUVATE?
    WHAT DOES THIS?
    WHAT ARE THE PRODUCTS?
    • CO2 IS REMOVED BY AND OXIDATION REACTION FROM COENZYME A.
    • THE END PRODUCTS ARE :
    • -NADH (FROM NAD+)
    • -ACETYLE CoA
    • -MOLECULE OF CO2
  43. IN AEROBIC RESP, ACETYL CoA REACTS WITH OXALOACETIC ACID TO FORM WHAT?
    HOW MANY CARBONS DOES THIS PRODUCT HAVE AND WHERE DID THEY COME FROM?
    • CITRIC ACID, THUS THE NAME CYTRIC ACID CYCLE (KREB'S CYCLE).
    • 2C ACETYLE CoA + 4C OXALOACETIC ACID
  44. REMEMBER TO REVIEW THE KREB'S (CYTIRC ACID) CYCLE!!!
  45. THE LECTRON TRANSPORT CHAIN IS ALSO KNOWN AS WHAT?
    RESPIRATORY CHAIN.
  46. THE ENZYMES AND THE [H+] AND e- CARRIERS IF THE ETC ARE BOUND TO THE ________ _______.
    MITROCHONDRIA CRISTAE.
  47. NAD+, FAD, COENZYME Q, CYT B, CYT C, CYT A.
    ALL OF THESE DO WHAT?
    ALL ARE PART OF A MULTIENZYME COMPLEX THAT ARE CARRIERS OF THE ETC.
  48. WHEN YOU WANT TO DETECT THE PRESENCE OF DNA IN A HOMOGENIZED CELL FRACTION OF0.25m SURCOSE, WHAT DO YOU USE?
    DIPHENYLAMINE. IT WILLL PRODUCE A BLUE COLOUR WHEN IT REACTS WITH DNA.
  49. MITOCHONDRIA CAN BE DETECTED USING WHAT?
    WHAT DOES IT REACT WITH AND WHAT IS THE RESULT?
    • MITO CONTAIN THE ENZYME SUCCINIC DEHYDROGENASE, WHICH IS CAPABLE OF CATALYZING THE REDUCTION OF MB DURING THE OXIDATION OF SUCCINIATE.
    • MB SERVES AS e- ACCEPTOR IN PLACE OF FAD.
  50. DEFINE OXIDATIVE PHOSPHORYLATION
    THE PROCESS IN WHICH ATP IS A RESULT OF THE OPERATION OF THE RESPIRATORY CHAIN.
  51. WHAT IS THE SUMMARY FORMULA FROM AEROBIC RESPIRATION?
    • C6H12O6 + 6 O2 + 36 ADP +36 P ------>
    • 6 CO2 + 6 H2O + 36 ATP
  52. HOW DOES ONE CALCULATE REQUIRED RPM?
    • 1000√ (RCF/ 11.2 * r)
    • RCF IS THE g UNITS
    • r IS RADUIS IN cm
  53. WHAT IS THE EFFECT OF SODIUM FLUORIDE ON FERMENTATION?
    The effect of sodium fluoride on fermentation process is that it severely inhibits anaerobic respiration and has some, if little or no effect, on aerobic respiration.
  54. the enzyme enolase catalyzes the dehydration of 2 phosphoglyceric acid. Sodium fluoride exerts its inhibitory effect by tying up the metal ions necessary for the activity of the enzyme. What metal is affected?
    Mg 2+
  55. Why is the lactic acid enzyme called a dehydrogenase rather than oxidase?
    oxidase will oxidize oxygen and dehydrogenase catalyzes both reactions.
  56. What subcellular particles could be obtained from a 12000 g supernatant?
    A 12000 g supernatant would produce a 57000S unit. Therefore ribosome organelles with similar molecular weighted particles would be obtained or anything smaller than mitochondria
  57. IF YOU HAVE NO Na SUCCINATE, WHAT WILL NOT HAPPEN IN THE MITOCHONDRIA?
    NO TCA CYCLE.
  58. Only two molecules of ATP are produced by the aerobic conversion of succinic acid to fumaric acid. Explain why.
    Only two of the ATP are produced because the net gain of only two ATP molecules is produced from the oxidative phosphorylation of succinic acid to fumaric acid via FAD to FADH2.FAD yields only two ATP whereas NAD+ will yield three ATP as a result of the process this is due to amount of protons that are pumped.
  59. What are the ultimate electronic accepters of the respiratory chain? How is the substance continually supplied to the mitochondria?
    The ultimate electronic acceptor of the respiratory chain is FAD. However methylene blue was utilized as acceptor during the experiment. The substance is continually supplied to the DNA of the mitochondria because it binds very easily to the phosphate backbones.
  60. WHAT IS THE PROCESS AND WHY.
    • GLYCOLSIS
    • MB and Yeast.... WHITE HAZE
    • MB, Preheated Yeast & Lactate..BLUE/GREEN LAYERS
    • MB, Yeast and Lactate...CLEAR W/PELLET.

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