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Provide introductory experience on the theory and application of centrifugation techniques in the biological sciences. Provide introductory experience on the theory and application of centrifugation techniques in the biological sciences. Observe (& work with) a protocol for isolation of plant mitochondria. Observe (& work with) a protocol for isolation of plant mitochondria. Provide experience with the theory and application of marker enzymes. Provide experience with the theory and application of marker enzymes. 2

Definition: A technique (or process) where centrifugal force is used to separate mixtures of particles or substances on the basis of their differential densities. A technique (or process) where centrifugal force is used to separate mixtures of particles or substances on the basis of their differential densities.Theory: Centrifugal force is applied in a “centrifuge”. Centrifugal force is applied in a “centrifuge”. Angular velocity (RPM) creates centrifugal force. Angular velocity (RPM) creates centrifugal force. centrifugal force is expressed relative to gravity. centrifugal force is expressed relative to gravity. Substances with greater densities will sediment faster than substances with lesser densities. Substances with greater densities will sediment faster than substances with lesser densities. 3

4 (Radius, r) in cm RCF = (1.12 x ) x (r, cm) x RPM 2 (r = 1 cm) (r = 5 cm) (r = 10 cm)  Note: RCF (Relative Centrifugal Force) is expressed as no. of x’s gravity.

5 Industrial Decanter

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George Stokes (1851) studied the frictional (drag) force exerted on spherical bodies as they fell through viscous liquids. George Stokes (1851) studied the frictional (drag) force exerted on spherical bodies as they fell through viscous liquids. Developed a mathematic expression that relates all factors that can affect sedimentation velocity. Developed a mathematic expression that relates all factors that can affect sedimentation velocity. 8 Where: V s = settling velocity* of a falling sphere. d = diameter of the sphere.  p = density of sphere.  l = density of the liquid medium.  = viscosity of liquid medium. g = gravitational (centrifugal) force. *V s is also known as “terminal velocity” (V t ) drag force gravitational force

The mathematical ratio of sedimentation velocity (v t ) of a particle to the centrifugal (gravitational) force causing it to sediment. The mathematical ratio of sedimentation velocity (v t ) of a particle to the centrifugal (gravitational) force causing it to sediment. Created by Theodor Svedberg during his analytical ultracentrifugation studies of proteins and ribosomes. Created by Theodor Svedberg during his analytical ultracentrifugation studies of proteins and ribosomes. Values of “s” range from to sec. Values of “s” range from to sec. 1 Svedberg (S) = sec. 1 Svedberg (S) = sec. 9 s = vtgvtg Where: v t = terminal velocity of a falling sphere (cm/sec). g = gravitational (centrifugal) force (cm/sec 2 ).

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1. Differential Centrifugation Cell-free extract and supernatants are centrifuged at progressively higher speeds and longer times. Cell-free extract and supernatants are centrifuged at progressively higher speeds and longer times. 2. Density Gradient Centrifugation Cell-free extract is centrifuged through a medium whose density gradually increases toward the bottom of the centrifuge tube. Cell-free extract is centrifuged through a medium whose density gradually increases toward the bottom of the centrifuge tube. a) Rate Zonal – sample is centrifuged until most dense component approaches bottom of centrifuge tube. b) Isopycnic – sample is centrifuged until all components reach their equilibrium buoyant density. 11

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15 “Shave” off approximately 15 g of floret tissue.

Definition: An enzyme that specifically occurs in only one type of tissue or cell type, or in only one subcellular compartment (or organelle), and whose activity can be used as a diagnostic tool to assess certain aspects related to the tissue, cell or organelle of origin. An enzyme that specifically occurs in only one type of tissue or cell type, or in only one subcellular compartment (or organelle), and whose activity can be used as a diagnostic tool to assess certain aspects related to the tissue, cell or organelle of origin. Example: Liver / Heart Function Tests. Alanine transaminase Alanine transaminase Aspartate transaminase Aspartate transaminase Alkaline phosphatase Alkaline phosphatase Gamma-glutamyl transferase Gamma-glutamyl transferase Lactate dehydrogenase Lactate dehydrogenase Creatine kinase Creatine kinase Troponin Troponin 16 Activities of one or more enzyme are measured in blood serum. Elevated levels indicate liver function abnormalities or damage. Elevated serum levels indicate recent heart (attack) damage.

Succinate dehydrogenase: Membrane-bound enzyme of mitochondrion. Membrane-bound enzyme of mitochondrion. Oxidizes succinate to fumarate in TCA Cycle. Oxidizes succinate to fumarate in TCA Cycle. 17 C I HCH I HCH I C OO - - C I CH II HC I C OO - - Succinate Dehydrogenase FADFADH 2 UQ UQH 2 Respiratory Electron Transport O2O2O2O2

Succinate dehydrogenase: Membrane-bound enzyme of mitochondrion. Membrane-bound enzyme of mitochondrion. Oxidizes succinate to fumarate in TCA Cycle. Oxidizes succinate to fumarate in TCA Cycle.Phosphoglucoisomerase: Soluble glycolytic enzyme of the cytosol. Soluble glycolytic enzyme of the cytosol. Converts glucose-6P to fructose-6P. Converts glucose-6P to fructose-6P. 18

Succinate dehydrogenase: Membrane-bound enzyme of mitochondrion. Membrane-bound enzyme of mitochondrion. Oxidizes succinate to fumarate in TCA Cycle. Oxidizes succinate to fumarate in TCA Cycle.Phosphoglucoisomerase: Soluble glycolytic enzyme of the cytosol. Soluble glycolytic enzyme of the cytosol. Converts glucose-6P to fructose-6P. Converts glucose-6P to fructose-6P. Can be coupled to glucose-6P dehydrogenase. Can be coupled to glucose-6P dehydrogenase. 19 PGI:Fructose-6P ↔ Glucose-6P G6PDH:Glucose-6P + NAD + + H + ↔ 6-Phosphogluconate + NADH PGI:Fructose-6P ↔ Glucose-6P G6PDH:Glucose-6P + NAD + + H + ↔ 6-Phosphogluconate + NADH

Measure activities of two marker enzymes in three cellular fractions (whole extract, mitochondrial fraction, cytosol fraction). Measure activities of two marker enzymes in three cellular fractions (whole extract, mitochondrial fraction, cytosol fraction). Examine the effects of Na-malonate on SDH. Examine the effects of Na-malonate on SDH. Make interpretations on purity of fractions. Make interpretations on purity of fractions. 20

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