Subcellular fractionation and marker proteins

Methods of preparation of cell lysate and identification of subcellular components by marker enzymes

1. Subcellular Fractionation and marker enzymes Pradeep Singh M.Sc. 2 nd Year Department of Biochemistry 14 August,2018

2. Contents Introduction History Homogenization of tissues & cells Isolation of Subcellular fractions Marker Enzymes Summary

3. I. Introduction Cell is the structural and functional unit of life. Cell contain organelles which perform a variety of specific functions. Electron micrographs explains only the structure but not functions of the cell organelles. To obtain precise information about the cell organelles, it is necessary to isolate them free from contaminating organelles.

4. Contd Hence subcellular fractionation using centrifugation technique is used. Individual organelle is identified using specific markers. This information is used to study potential cellular irregularities and methods to correct them.

5. II. History Albert Claude in 1930 developed the technique of cell fractionation & identified the different organelles using the technique of centrifugation. He received Nobel Prize for the same in 1974.

6. III. Homogenization of tissues & cells

7. Methods of homogenization Homogenizers fall broadly into two categories on the basis of disruptive forces: Type 1 homogenizers: The material is subjected to the disruptive force once. Eg : The French press is commonly used in the laboratory for the homogenization of microorganisms particularly those with a tough outer wall.

8. Type 2 homogenizers: The material is repeatedly or continuously exposed to the disruptive force. Liquid Shear Mechanical Shear Sonication Osmotic lysis Other abrasive methods

9. Homogenization of cells and tissues Homogenization just means to “break open”. Homogenization is concerned only with rupture of the surface membrane. The principal aim is to achieve highest degree of cell breakage using the minimum of disruptive forces without damage to any of the organelles of interest.

10. Cell Disruption Methods

11. 1. Bead Mill It consists of a jacketed chamber with a rotating shaft, running in its centre. Agitators are fitted with the shaft which provide kinetic energy to the small beads present in the chamber. Glass or ceramic beads often used to crack open cells. The choice of bead size and weight is greatly dependent on the type of cells.

12. Disruption takes place due to the grinding action of the rolling beads and the impact resulting from the cascading ones. Bead milling can generate enormous amounts of heat. Cryogenic bead milling : Liquid nitrogen or glycol cooled unit. Application: Yeast, animal and plant tissue. Small scale: Few kilograms of yeast cells per hour. Large scale: Hundreds of kilograms per hour.

13. 2. Ultra Sonication •Ultrasonic homogenizers work by inducing vibration in a titanium probe that is immersed in the cell suspension. • A process called cavitation occurs, in which tiny bubbles are formed and explode, producing a local shockwave and disrupting cell walls by pressure change. • This method is very popular for plant and fungal cells but comes at a disadvantage: It’s very loud and has to be performed in an extra room. •Used in conjunction with chemical methods

14. 3. French Press •Primary mechanism: High shear rates within the orifice •Secondary mechanism: Impingement •Operating pressure: 10,000 to 50,000 psi •Application: Small-scale recovery of intracellular proteins and DNA from bacterial and plant cells

15. 4. Thermolysis More common method in large scale release of proteins from cells. Cells are exposed to high temperature shocks for short durations immediately followed by longer exposure to lower values in presence of buffer (1 mM MgCI2, 10 mM Tris -HCI pH 7.4) Periplasmic proteins in Gram Negative bacteria are released when the cells are heated up to 50ºC. Cytoplasmic proteins can be released from E.coli within 10min at 90 ºC.

16. 5. Osmotic shock

17. By changing the solute concentration of the liquid surrounding the cell. Through the process of osmosis, water can be moved into/out the cell causing its volume to increase/decrease to the point that results in cell bursts. Note that this method can only work with animal cells and protozoa, since they do not have cell walls.

18. 6. Chemical Solvents Often used with plant cells, organic solvents such as toluene, ether, benzene, methanol, surfactants, and phenyl ethyl alcohol, DMSO ( Dimethyl sulphoxide ) can be used to permeate cell walls. EDTA can be used specifically to disrupt the cell walls of gram negative bacteria, whose cell walls contain lipopolysaccharides that are stabilized by cations like Mg2+ and Ca2+. EDTA will chelate the cations leaving holes in the cell walls. This method can be used with wide range of production organisms but the problem can be that some proteins are denatured.

19. 7. Detergents Directly damage the cell wall or membrane, and this will lead to release of intracellular content. Its mechanism of action is to solubilize membrane proteins. Detergents can be anionic, cationic and non-ionic detergents. Most commonly used anionic detergent is sodium dodecyl sulfate (SDS) which reorganizes the cell membrane by disturbing protein-protein interactions.

20. 8. Enzymes Enzymes degrade the cell wall components which will lead to release of intracellular compounds. Enzymes that are commonly used for degradation of cell wall of plants, yeast and fungi include cellulases , pectinases , xylanases and chitinases . The enzyme’s high price and limited availability limits their utilization in large scale processes.

21. IV. Isolation of Subcellular fractions

22. The aim of subcellular fractionation is to separate organelles with as little damage as possible. The methods of separation of cell organelles differ from tissue to tissue. Approaches of subcellular fractionation: Size: Difference in size result in differences in rate of sedimentation. Problem: Individual mitochondria and microsomes derived from different membrane systems are similar in size. Surface Charge – Very limited use in practice Density – Currently the most useful property for separation of cell organelles.

23. Factors affecting organelle density and size Density = Mass___ Volume Neither mass nor volume of cell organelle are necessarily constant. The easiest method for separating cell organelles according to their density is to form a concentration gradient of some suitable material, known as density gradient solute. A particle suspended in a liquid of its own density neither floats nor sinks whatever the centrifugal field is applied.

24. Centrifugation The first analytical ultracentrifuge was developed by Theodore Swedberg in 1925.

25. Principle of centrifugation Particles suspended in a solution are pulled downward by Earth’s gravitational force. The  centrifuge  works using the sedimentation principle , where the centrifugal acceleration causes denser substances and particles to move outward in the radial direction . In a solution , particles whose mass or density is higher than that of the solvent sink or sediment and particles that are lighter than it float on the top. Centrifugal force = m ω 2 r ω is the angular velocity r is the distance from the centre of rotation

26. Centrifugal methods for the separation of organelles Separation by size – Differential centrifugation Separation by density – Density gradient centrifugation

27. Differential Centrifugation Differential centrifugation separates particles based on difference in sedimentation rate, which reflect differences in sizes and densities. Steps: Preparation of broken cells is poured in a centrifuge tube. The preparation is initially centrifuged at low speeds to completely sediment the largest and heaviest sub-cellular component. The supernatent is carefully decanted and is again centrifuged at a higher speeds till the desired portion of cell lysate is obtained.

28. Density Gradient Centrifugation Density gradient centrifugation is a variation of differential centrifugation in which the sample is centrifuged in a medium that gradually increases in density from top to bottom. Two types of density gradient centrifugation are: Rate Zonal Centrifugation Isopycnic Centrifugation

29. 1. Rate Zonal Centrifugation The particles are separated according to their size, shape, and density or the sedimentation coefficient(s). Methods used for preparation of density gradients are sucrose, glycerol, ficoll etc. 5-20% sucrose solution is commonly used to form density gradient. The sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. If the density of the particle at any point in the gradient is same to that of the gradient, then these particles will stop otherwise it will move downward towards more denser region. Separation of particles depends on the duration of centrifugation.***

30. 2. Isopycnic Centrifugation Isopycnic centrifugation separates the particles solely on the basis of buoyant density. Cesium chloride is used as a density gradient. This technique is used to separate particles of similar size but different densities. It is also independent of time of centrifugation. Sedimentation of particles occur until the buoyant density of particle is similar to the density of the gradient.

31. Low Density Medium Density High Density

32. V. Marker Enzymes

33. An enzyme that is known to be localized exclusively in the particular organelle. Examples-Acid phosphatase in lysosomes ; Succinate dehydrogenase in mitochondria. By monitoring where each enzyme activity is found during a cell fractionation protocol, one can monitor the fractionation of organelle protocol. Marker enzymes also provide information on the biochemical purity of the fractionated organelles. The presence of unwanted marker enzyme activity in the preparation indicates the level of contamination by other organelles.

34. Summary

35. References Subcellular fractionation - A Cellular Approach by J.M. Graham and D. Rickwood , Oxford University Press Wilson & Walker - Principles and Techniques of Biochemistry and Molecular Biology, 7th Edition Stryer Biochemistry, 8 th Edition Textbook of Biochemistry – DM Vasudevan , 8 th Edition

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