cell & molecular biology

Experiment 3

1.Isolation of Chloroplast from spinach leaves

Introduction

Chloroplasts are organelles 5-10 um in size in plants which perform the function of photosynthesis. These structures are mainly located in the palisade parenchyma of the mesophyll cells in the leaf. Chloroplasts belong to a family of specialised organelle called the plastid. The stem, leaves and unripened fruit in all the plants contain chloroplast. But leaves are major part of plants performing the process of photosynthesis. The green color of these structures in plants is due to the presence of a pigment called chlorophyll which resides in the chloroplasts.

Chloroplasts like mitochondria are evolved by endosymbiosis. Both chloroplasts and mitochondria are involved in generating metabolic energy. Both organelles have their own genetic systems. During photosynthesis, the conversion of light energy derived from sunlight is converted into chemical energy in the form of carbohydrates and during this process ATPs are generated. In spite of having their own genetic system, most of the chloroplast proteins are encoded by the cell’s nuclear DNA. These proteins are synthesised in the ribosomes of the cytosol and are then imported to the organelle. Approximately 30 proteins are encoded by the chloroplast genome and some of these proteins are involved in photosynthesis.

Structure of chloroplast

Chloroplasts are found in the mesophyll cells of the leaves. The chloroplast is divided into three compartments bounded by three membrane systems: an intermembrane space between the inner and outer membranes, the stroma and the thylakoid lumen. Chloroplasts have a double membrane structure called the chloroplast envelop. The chloroplast envelop has an inner membrane and an outer membrane. A third membrane system called the thylakoid membrane surrounds the thylakoids in the stroma.

Chloroplasts are located inside the thylakoid membranes. Thylakoid membrane consists of the thylakoids which are flattened discs arranged on top of the other and they are termed as grana. The thylakoids are located inside the stroma. Photosynthesis takes place in the thylakoid membranes.The chlorophyll molecules absorb light in the form of photons and this leads to the emission of electrons by the chlorophyll molecules. This drives the hydrogen ions across the membrane surrounding the thylakoid stack. This leads to the formation of an electrochemical gradient which drives the production of ATP.

Isolation of Chloroplast

In chloroplast isolation method, the cell wall is broken mechanically using a blender or homogenizer. Then the unbroken leaf tissue and the cellular debris are removed by filtration. The chloroplasts are collected by centrifugation using a percoll gradient. Leaves of spinach, lettuce are commonly used for the isolation of chloroplasts. Both intact and broken chloroplasts are formed after centrifugation. The broken chloroplasts are removed and the intact chloroplasts are taken for further studies. Intact chloroplasts are the best source for studying the processes like carbon assimilation, electron flow and phosphorylation. The concentration of the chlorophyll molecules can be estimated from the isolated chloroplasts. DNA and RNA of chloroplasts can also be extracted.

Materials

1.Spinach leaves 30 grams.

2.Knife and scissors.

3.Cutting board.

4.Kitchen blender.

5.Muslin cloth.

6.Glass beaker.

7.50 ml centrifuge tubes.

8.1.5 ml centrifuge tubes.

9.Micropipette.

10.Glass pipette.

11.Cooling centrifuges.

12.Spectrophotometer.

Reagents and buffers

1. 1x Chloroplast isolation buffer without BSA:- 0.33M sorbitol, 0.1M tris-Cl ph 7.8, 5mM MgCl2, 10mM NaCl, 2mM EDTA.

2. 1x Chloroplast isolation buffer with BSA (0.1%w/v):

3. 40% percoll: 4ml percoll and 6 ml 1x CIB buffer with BSA to make 10 ml of 40% percoll. (Use 10 ml of 40% percoll for 6ml of chloroplast suspension)

4. 80% acetone.

Procedure

1. Wash 30 gms of spinach leaves thoroughly first with tap water and then with distill water.

2. Remove the midrib veins of the leaves and cut into small pieces.

3. Add 120 ml of 1xCIB buffer with BSA to the cut leaves in a blender. Blend with 2-3 strokes.

4. Filter the blended leaves through 6 layers of muslin cloth.

5. The filtrate is then evenly divided into four 50 ml centrifuge tubes.

6. Centrifuge the tubes for 3 minutes at 200xg. A white pellet will be obtained.

7. Transfer the supernatant into chilled 50 ml centrifuge tubes and centrifuge at 1000xg for 7 minutes. A green pellet will be obtained.

8. Discard the supernatant and break the green pellet gently by finger tapping.

9. Resuspend the pellet in 2 ml of 1x CIB buffer with BSA and mix gently by pipetting up and down.

10.Pool the suspended pellet into one centrifuge tube.

11.Preparation of 40% percoll layer: Mix 4 ml percoll with 6 ml of 1x CIB buffer with BSA.

12.Gently overlay 6ml of the chloroplast suspension over this 40% percoll layer.

13.Centrifuge at 1700 xg for 6 minutes. The intact chloroplast will sediment to the bottom of the tube as a green pellet and the broken chloroplast will form the upper layer.

14.Carefully remove the upper layer of the chloroplast suspension leaving only the pellet containing the intact chloroplast.

15.Mix the pellet with 500 ul of 1x CIB buffer without BSA.

Estimation of chlorophyll concentration

16.Add 10 ul of chloroplast suspension to 990ul of 80% acetone solution and mix gently.

17.Centrifuge at 3000xg for 2 minutes.

18.Take 100ul of the supernatant and transfer into a cuvette and measure the absorbance at 650 nm. Use 100 ul of 80% acetone as blank.

19.Take duplicate OD 650 values.

20.Take the average of the two values and estimate the mg/ml chlorophyll concentration using the following formula:

A 650 x 100/36 = mg/ml chlorophyll.

Where A 650 is the absorbance at 650 nm, 100 is the dilution factor and 36 is the extinction coefficient of chlorophyll.

Result

Concentration of chlorophyll = ---------------------mg/ml

2.Isolation of mitochondria

Introduction

Mitochondria are rod-shaped structures ranging from 2 to 8 μm in length. They are found throughout the cytoplasm and may account for up to 20% of the cell's volume. The number of mitochondria in a cell depends upon the metabolic requirements of that cell, and may range from a single large mitochondrion to thousands of the organelles. Mitochondria are considered as the "power house of the cell" because It produces Adenosine Tri Phosphate (ATP), the energy currency by extracting energy from nutrient molecules. Number of enzymes and proteins present in the mitochondria, which helps in processing fats and carbohydrates obtained from food. ATP powers the cell's metabolic activities. This process is called aerobic respiration, that is the reason for animals breathing oxygen. The cells in the higher animals obtain energy from anaerobic respiration(in the absence of oxygen), so it does exist without mitochondria.

The discovery of mitochondria came in 1886 when Richard Altman, a cytologist, identified the organelles by dye technique, and dubbed them as "bioblasts". He postulated that these structures were the basic units of cellular activity. Carl Benda, in 1898, coined out the term mitochondria. Actually Albert von Kolliker should be credited with discovery of the existence of mitochondria around 1857. He was studying human muscle cells when he noted strange granules in them.

Structure of Mitochondria

Mitochondria are a double membranous organelle found in the cytoplasm of all eukaryotic cells. It contains the outer membrane and the inner membrane which is made up of proteins and phospholipids The space between the two membranes is called the inter-membrane space. The protein content in this space differs from that in the cytoplasm.

The outer membrane of mitochondria is smooth and it contains many of special proteins called porins, that allow the molecules to enter 5000 Daltons or less in weight to pass through it. Outer membrane is very permeable to nutrient molecules, ions, ATP and ADP molecules. The inner membrane is more complex than the outer membrane in structure because it contains the complexes of the electron transport chain and the ATP synthetase complex. It is selectively permeable and allows the passage of oxygen, carbon dioxide and water. It is composed of a large number of proteins that play an important role of producing ATP, and regulating transfer of metabolites across the membrane. Cristae, the infoldings in the inner membrane increase the surface area for maintaining the complexes and proteins that help in the production of ATP, the energy rich molecules. The matrix is a complex mixture of enzymes, play an important role for the synthesis of ATP molecules, special mitochondrial ribosomes, tRNAs and the mitochondrial DNA. It also contain carbon dioxide,oxygen and other recyclable intermediates. Many of the critical metabolic steps of cellular respiration are catalyzed by enzymes that are able to diffuse through the mitochondrial matrix. The mitochondrial inner membrane embedded with other proteins involved in respiration, including the enzyme that generates ATP.

Isolation of Mitochondria

Isolation of mitochondria involves cell disruption and centrifugation. The process of cell disruption involves breaking open of cell so as to spill out the contents within the cell. Centrifugation is the process by which mixtures of cell components are separated by centrifugal force. The more dense particles migrate away from the axis, while less dense components of the mixture migrates towards the axis of centrifuge. The centrifugal technique which is used to separate the cell components from whole cell is called differential centrifugation. Differential centrifugation gives only a crude extract.

Cell Disruption Method

The process by which cell contents are spilled out of the plasma membrane barrier is called cell disruption. The cell disruption step should be gentle enough not as to mutilate the structure of the organelles. There are several techniques involved in cell disruption. The cell disruption method used in the experiment is grinding.

1. Grinding.

2. Cutting.

3. Ultrasonic vibrations.

4. High pressure.

5. Enzymatic method.

Differential Centrifugation

Differential centrifugation is the most common method of fractionating cell. Fractionation is separation of different organelles within a cell. It is a classical procedure used to isolate different particles by step wise successive centrifugations at increasing RCF's (Relative Centrifugal Forces).

Centrifugation separates particles in a suspension based on differences in size, shape and density that together define their sedimentation coefficient. The tube containing the suspension of particles is rotated at a high speed, which exerts a centrifugal force directed from the center of the rotor towards the bottom of the tube. Centrifugal Force 'G' is more commonly expressed as the Relative Centrifugal Force (RCF) or g value in multiples of the earth's gravitational field 'g'.

When doing differential centrifugation, density of the liquid in which the centrifugation is carried out should be uniform and its density must be far lower than that of the particles to be separated. The viscosity of the particles should also be very low. As a consequence, the rate of particle sedimentation depends on its size and the applied g force. Differential centrifugation gives a crude resolution of sub cellular fraction. This centrifugation is usually carried out using fixed angle rotor.

The process of differential centrifugation can be illustrated as below:

Materials

1. Yeast culture.

2. Refrigerated Centrifuge.

3. 15ml centrifuge tubes.

4. Sodium Chloride (0.9%).

5. Micropipette.

6. Ice cold lysis buffer.

7. Shaker.

8. Mitochondria storage buffer.

9. Refrigerator.

10. 15 ml micro centrifuge tube.

Procedure

1. Aseptically transfer the overnight yeast culture into two 15ml centrifugation tubes.

2. Centrifuge it at 500g for 10 minutes at 4^oC.

3. Carefully remove the supernatant without disturbing the pellet.

4. Carefully rinse the pellet in 1ml sodium chloride (0.9%) using a micropipette.

5. The sodium chloride from the centrifugation tube is discarded using a micropipette.

6. Resuspend the pellet in 1ml of ice cold lysis buffer and mix well using a micropipette.

7. Incubate it at 4^oC on a shaker for 10 minutes.

8. Centrifuge it at 1000g for 10 minutes at 4^oC and carefully remove the supernatant.

9. Resuspend the cell pellet in 1.5 ml ice cold disruption buffer and complete cell disruption by using the blunt end of a needle.

10. Centrifuge the lysate at 1000g for 10 minutes at 4^oC.

11. Transfer the supernatant to a fresh 15mL tube and also mix the supernatant obtained from the step 7.

12. Centrifuge it at 6000g for 10 minutes at 4^oC and discard the supernatant.

13. Wash the pellet with mitochondria storage buffer.

14. Centrifuge it at 6000g for 20 minutes at 4^oC.

15. Resuspend the pellet in mitochondria storage buffer and store at -20^oC.

Reference Material

Video link for isolation of mitochondria: http://vlab.amrita.edu/?sub=3&brch=187&sim=327&cnt=2135

Isolation of Mitochondria: https://www.youtube.com/watch?v=yaW-aPvriX4&t=34s

Isolation of chloroplast: http://vlab.amrita.edu/?sub=3&brch=187&sim=878&cnt=1

Questions

1. What is endosymbiotic theory ?

2. Light dependent reaction takes place where in the chloroplast?

3. Which organelle is considered the power house of the cell?

4. Who coined the term mitochondria?

5.Which is the most important function of mitochondria?