Objectives

 

To isolate mitochondria from the cell through differential and density gradient centrifugation.

 

Theory

 

Mitochondria

 

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.

Unique Nature

 

Although most of the genetic material of a cell is contained within the nucleus, the mitochondria have their own circular DNA. They have their own machinery for protein synthesis and reproduce by fission similarly bacteria can do. It is hypothesized that mitochondria have originated from bacteria by endosymbiosis because of the independence from the nuclear DNA and similarities with bacteria. Mitochondrial DNA is localized to the matrix, which also contains a host of enzymes, as well as ribosomes for protein synthesis.

This molecule was caught in the act of replication; the arrows indicate the points at which replication was proceeding when the molecules was fixed for electron microscopy. The genome of the human mitochondrion, for example, consists of a circular DNA molecule containing 16,569 base pairs and measuring about 5μm in length. The RNA and the polypeptides encoded by this DNA are just a small fraction (about 5%) of the number of RNA molecules and proteins needed by the mitochondrion.

The size of the mitochondrial genome varies considerably among organisms. Mammalian mitochondria typically have about 16,500bp of DNA, whereas yeast mitochondrial DNA is roughly five times larger and plant mitochondrial DNA is even bigger than that. A comparison of yeast and human mitochondrial DNA, for example, suggest that most of the additional DNA present in the yeast mitochondrian consists of noncoding sequences.

 

Function

 

Energy production is the important function of mitochondria. The food  is broken into simpler molecules like carbohydrates, fats, etc. These are entered to  mitochondrion where they are further processed to produce charged molecules that combine with oxygen and produce ATP molecules. This process is known as  oxidative phosphorylation. The maintainance of proper concentration of calcium ions present in  the various compartments of the cell is achieved by mitochondria by serving as storage tanks of calcium ions. Mitochondria has a role for the building of certain parts of the blood, and hormones like testosterone and estrogen. The ammonia in the liver cells that detoxified by the presence of mitochondria.

 

Mitochondrial membranes contain numerous transport systems for the import of metabolites and high energy intermediates, export of ATP which is utilized in the cytosol, and inorganic phosphate, which is returned to the matrix via a phosphate-proton symport that is driven by the chemiosmotic gradient. Thus some of the gradient energy is always used for purposes other than synthesis of ATP. It is in the inner mitochondrial membrane were the three enzyme complexes (NADH dehydrogenase, b-c1 cytochrome, and cytochrome oxidase) and the three electron carriers (iron-sulfur centers, ubiquinone, cytochrome c) of the respiratory chain are located. The electron transfer through this chain creates a high proton concentration in the outer mitochondrial compartment, resulting in an electro-chemical gradient. The passage of these protons to the inner mitochondrial compartment through the ATP synthase complex drives the synthesis of ATP. This is a very efficient energy obtaining machinery that results in fifteen times more ATP molecules than anaerobic glycolysis.

 

Mitochondrial Plasticity

 

Mitochondria are able to modify their structure to meet the changing requirements of the cell. Some of these changes are typical of specialized cells, i.e. tubulo-vesicular cristae in steroid-producing cells. In other instances, there is an increase in the number of cristae or a change in their shape that results in a larger active surface for energy conversion, such as in zigzag, longitudinal or prismatic cristae, the latter resulting in a 75% increase in active membrane surface. Mitochondria may fuse or increase in size to form giant mitochondria or megamitochondria, and they are also able to divide in a sequence that morphologically resembles bacterial division. Thus, increased number of mitochondria are generated in situations with high metabolic activity.

 

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 stepwise 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'.

 

Relative centrifugal force or g value is calculated using the following formula:

 

 

RCF =Relative Centrifugal Force,
r = radius in centimeter,
Q = revolutions per minute.

 

Thus depending on the radius of centrifuge being used the Q value will vary for different centrifuge to obtain the same g value. 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: