Isolation of Mitochondria

ISOLATION OF MITOCHONDRIA

Mitochondria have been studied by following three types of methods:

1.The examination of mitochondria in living cells is somewhat difficult because of their low refractive index.However,they can be observed easily in cells cultured in vitro,particularly under darkfield illumination and phase contrast microscope.Such an examination has been greatly facilitated by colouration with vital stain Janus green which stains living mitochondria greenish blue due to its action with cytochrome oxidase system present in the mitochondria.This system maintains the vital dye in its oxidized(coloured)state.In the surrounding cytoplasm, the stain is reduced to a colourless leek base.

Fluorescent dyes, which are more sensitive,have been used in isolated mitochondria and intact cultured cells.Such stains are more suitable for in situ metabolic structure of mitochondria.

2.Cytochemical Marking of Mitochondrial Enzymes

Different parts of mitochondria have distinct marker enzymes for histochemical marking such as cytochrome oxidase for inner membrane ,monoamine oxidase for outer membrane,malate dehydrogenase for matrix and adenylate kinase for the outer chamber.

3.Isolation

Mitochondria can be easily isolated by cell fractionation brought about by differential centrifugation.The homogeneous fraction of mitochondria has been obtained from liver,skeletal muscle.heart and some other tissues.In differential centrifugation mitochondria sediment at 5000 to 24000g,while in living cells at the ultracentrifugation (20,000 to 400,000g)mitochondria are deposited intact at the centrifugal pole.

The two mitochondrial membranes have been separated by density gradient centrifugation.The outer membrane is separated by causing a swelling which can be brought about by breakage followed by contraction of inner membrane and matrix.Certain detergents such as digitonin and lubrol are often used for this purpose.Since the outer membrane is lighter and much stronger,centrifugal force is needed to separate it.When the outer membrane is removed with digitonin,the so-called mitoplast is formed.Mitoplast includes inner membrane with unfolded cristae and matrix.Mitoplast found to carry out oxidative phosphorylation.The isolated outer membrane as revealed by negative staining and shows a “folded-bag”appearance.Such isolation of two membranes and compartments has enabled localization of various enzymes system of mitochondria

CHEMICAL COMPOSITION

The gross chemical composition of mitochondria varies in different animal and plant cells.However, the mitochondria are found to contains 65 to 70 percent proteins,25 to 30 percent lipids,0.5 percent RNA and a small amount of the DNA.The lipid contents of the mitochondria are composed of 90 percent phospholipids (lecithin and cephalin),5 percent or less cholesterol and 5 percent free fatty acids and triglycerides.The inner membrane is rich in one type of phospholipid,called cardiolipin which makes this membrane impermeable to a variety of ions and small molecules(e.g.,Na+,K+,Cl-,NAD+,AMP,GTP,CoA and so on).

The outer mitochondrial membrane has the typical ratio of 50 percent proteins and 50 percent phospholipids of ‘unit membrane’.However,it contains more unsaturated fatty acids and less cholesterol.It has been estimated that in the mitochondria of liver 67 percent of the total mitochondrial protein is located in the matrix,21percent is located in the inner membrane,6 percent is situated in the outer membrane and 6 percent is found in the outer chamber.Each of these four mitochondrial regions contains special sets of proteins that mediate distinct functions:

1.Enzymes of the outer membrane.Besides porin, other proteins of this membrane include

enzymes involved in mitochondrial lipid synthesis and those enzymes that convert lipid substrates into forms that are subsequently metabolized in the matrix.Certain important enzymes of this membrane are monoamine oxidase,retune-insensitive NADH-cytochrome-C-reductase,kynurenine hydroxylase ,and fatty acid CoA ligase.

2.Enzymes of intermembrane space.This space contains several enzymes that use the ATP molecules passing out of the matrix to phosphorylate other nucleotides.The main enzymes of this part are adenylate kinase and nucleoside diphosphokinase.

3.Enzymes of the inner membrane.This membrane contains proteins with three types of functions:

1.those that carries out the oxidation reactions of the respiratory chain;2.an enzymes complex,called ATP synthetase that makes ATP in the matrix ;3.specific transport proteins that regulate the passage of metabolites into and out of the matrix.Since an electrochemical gradient,that drives ATP synthetase,is established across this membrane by the respiratory chain,it is important that the membrane is impermeable to small ions.The significance enzymes of inner membrane are enzymes of electron transport pathways,viz.,nicotinamide adenine dinucleotide(NAD),flavin adenine dinucleotide (FAD),diphosphopyridine nucleotide (DPN)dehydrogenase, four cytochromes (Cyt. B,Cyt. c,Cyt.c1,Cyt.a and Cyt.a3).ubiquinone or enzymes Q10,non-heme copper and iron,ATP synthetase,succinate dehydrogenase;B-hydroxybutyrate dehydrogenase; carnitine fatty acid acyltransferase.

4.Enzymes of the mitochondrial matrix.The mitochondrial matrix contains a highly concentrated mixture of hundreds of enzymes,including those required for the oxidation of pyruvate fatty acids and for the citric acid cycle or Krebs cycle.The matrix also contains several identical copies of the mitochondrial DNA ,special 55S mitochondrial ribosomes,tRNAs and various enzymes required for the expression of mitochondrial genes.Thus,the mitochondrial matrix contains the following enzymes : malate dehydrogenase,isocitrate dehydrogenase,fumarase,aconitase,citrate synthetase,a-keto acid dehydrogenase,B-oxidation enzymes.Moreover,the mitochondrial matrix contains different nucleotides,nucleotide coenzymes and inorganic electrolytes –K+,HPO4-,Mg++,Cl, and SO4-.

MITOCHONDRIA AND CHLOROPLAST AS TRANSDUCING SYSTEMS

In cells,energy transformation takes place through the agency of two main transducing systems (i.e.,systems that produce energy transformation)represented by mitochondria and chloroplasts.These two organelles of eukaryotic cells in some respects operate in opposite directions.For example,chloroplasts are present only in plant cells and especially adapted to capture light energy and to transduce it into chemical energy,which is stored in covalent bonds between atoms in the different nutrients or fuel molecules.In contrast,the mitochondria are the “power plants”or “powerhouses”that by oxidation,release the energy contained in the fuel molecules and make other forms of chemical energy.The main function of chloroplasts is photosynthesis,while that of mitochondria is oxidative phosphorylation.Finally, photosynthesis is an endergonic reaction,which means that it captures energy;oxidative phosphorylation is an exergonic reaction,meaning that it releases energy.

FUNCTIONS

The mitochondria perform most important such as oxidation,dehydrogenation,oxidative phosphorylation and respiratory chain of the cell.Their structure and enzymatic system are fully adapted for their different functions.They are the actual respiratory organs of the cells where the oxidation foodstuffs,i.e.,carbohydrates and fats are completely oxidized into CO2 AND H2O.During the biological oxidation of the carbohydrates and fats, large amount of energy is released which is utilized by the mitochondria for the synthesis of the energy-rich compound known as adenosine triphosphate or ATP.Because mitochondria synthesize energy-rich compound ATP,they are also known as “powerhouses”of the cell.In animal cell mitochondria produce 95 percent of ATP molecules,remaining 5 percent is being produced during anaerobic respiration outside the mitochondria.In plant cells,ATP is also produced by the chloroplasts.

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