Isolation of Mitochondria

Sausage-shaped

organelle Power house of the cell Tends to localize at intracellular sites of maximum energy requirement Number of mitochondria varies greatly: depends on the type of cell Capable of division and fusion (has their own DNA)

 Outer

membrane

Relatively permeable
Contains a pore-forming protein called porin which allows free passage of small molecules Allow molecules such as ATP NAD, CoA to enter and , plays a key role in energy metabolism inside the mitochondria

Contains enzymes that convert certain lipid substrates into forms that can be metabolized within the mitochondrion

 Inner

membrane

Highly impermeable
Almost all molecules and ions require special membrane transporters to gain entrance to the matrix i.e. Calcium ions Permeable only to oxygen, CO2, and water

Contains a series of double-layered membranous sheets called cristae that projects into the inner cavity CRISTAE

Contains large amount of membrane surface that houses the machinery needed for aerobic respiration and ATP formation Increase surface area of the inner membrane

 Intermebrane

Space

Space between the inner and outer membrane Primarily responsible for oxidative phosphorylation

Mitochondrial

Matrix

Has a gel-like consistency due to the high amount of water-soluble proteins and enzymes

Used for citric acid cycle Mitochondria process their own genetic material and is capable of synthesizing their own RNAs and proteins Mitochondrial DNA encodes a small number of mitochondrial polypeptides (13) Also encodes: 2 rRNAs and 22 tRNAs used in protein synthesis within the organelle

Contains ribosomes and several molecules of DNA

 ATP

synthesis (oxidative phosphorylation) Programmed Cell Death Cell-specific functions

Building, breaking down, and recycling products needed for proper cell functioning Required for cholesterol metabolism, neurotransmitter metabolism, and detoxification of ammonia in the urea cycle

 Cell

Fractionation

separation of different organelles within the cell

Differential

Centrifugation

Separates the parts of cell by size and density The faster the centrifuge spins, the smaller the components that settle out. Mannitol used to obtained pure preparations of almost any cell component and does not damage the sample by chemical reactions or osmosis

 What

is the purpose of using pre-cooled materials during the preparation for centrifugation?

Pre-cooled materials were used in order for the enzymes in the mitochondria remain inactive and not destroyed so that the vital processes in mitochondria can be observed later when stained with Janus green.

 Draw

the morphology of the mitochondria observed.

 What

is the function of the Janus green stain in mitochondria staining?

Janus Green Stain is a supravital stain for mitochondria. It means that, by using this stain, the vital processes of mitochondria can be studied. The dye will indicate whether the oxygen is present or absent:

Oxygen present: indicator oxidized to blue color Oxygen absent: indicator reduced to pink color

Table 1. Size of Isolated Mitochondria

TRIAL 1 TRIAL 2 TRIAL 3 TRIAL 4 TRIAL 5 MEAN

2.5m

3.75m

1.25m

1.25m

2.5m

2.25m