Macromolecular composition of a liver cell

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Essentials of Living Systems

Macromolecular Composition of the Liver cell

Abstract

A liver cell is to be homogenised and fractionated into a nuclei rich sediment and a nuclei free supernatant using centrifugation. After treatment with perchloric acid the samples are centrifuged producing supernatants containing glycogen, and these are decanted and stored. The sediments are washed, then treated with KOH and perchloric acid and centrifuged again. This supernatant contains ribonucleotides and it is also stored. The remaining precipitates are suspended in KOH and incubated to ensure it is fully dissolved. The addition of various reagents to each of the supernatants and suspended sediments will allow for an examination of the distribution of RNA, DNA, glycogen and protein, and for an explanation of why this is so.

Introduction

For supernatants to be produced for examination of this kind, the liver cells must be fractionated to allow specific organelles and molecules to be collected. This is done through homogenisation and differential centrifugation. During homogenisation citric acid is added and in put in a pre-cooled homogeniser; liver is easily broken up. It would be relatively much more difficult to homogenise a plant cell due to the presence of a cell wall, an outer layer that maintains cell shape and is made of cellulose, other polysaccharides and protein (Campbell and Reece, 2005).

A centrifuge spins at many revolutions per minute (rpm), producing forces many times the size of gravity, g. The resulting force separates the cell components by size and density (Campbell and Reece, 2005). The larger organelles such as nuclei are separated first since only about 1000 g is needed in order to do so, while smaller organelles such as ribosomes can require up to 150 000 g .  

In this instance, the first centrifugation cycle will reach 2500 rpm, allowing the separation of the nuclei from the rest of the relevant organelles, creating a nuclear precipitate (N) and a nuclei free supernatant (C). The precipitate is suspended in water, and both samples are treated identically from this point onwards.

First 1M HClO4 (perchloric acid) is added, which precipitates protein and nucleic acid but not glycogen. This is then put into the centrifuge at 2000 rpm for 5 minutes at 4 degrees Celsius. When the cycle is finished the supernatants from each fraction will contain glycogen and these supernatants will be decanted and known as N-S1 (nuclear supernatant, fraction 1) and C-S1(cytoplasmic supernatant, fraction 1). Glycogen is a dense granule that is used as a way of storing glucose in muscle and liver cells, making it easily accessible for all the nearby metabolic reactions. There should be little if any glycogen present in a nucleic sample, since many of the processes inside a nucleus are chemical reactions that do not require energy. They will be used in a test for glycogen where 0.01M iodine is added and a colour change observed. Glycogen and water solutions are also prepared for comparison purposes. The sediments will then be treated with 0.3M perchloric acid and recentrifuged, so that any existing glycogen will be thrown away when this supernatant is discarded. It is important to note at this point if a plant cell was involved in this investigation, then a glycogen test could not be performed, as plant cells do not use glycogen as a store of energy. Instead it uses starch, but this can also be tested for using iodine.

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To obtain the RNA molecules necessary for examination 0.3M KOH is added, which when incubated at 45 degrees Celsius for 30 minutes will break the RNA down into ribonucleotides, substances that will not precipitate during the next addition of HClO4. After incubation and cooling 1M perchloric acid is added, and the solutions are centrifuged at 2000 rpm for 5 minutes at room temperature. These supernatants will be decanted and known as N-S2 and C-S2. RNA is present in the nucleus because this is where mRNA is made, but it is also present in the cytoplasm since tRNA and mRNA are used ...

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