Macromolecular Composition Of The Liver Cells Biology Essay

Published:

A liver cell is to be homogenised and fractionated into sediments that contain nuclei and intact cells and a nuclei free supernatant using centrifugation. The samples will then be washed in perchloric acid and centrifuged to produce supernatants glycogen that will be decanted and stored. The sediments will then be washed with KOH and perchloric acid and centrifuged again which will produce supernatants that contains ribonucleotides that will also be stored. The remaining precipitates will be suspended in KOH and incubated to ensure that precipitate is dissolved and contain the DNA and protein.

The aim of this experiment is to illustrate how the addition of various reagents to each of the supernatants and sediments will allow for an examination of the distribution of RNA, DNA, glycogen and protein.

Introduction

Comparison of Animal and Plant Cells

All living organisms except viruses are made up of cells. There are two types of cells, prokaryotic cells that consist of bacteria and the more complex cell type known as the eukaryotes, which consist of plant, animal and fungal cells. (Robertis & Robertis Jr., 1981) Eukaryotic means 'true nucleus' and these cells all have a double membrane, which surrounds an area containing the DNA, i.e. the chromosomes are surrounded by a nuclear envelope. Unlike prokaryotes, eukaryotes have larger ribosomes (80S) and they also contain membrane bound organelles. These three features generally help distinguish between prokaryote and eukaryotes. (Singh & Tomer, 2007)

Lady using a tablet
Lady using a tablet

Professional

Essay Writers

Lady Using Tablet

Get your grade
or your money back

using our Essay Writing Service!

Essay Writing Service

Karp (1988) has reported that when comparing animal cells to plant cells the differences are quite evident as plant cells provide different functions for the plant and animal cells provide for the body even though they share some of the same organelles as you can see in the diagram below.

(Cell Diagram, 2009)

One of the primary differences between animal and plant cells is that plant cells have a rigid cell wall made of fibres of cellulose where the arrangement of these micro fibrils may be regular or irregular. This helps the plant cells to maintain its rectangular shape and resist expansion of the cell when water enters (turgidity). However a cell wall is not present in animal cells which gives it the circular shape, therefore the cell can burst if too much water is present under high pressure, on the other hand this allows animal cells to adopt to different shapes. (Robertis & Robertis Jr., 1981)

Plant cells are also different from animal cells as they contain chloroplast and inside this chloroplast is the green pigment known as chlorophyll. Chlorophyll is the pigment that traps the suns energy, which is utilised by plants to make food through the process of photosynthesis. Chloroplasts contain their own DNA, which is used to control how the chloroplast works. In animal cells though the chloroplast is absent and as they lack this pigment, they cannot make their own food. (Karp, 1988)

In animal cells, vacuoles are hardly present but if present then there are a number of small vacuoles spread throughout the cytoplasm that store water, ions and waste materials. Conversely the space inside a plant cell is filled with cell sap in a large single vacuole that stores water and maintains turgidity of the cell. (Robertis & Robertis Jr., 1981)

Differential Centrifugation

To produce supernatants for examination, liver cells must be fractionated to allow specific organelles and molecules to be collected which is done through homogenisation and differential centrifugation. During homogenisation citric acid is added and in put in a pre-cooled homogeniser. It would be relatively much more difficult to homogenise a plant cell due to the presence of a cell wall which is made of cellulose and maintains cell shape. (Robertis & Robertis Jr., 1981)

A centrifuge spins at many revolutions per minute (rpm), producing forces many times the size of gravity. The resulting force separates cell components by size and density therefore the larger organelles such as nuclei are separated first since only about 1000g is needed, while smaller organelles such as ribosomes can require up to 150000g. (Campbell and Reece, 2005)

DNA, RNA and Glycogen Content of Cells

Lodish et al., (2003) indicate that nucleic acids are polymers composed of monomers called nucleotides. These nucleotide molecules are composed of an organic base, a pentose sugar and a phosphate group. The bases have molecules shaped in a ring containing nitrogen, a pyrimidine base has one ring, and a purine has two. There are five bases in total: Adenine, Thymine, Guanine, Cytosine and Uracil that are often abbreviated in the form A, T, G, C and U. Purines consist of A and G, pyrimidines consist of T, C and U.

Lady using a tablet
Lady using a tablet

Comprehensive

Writing Services

Lady Using Tablet

Plagiarism-free
Always on Time

Marked to Standard

Order Now

The pentose sugar can either be ribose or deoxyribose so if a nucleotide contains ribose then it is a ribonucleotide while one containing deoxyribose is known as a deoxyribonucleotide. Nucleotides can join together by condensation reactions to form polymers called polynucleotides, which can be ribonucleic acid, RNA if the monomer contains ribose or deoxyribonucleic acid, DNA if the monomer contains deoxyribose. (Lodish et al., 2003)

A DNA molecule is made of two polynucleotide strands running in opposite directions, wound around each other to form a double helix. Each strand has a backbone of alternating deoxyribose and phosphate groups with the bases projecting into the centre of the helix. The bases are held together by hydrogen bonds. The purine bases always pair with pyrimidines so A pairs with T and C pairs with G which is known as complimentary base pairings. There are two hydrogen bonds between A and T and three between C and G so the CG pair is more stable. An RNA molecule is similar in structure to that of DNA except in a RNA molecule there is only one polynucleotide strand not two, RNA contains ribose, not deoxyribose and RNA contains the base Uracil whereas in DNA Thymine is present. (Robertis & Robertis Jr., 1981)

Protein synthesis has two stages: transcription and translation. In transcription, part of the DNA molecule unwinds exposing the bases. Free nucleotides that are already present in the nucleus slot into place against their complimentary bases on one of the DNA strands. The phosphate and ribose groups of the RNA nucleotides are linked together by RNA polymerase to form mRNA. The base triplets on the mRNA are called codons, and these mRNA lie against a ribosome where the protein will be synthesised in the process of translation.

Already present in the cytoplasm are different types of amino acids and RNA molecules. One molecule in particular is tRNA, which has a sequence of three bases; an anticodon at one end and at the other end of each tRNA molecule an amino acid can bond. A tRNA molecule with complimentary anticodon to the mRNA's first codon binds to the mRNA. A second tRNA molecule binds in a similar way forming hydrogen bonds. The amino acids that the two tRNAs are carrying are joined together forming peptide bonds. The first tRNA molecule then leaves the ribosome and a third tRNA molecule binds and adds to the growing chain until a 'stop' codon is reached, the peptide chain then leaves the ribosome. (Axford and O'Callaghan, 2004)

Polysaccharides are carbohydrates whose molecules are polymers; glycogen has a similar structure to amylopectin where it is a branched polymer. The glycosidic bonds are found between carbons 1 and 4 of glucose, and branches between carbons 1 and 6. Glycogen is one of most importance in living organisms, as it is a long-term energy store in animal cells and is found in liver and muscle cells. However when glycogen is stored within these cells, it retains water along with it. The primary role of glycogen in liver cells is to provide a reserve supply of glucose in order to maintain blood glucose concentration. Conversion of glucose to glycogen is called glycogenesis and hydrolysis of glycogen to glucose is glycogenolysis. Glycogen is also formed by and stored in muscle cells and during vigorous activity; energy is released in the muscles by direct conversion of glycogen to lactic acid. During normal activity, energy is released by metabolic oxidation of glucose to lactic acid. (Nelson & Cox, 2008)

Tabulated Results

Test for Glycogen

Table 1 is showing colour change in solutions after adding iodine to test for glycogen content.

Solution

Colour Before

Colour After

N-S1

Pale Yellow

Honey Orange

C-S1

Pale Yellow

Dark Brown

Glycogen

Colourless

Red/Brown

Distilled Water

Colourless

Honey Orange

.

These results suggest that the C-S1 supernatant has a greater level of glycogen.

Test NP and CP for DNA

Table 2 is showing colour change in solutions after testing for DNA using diphenylamine reagent.

Solution

Colour Before

Colour After

NP

Light Green

Dark Grey

CP

Brown

Lady using a tablet
Lady using a tablet

This Essay is

a Student's Work

Lady Using Tablet

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Examples of our work

Light Brown

DNA

Colourless

Dark Blue

Water

Colourless

Colourless

Results suggest that there is none to very little DNA in the CP but a high amount in the NP.

Test NP and CP for Protein

Table 3 is showing colour change in solutions after testing for protein using biuret reagent.

Solution

Colour Before

Colour After

NP

Light Brown

Purple/Brown

CP

Light Brown

Purple/Brown

Protein

Colourless

Purple

Water

Colourless

Light Blue

.

Test results do not show enough difference in colour to give a conclusive result.

Test NS2 and CS2 for RNA fragments

Table 4 is showing colour change in solutions after testing for RNA using Orcinal reagent.

Solution

Colour Before

Colour After

NS2

Pale Yellow

Green

CS2

Pale Yellow

Light Green

RNA

Pale Yellow

Dark Green

Distilled Water

Pale Yellow

Light Orange

Results suggest that there is a higher presence of RNA in NS2.

Quantitative Assay of Protein

Table 5 is showing the quantitative protein assay of various protein solutions, NP and CP, showing absorbance rates and protein mass per tube (mg).

Test Tube

Standard protein, (10mg/ml)

Water (ml)

CP (ml)

NP (ml)

Biuret reagent (ml)

Absorbance (550nm)

Mass

Protein

(Mg)

1

0.0

2.0

0

0

6

0.000

0

2

0.4

1.6

0

0

6

0.076

4

3

0.8

1.2

0

0

6

0.193

8

4

1.2

0.8

0

0

6

0.299

12

5

1.6

0.4

0

0

6

0.387

16

6

2.0

0.0

0

0

6

0.532

20

7

0

1.5

0

0.5

6

0.456

18

8

0

1.5

0

0.5

6

0.435

17.1

9

0

1.5

0.5

0

6

0.442

17.4

10

0

1.5

0.5

0

6

0.431

17

As you can see from the calibration curve of the A550 measurements against protein content, the mass of protein in NP for test tubes 7 and 8 is 18mg and 17.1mg respectively. The mass of protein in CP for test tubes 9 and 10 is 17.4mg and 17mg respectively.

To calculate how much mass protein is in the total volume (10ml) of NP, you need to add together the values of mass protein for test tubes 7 and 8 and work out the average. Once the average has been worked out, you then need to multiply the value by 20 because only 0.5ml of NP was tested. The same is done for calculating the protein content of CP. The calculations and graph are on separate pieces of paper attached.

Discussion

In animals, glycogen is used as storage for glucose in muscle and liver cells. The glucose that is hydrolysed from glycogen via glycogenolysis is used to raise blood glucose level back to its normal state and metabolic reactions of cells to provide muscles with energy when exercising. (Nelson & Cox, 2008) Therefore looking at result table 1, the level of glycogen is higher in the cytoplasmic fraction where it turned from pale yellow to dark brown upon the addition of iodine whereas is in the nucleic fraction the colour change was slight.

Table 2 shows the colour change in solutions after testing for DNA by adding diphenylamine reagent. The addition of this reagent turned the nucleic sample from light green to dark grey, which suggests that the level of DNA content is high whereas the colour change that occurred in the cytoplasmic fraction is minimal therefore a very low amount if any is present. This theory is supported because in a cell, the nucleus is where all the DNA information is stored as chromosomes. Singh and Tomer (2007) have also stated that the nucleus is surrounded by a double membrane called the nuclear envelope that keeps the DNA away from the rest of the activities going on in the cell. This supports the results no DNA content was found in the cytoplasmic fraction.

The presence of protein was tested by using biuret reagent, the results from table 3 show that when the reagent was added to the nucleic sample, the colour change went from light brown to purple/brown and the same happened with the cytoplasmic sample. The same colour changes in both samples suggest that protein is present in both samples however the test does not show enough difference in colour to give a conclusive result. The reason both samples showed a colour change is because protein is present in DNA, RNA and enzymes, which are globular proteins that are present in the cells. Lodish et al., (2003) have supported this by stating that nucleic acids, which are used in the formation of DNA and RNA, are polymers made of monomers called amino acids.

Table 4 is showing the colour change in solutions after testing for RNA using Orcinal reagent. When the Orcinal reagent was added to NS2 the colour of the solution changed from pale yellow to green, and when the reagent was added to CS2 the solution changed from pale yellow to light green. These results show that a higher concentration of RNA is present in the nucleic sample but is also present in the cytoplasmic sample. Due to transcription taking place in the nucleus (Axford and O'Callaghan, 2004) the concentration of free RNA nucleotides needs to be high because these nucleotides need to slot into place against their complimentary bases on one of the DNA strands. However RNA is present in CS2 because after transcription comes translation that occurs in the cytoplasm and the RNA molecules required for this are tRNA.

Conclusion

When testing for glycogen by adding iodine to the solutions the results showed a high presence and colour change from pale yellow to dark brown in the cytoplasm and a small amount in the nucleus. The diphenylamine test showed that no DNA was present in the cytoplasmic fraction, however the concentration of DNA was greater in the nucleic fraction as the colour change was vast. Adding Orcinal to both samples showed that they both had the presence of RNA although the concentration was greater in the nucleic sample. The test for protein by adding biuret reagents to the samples proved to be inconclusive as the change in colour of the samples was not significant enough.

The quantitative assay allowed for the observation that there is more protein in the nucleus than in the cytoplasm, with the percentage of protein per 1000mg of liver being approximately 17.4%.