Macromolecular Compsition of the Liver Cell

Macromolecular Composition of the Liver Cell
Formal Report
Aims
The aim of the experiment was to test for the presence of DNA, RNA, protein and glycogen in the cytoplasm and the nucleus of bovine liver cells. From the findings of the results the distribution of these macromolecules can be shown within the liver cell. This was carried out by undertaking qualitative experiments, where the observation of a colour change was noted and a quantitative experiment, where numerical data was recorded from the measurements of light absorbance.
Introduction
The liver is the largest gland in the human body, it is a vital organ which is essential for human survival. The liver cells serve for over 500 functions within the body, their main functions are to process and store various nutrients from food, the release energy, to clean the blood of toxins and to make proteins. (Britishlivertrust.org.uk)
Glycogen
Glycogen is sugar composed of multiple units of glucose linked together by α-1.4. glycosidic bond. These large units are called polymers, they also contain branched links every 8 – 10 units by a α-1.6. glycosidic bond (fig 1). These polymers form granules ranging in size from 10 – 40 nm (fig 2).

Fig 1. A section of glycogen showing fig 2. Structure of a glycogen granule the α-1.4. and α-1.6. Glycosidic Bonds http://www.scientificpsychic.com/fitness/carbohydrates1.html http://themedicalbiochemistrypage.org/carbohydrates.html Glucose is consumed in the diet and is required by the bodily tissues and the brain as a source of energy. The smooth endoplasmic reticulum (ER) in the liver and muscle cells synthesise glucose into glycogen and store this as an energy reserve in the cytoplasm. When this energy is required by the body, the smooth ER can quickly convert it back into glucose and release it into the bloodstream. The liver cells act as a buffer by keeping the blood sugar levels at a constant rate of approximately 0.1% at all times. The muscle cells differ from the liver cells as they do not release their reserves of glycogen into the blood stream, they keep it purely for their own use. (Berg et al.. 2002)
Protein
A protein is a polypeptide chain which consists of amino acid molecules joined together with peptide bonds. Protein is an essential ingredient of living organisms and plays a fundamental role in nearly all cellular processes. Each cell consists of thousands of different proteins, each protein has its own structure and function which is important for its specific attributes. Proteins serve many functions within the body, fig 3 shows a table explaining the overview of some common functions:- Protein Type | Protein Function | Enzymes (most common type) | Proteins that speed up a chemical reaction whilst not being changed themselves | Defence proteins | Proteins that fight off viruses and bacteria | Transport proteins | Proteins that transport molecules across a cell or to different locations in the body | Storage proteins | Proteins that store amino acids, nutrients or other proteins for later use by the cell | Structural proteins | Proteins that provide support to the cell, cell-cell connections, tissue connections and skin appendages | Hormonal proteins | Proteins that produce hormones in homeostasis | fig 3. Table of common protein functions
Proteins are constructed in the cell from only 20 different amino acids. The order in which the amino acids occur will determine the primary structure of the protein, the polypeptide chain will progress through further modifications within the cell by spiralling and folding due to the interactions and chemical bonds between the different amino acid molecules and hydrogen bonding.
Some amino acids are necessary to be consumed within the diet as the body cannot make these. Once the body has consumed these within the diet it will go about breaking them down into their individual amino acid building blocks and reassembling them into the protein structures required. (Campbell et al..2005)
Protein synthesis begins in the nucleus of the cell, the DNA contains the instructions in the form of a code of how to assemble the amino acids in a certain order to make a particular protein. Proteins within the nucleus and cytoplasm take part in the synthesis of new proteins. Within the nucleus the most abundant proteins present are called histones, the role these undertake is for the DNA to coil around them to remain tightly bound and to prevent it from fragmenting and breaking down. Also proteins such as DNA primase, DNA helicase and DNA polymerase are present, these take part in the reading and copying of the DNA strand alongside RNA. (Campbell et al.. 2002) The main site of protein synthesis is on the ribosome, these are located freely in the cytoplasm and also attached to the endoplasmic reticulum (ER). The ribosome consists of proteins and RNA called ribonucleoproteins, they synthesize proteins by translating the genetic code that has been delivered from the messenger RNA (from the nucleus) and combining it with the amino acids present on the transfer RNA to make a polypeptide chain. Ribosomes that are attached to the ER tend to synthesize proteins that are for use in the plasma membrane, packaged for storage or to be exported and used outside of the cell. Proteins that are synthesized in free cytosolic ribosomes are mainly used within the cell. (Brooker et al.. 2011)

DNA
DNA stands for deoxyribonucleic acid it consists of a double helix strand. A single unit is called a nucleoside which consists of a pentose sugar named Deoxyribose, a phosphate group and a nitrogenous base. The nucleosides join together to form a single strand by the 3’ carbon atom on the sugar of one nucleoside to the phosphate group from another nucleoside by a phosphodiester bond. Two strands then join together by the nitrogenous bases with hydrogen bonds. There are 4 nitrogenous bases: adenine (A), guanine (G), thymine (T), and cytosine (C). A will always bond with T and C will always bond with G, thousands of these bases will pair up to form a gene. It is the DNA that provides the cell the information to make proteins and to self replicate, which in turn will be passed down from one generation of cells to the next.
DNA is located within the nucleus of a cell in the form of chromosomes, these are extremely long molecules that contain anything from several hundred to several thousands of genes. Each cell within the body contains the same DNA, although cells differ within the body as some of the genes are turned on and off. (Campbell et al.. 2002)
RNA
RNA stands for ribonucleic acid it consists of a single strand. It has many similarities to DNA in the way that its single unit is called a nucleoside which consists of a pentose sugar named ribose, a phosphate group and a nitrogenous base. The pentose sugar differs on the RNA as it contains an additional hydroxyl group on the 2’ carbon atom. Also one of the bases differ in RNA, the base thymine (T) is replaced by uracil (U).
The sequence of the bases allows RNA to encode genetic information. The major function of RNA is to carry the genetic information from the DNA in the nucleus to the ribosome in the cytoplasm for the synthesis of protein. There are many different types of RNA within the nucleus and cytoplasm, the three main types involved in protein synthesis are:

Messenger RNA - mRNA : takes the transcribed information copied from the DNA in the nucleus in the form of 3 base codons, each of these represents a particular amino acid.
Transfer RNA – tRNA : each amino acids has its own tRNA on it, this contains an anti-codon of which will bind to the growing end of the polypeptide chain.
Ribosomal - rRNA : these make up the ribosomes which unite the tRNA and mRNA. (Baltimore et al.. 2000)

Materials and Methods
Please refer to the lab protocol

Results
The first 4 experiments were qualitative tests for glycogen, DNA, protein and RNA. The end results to be observed was a colour change within the sample.
In all 4 tests the use of a positive and negative control was included. This was to be used as a reference point for the observation of the colour change. The reason for including positive and negative controls is to ensure that the colour change in the sample was due to the biological molecule being tested for and not an interfering molecule of a different kind. The experimental use of complex living organisms includes a lot of components in the cell all of which can possibly cause a colour change, so it is a good measure of ensuring it is the only biological molecule present that the reagent will react with.

1 – Test for glycogen
Iodine reagent is used to detect the presence of glycogen. Glycogen molecules coil up to form helices, the iodine atoms will bond into these helices and form a glycogen-iodine complex, this results in a colour change ranging from yellow to dark brown.
The outcome from the experiment showed the Iodine reacted with the cytosol supernatant and yielded a dark brown colour change. This confirms the presence of glycogen in the cytoplasm of the liver cell. It is noted that no colour change occurred in the nuclear supernatant, so therefore this indicates that no glycogen is present within the nucleus of the liver cell. fig 4

Fig 4. Test for the presence of glycogen in nuclear and cytosol supernatant of bovine liver cells Sample | Colour Before the Addition of Iodine | Colour Change after the Addition of Iodine | Presence of Glycogen | Water – Negative control | Clear | Light brown | No – negative | Glycogen – Positive control | Clear | Dark brown | Yes – positive | NS1 – Nuclear Supernatant | Clear | Light brown | No – negative | CS1 – Cytosol Supernatant | Very pale yellow | Dark brown | Yes – positive |

2 – Test for Protein
Biuret reagent is used to detect the presence of protein. Thus is a blue solution consisting of Potassium hydroxide and Copper (II) sulfate. The Biuret reagent works by firstly the Potassium hydroxide raising the pH levels in the solution, if any peptide bonds are present the Copper (II) ions will bond with the nitrogen atoms on the peptide bonds. It is the formation of the new bonds that causes a colour change to violet, the more protein present will give a more intense violet colour change. (Pentz, 1989)
The results from the experiment showed that the biuret reagent reacted with peptide bonds present in both solutions of nuclear and cytosol pellet and yielded a colour change to violet in both samples. This confirms the presence of protein is present in both the nucleus and cytoplasm of the liver cell. Fig 5

Fig 5. Test for the presence of protein in nuclear and cytosol pellet in bovine liver cells Sample | Colour Change after the Addition of Biuret Reagent | Presence of Protein | Water – Negative control | Blue | No – negative | Protein solution – Positive control | Violet | Yes – positive | NS – Nuclear pellet | Violet | Yes – positive | CS – Cytosol pellet | Violet | Yes – positive |

3 – Test for DNA
Diphenylamine reagent is used to detect the presence of DNA, it reacts with the 2’ carbon atom on the deoxyribose (pentose sugar). The solution and reagent require heating to above 95°C to break the hydrogen bonds in the double helix chain, also upon heating the 2-deoxyribose is hydrolyzed and is converted into a aldehyde. It is this that reacts with the diphenylamine to produce a blue colour change. (Pentz, 1989)
The results from the experiment showed that the diphenylamine reagent reacted with the nuclear pellet to yield a colour change of dark blue. The reagent did not react with the cytosol pellet. This confirms that the presence of DNA is contained only the nucleus of the liver cell and does not migrate out to the cytoplasm. Fig 6
Fig 6. Test for the presence of DNA in nuclear and cytosol pellet of bovine liver cells Sample | Colour Change after the addition of Diphenylamine and heating <95°C | Presence of DNA | Water – Negative control | Clear | No – negative | DNA solution – Positive control | Dark blue | Yes – positive | NS – Nuclear pellet | Dark blue | Yes – positive | CS – Cytosol pellet | Clear | No – negative |

4 – Test for RNA
Orcinol reagent is used to detect the presence of RNA. Orcinol contain hydrochloric acid, this converts the ribose sugar on the RNA chain into furfural. The furfural reacts with the orcinal to produce a green colour change. (Pentz, 1989)
The results from the experiment showed that the orcinol reagent reacted with both the nuclear and cytosol supernatant to yield a colour change of green. This confirms the presence of RNA in both the nucleus and cytoplasm of the cell. Fig 7

Fig 7. Test for the presence of RNA in nuclear and cytosol supernatant of bovine liver cells Testing Solution | Colour Change after the Addition of Oricinol reagent and heating | Presence of RNA | Water – Negative control | Clear | No – negative | RNA solution – Positive control | Green | Yes – positive | NS2 – Nuclear Supernatant | Green | Yes – positive | CS2 – Cytosol Supernatant | Green | Yes – positive |

5 – Quantitative Assay of Protein Test
The quantitative test of protein in the cytoplasm and nucleus of the liver is devised by studying the colour intensity of the biuret reagent. As explained in test 2 the violet colour change becomes more intense with the greater presence of protein in the cell. The intensity of the colour is measured with a spectrophotometer from known values of protein in tubes 1 – 6. From these known values a calibration curve can be devised and the unknown amount of protein in tubes 7 – 10 can be calculated Fig 8. The quantitative method uses data in a numerical form that can then be calculated to provide a result. Please refer to Fig 7. A calibration curve to show the quantitative assay of protein in bovine liver cells.

Fig 8. Table of Results for the Quantitative Assay of Protein | Known values | Unknown values | Tube Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Absorbance at 550 nm | 0 | 0.220 | 0.331 | 0.445 | 0.556 | 0.592 | 0.150 | 0.151 | 0.248 | 0.254 | Mass protein per tube, mg | 0 | 4 | 8 | 12 | 16 | 20 | 4.25 | 4.25 | 9 | 9 |

From plotting the known values in tubes 1-7 a calibration curve of light absorbencies against mass of protein been calculated. From this curve the values of the unknown samples 7 – 10 have been calculated to show the following:- * Tubes 7 and 8 both contained 0.5ml of cytosol pellet, according to the calibration curve both tubes contained 4.25mg of protein.

* Tubes 9 and 10 both contained 1ml of nuclear pellet, according to the calibration curve both tubes contained 9mg of protein.

These fractions were derived from a 4g sample of liver. Simple calculations were then required to find the protein content in 1000mg of liver and the protein content by percentage of the liver as follows:-
Calculation to obtain the protein content in 1000mg of bovine liver cells
4.25mg of protein present in 1ml of nuclear pellet (1ml x 10 = 10ml) 10 x 4.25mg 42.5mg of protein in 10ml of nuclear pellet
9mg of protein present in 0.5ml of cytosol pellet (0.5ml x 20 = 10ml) 20 x 9mg 180mg of protein in 10ml of cytosol pellet
180mg+42.5mg = 225mg of protein present in 4000mg of protein 225mg / 4 (to obtain per 1000mg) = 56.375mg of protein present in 1000mg of liver cells
Calculation to obtain the percentage by weight of protein 56.375/10 = 5.6% of the liver is protein.

Discussion
The liver is a very active metabolically, it serves for over 500 functions in the body and provides the whole body (with the exception of the muscles) with a supply of glucose when dietary glucose is no longer available. It is this reason that can support my findings and expectations from the experiment that the liver contains a rich content of glycogen. From studying pictures of the liver cell, glycogen granules are clearly present within the cytoplasm and not contained within the nucleus. Glycogen granules are constructed within the smooth endoplasmic reticulum of which is present in the cytoplasm of the cell, the endoplasmic reticulum also provides the enzymes that quickly transform the glycogen back into glucose to be released into the bloodstream. (Cox et al.. 2008) I did not expect to find any presence of glycogen in the nucleus as these enzymes are not present there.
Protein is present all throughout the cell both in the nucleus and in the cytoplasm. The most abundant proteins present in the nucleus are called histones, also proteins such as DNA primase, DNA helicase and DNA Polymerase are examples of proteins within the nucleus of the cell. The cytosol is the main site for the synthesis of proteins as this is where the ribosomes are present. From the outcome of the experiment I did expect to find evidence of protein in both the nucleus and cytosol, although I thought it would have been at a higher percentage. According to literature in the book Molecular Cell Biology (Baltimote et al.. 2000) protein counts for an average 15% wet content of the liver cell. A reason why I could assume my results may have shown lower protein content may have been due to certain organelles of the cell of which contain proteins not being included. Organelles such as mitochondria, lysosomes and peroxisomes all contain enzymes. (Brooker et al.. 2011)A reason for these not being included may have been due to the lower rpm of the centrifuge in the preparation of the liver cells, as in order to expose the contents of these organelles a much higher rpm would of been required for a longer period of time due to their much lighter density.
I did expect to find DNA only present within the nucleus of the cell. This evidence of DNA can be seen clearly on microscopic images. Also according to the Central Dogma of Biology information travels in one direction only DNA RNA Protein. This is evidence to support the theory behind my findings of why DNA is only located within the nucleus, as the RNA will copy the genetic code and travel out of the nucleus to the cytoplasm. I did also expect to find DNA within the cytoplasm of the cell as there is DNA present within the mitochondria (of which is located in the cytoplasm) but on this occasion none appeared. I assume this may of been due to the lower rpm of the centrifuge in the preparation of the liver cells, as in order to break open membrane of the mitochondria to expose the DNA a much higher speed for a longer period of time would of been required (Brooker et al.. 2011)
This also supports my assumption that RNA is present both within the nucleus and the cytoplasm. As the RNA begins to be formed in the nucleus from the DNA strain, it then travels out to the cytoplasm to the ribosome where it synthesizes the polypeptide chain.

Conclusion
I can conclude from the experiment the following findings:-
Glycogen is only located in the cytoplasm of the cell as the enzymes responsible for the construction and breaking down of glycogen are located on the ER in the cytoplasm and not in the nucleus.
Protein is present in both the nucleus and cytoplasm of the cell, this was clearly detected by the detection of peptide bonds. Proteins are involved in almost all chemical reaction within the cell this is evidence alone that protein is present in both the nucleus and cytoplasm.
DNA is only present in the nucleus of the cell, this evidence is supported by the Central Dogma of Biology stating that information moves in one direction only. The DNA will remain in the nucleus as the role of the RNA is to copy its information to take it out of the nucleus to the cytoplasm.
RNA is present in both the nucleus and the cytoplasm. As mentioned above with the DNA the role of the RNA is to copy the genetic information from the DNA within the nucleus and then form a protein with this information in the cytoplasm.

Bibliography
Books

Baltimore, David. Berk, Arnold. Darnell, James. Lodish, Harvey. Zipursky, S Lawrence. 2000. Molecular Cell biology 4th Edition. New York. W H Freeman & Company

Berg J M. Stryer L. Tymoczko JL. Biochemistry 5th edition. 2002 New York. W H Freeman & Company

Brooker, Robert J. Graham, Linda E. Stiling, Peter D. Widmaier, Eric P. 2011. Biology 2nd Edition. New York. The McGraw-Hill Companies Inc

Campbell, Neil A. Reece, Jane B (2005). Biology. 7th Edition. San Francisco. Pearson Education Ltd.

Cox, Micheal M. Nelson, David L. 2008. Lehninger Principles of Biochemistry 5th Edition. USA. W H Freeman & Company

Pentz, Lundy. The Biolab book 2nd Edition. 1989. USA. The Johns Hopkins University Press. P105 – 107.

Website
Britishlivertrust.org.uk 2011 Fighting Liver Disease http://www.britishlivertrust.org.uk/home/the-liver/summary-of-the-livers-functions.aspx (accessed 12.11.2011 19:50)

Bibliography: Books Baltimore, David. Berk, Arnold. Darnell, James. Lodish, Harvey. Zipursky, S Lawrence. 2000. Molecular Cell biology 4th Edition. New York. W H Freeman &amp; Company Berg J M. Stryer L. Tymoczko JL. Biochemistry 5th edition. 2002 New York. W H Freeman &amp; Company Brooker, Robert J. Graham, Linda E. Stiling, Peter D. Widmaier, Eric P. 2011. Biology 2nd Edition. New York. The McGraw-Hill Companies Inc Campbell, Neil A. Reece, Jane B (2005). Biology. 7th Edition. San Francisco. Pearson Education Ltd. Cox, Micheal M. Nelson, David L. 2008. Lehninger Principles of Biochemistry 5th Edition. USA. W H Freeman &amp; Company Pentz, Lundy. The Biolab book 2nd Edition. 1989. USA. The Johns Hopkins University Press. P105 – 107. Website Britishlivertrust.org.uk 2011 Fighting Liver Disease http://www.britishlivertrust.org.uk/home/the-liver/summary-of-the-livers-functions.aspx (accessed 12.11.2011 19:50)