To examine the Glycogen and Protein concentration of a rat liver cell sample, using Iodine solution, Diphenylamine reagent, Biuret reagent and by applying heat through boiling.
This experiment was designed to illustrate and calculate the distribution of RNA, DNA, protein and Glycogen between the nucleus and cytolplasim of the liver cell. The liver is the largest visceral organ in the body and forms about 3-5% of the body weight. It probably originated as a digestive gland but is now also used involved in excretion, storage, homeostasis and the maintenance of consistency in the internal environment of the body. The liver is present in amphioxus and vertebrates, but the structure called the liver in some invertebrates, such as gasteroids, has few properties of the vertebraic liver. (W. H. Horner Andrews, 1979)
In this experiment rat liver cells were used. There are many differences between an animal and plant cell. Plant cells are surrounded by a thick cell wall for protection which contains cellulose. This helps the plant cells to allow high pressure to build inside of it, without bursting. A plant cell has to be able to accept large amounts of liquid through osmosis, without being destroyed. An animal cell does not have a cell wall in their structure. If you were to fill the animal cell with too much fluid, it will eventually burst. (Wise Geek, 2010) Also plant cells, when viewed under the microscope, appear extremely different than an animal cell because of the presence of a large vacuole, which exists in the cell s cytoplasm. It usually takes up most of the room in the cell, and the membrane of the cell encircles it. It contains waste materials, water, and nutrients that can be used or secreted as necessary. There are also many other components present in the animal cell, such as ribosome s, mitochondria and lipid droplets. (Wise Geek, 2010)
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In every cell nucleus in the body there is DNA within the cell nucleus. The DNA s main functional role is to carry the cell s information on strands of genes, which are found along chromosomes. Ribonucleic acid or RNA is also present in the liver cell. RNA is one of the three major macromolecules (along with DNA and proteins) that are essential for all forms of life. RNA is made up of components called nucleotides. The sequence of nucleotides allows RNA to encode genetic information. There are different types of RNA, such as messenger RNA, ribosomal RNA and Transfer RNA. (C. E. Ophardt, 2003) These all carry out different functions to transfer and organize information and codes within the cell. Glycogen is also present in the liver cell. Glycogen is found as a polysaccharide and is derived from the carbohydrate group. Its main function is to be readily converted to glucose as needed by the body to satisfy its energy needs. (Wise Geek, 2010) Proteins are also found within the liver cells. Proteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The liver is0 not a protein store but it is exceedingly active in protein metabolism.
Test for Glycogen
Test tube containing:
Test NP and CP for DNA
Test Tube Containinglight brown
Test NP and CP for Protein
Test tube containing
Test NS2 and CS2 for RNA fragments
Test tube containing
Quantitative Assay of Protein
Test tube number
CP = 10.5 (mg)
Table 1-5: The absorbance levels of 10 test tubes containing different solutions after 30 minutes at room temperature, as measured against a blank sample in the spectrophotometer at a wavelength of 550nm. The large difference in values for NP and CP, shown in the text box beside the table, implies inaccurate pippeting.
The attached calibration curve plots the A550 measurements against protein content. The curve is well shaped, reflecting appropriate and accurate results obtained.
The liver is a large, soft organ which alters in size and shape according to the amount of blood present. Cholesterol may be found in large quantities in the liver. Bile is the main vehicle of cholesterol excretion. Cholesterol may be secreted to carry out several functions, part of its synthesis is to replace the cholesterol lost from the membrane when the bile salts are secreted. It is also used to form micelles in the bile. Phospholipids are also found in the bile. Again, they possibly may have been torn out of the membrane when bile salts are secreted and it is involved in micelle formation. (W. H. Horner Andrews, 1979)
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Within the cytolpasim of the liver cell many organelles can be found. These include mitochronria for cell respiration and energy formation. The endoplasmic reticulum (ER) is an eukaryotic organelle that forms an interconnected network of tubules, vesicles, and cisternae within cells. The endoplasmic reticulum has two parts to it. The Rough ER has many Ribosomes lining its inside and creates proteins for the cell which are either used or sent to the Golgi Apparatus for transportation. Proteins that are created in the Rough ER have a higher chance of being secreted. The smooth ER's function is to remove toxins from the system, which is why a high concentration of Smooth ER is found in the liver. Lysosomes are also found in the liver cell. A Lysosome is an organelle found in all types of animal cells which contains a large range of digestive enzymes capable of splitting most biological macromolecules. The Golgi Apparatus is an organelle found in most eukaryotic cells, including those of plants, animals, and fungi. This organelle's primary function is to process proteins targeted to the plasma membrane, lysosomes or endosomes and those that will be formed from the cell, and sort them within vesicles. Thus, it functions as a central delivery system for the cell. (M. W. King, 2008) Peroxisomes are a cell organelle containing enzymes, such as catalase and oxidase, that catalyze the production and breakdown of hydrogen peroxide. (The Medical Dictionary, 2010) There are also lipid drolets present in the liver cells cytoplasm. Ribosomes are present in the macromolecular complex but are not classified as organelles.
The concentration of Sodium and of Potassium is similar in both bile and plasma. This is probably due to diffusion through, or between, cells of the biliary epithelium. The actual and relative concentration of ion may vary with the secretory state of the liver. When secretion of bicarbonate is stimulated, there is a fall in chloride concentration. With a stimulation of flow with bile salts, there is usually a fall in bicarbonate concentration, though there is an increase of out put per minute. (W. H. Horner Andrews, 1979) Possible interpretation of this is that normally there is an equilibrium between plasma and bile, so when bile flow increases there is a greater concentration gradient, favouring a faster passage of bicarbonate into bile. (W. H. Horner Andrews, 1979)
The body stored carbohydrates as Glycogen, a compound formed by the polymerisation of glucose. Other hexoses, such as galactose and fructose, are converted into glucose by the liver before being stored. Storage in the liver is aided by insulin, the hormone from the beta cells of the pancreas, but is not essential when the concentration of sugar in the blood is high. Up to 10% of the liver weight (wet) may be glycogen, but 3-5% is more common, about half of the bodies store of glycogen is in the liver. Enzymes in the liver may break down glycogen (glycogenesis) with great rapidity and glycogen disappears soon after death unless steps are taken to inhibit the enzymes. Adrenaline, noradrenaline and the stimulation of the sympathic nerves induce the breakdown of glycogen by the hepatocytes and a sudden fright produces a surge of glucose into the blood. During violent muscular excercise, the glycogen of the muscles is broken down into glucose and in turn, into the lactic acid. This creates the Cori Cycle. The role of the liver is to keep the concentration of glucose in the blood as constant as possible, the brain especially suffering damage if the concentration falls too low. (W. H. Horner Andrews, 1979)
Figure 1-1: The structure of Glycogen
M. W. King
Although the liver is not a protein store, it is exceedingly active in protein metabolism and almost all of the proteins in plasma have an hepatic origin. Proteins are built up from amino acid nucleotides, from food or coming from other tissues. Plasma proteins can be classified into Albumins and Globulins. Plasma contains several carrier proteins, such as transferran for iron, ceruloplasmin for copper and yet another to bind adrenal cartiel hormones. The blood contains many blood clotting factors which are proteins synthesised by the liver, also known as fibrinolysinogen. This can be split to form plasmin (fibrinolysin), a substance which prevents intravascular clotting. (W. H. Horner Andrews, 1979)
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Within the liver there are several different types of RNA present. The messenger RNA carries the genetic code into the cytoplasm where protein synthesis occurs. Each gene (or distinct segment) on DNA contains instructions for making one specific protein with order of amino acids coded by the precise sequence of heterocyclic amines on the nucleotides. It has been determined that the genetic code is actually based upon triplets of nucleotides which provide 64 different codes using the four nucleotides. (C. E. Ophardt, 2003) These 4 nucleotides are Adenosine, Thymine, Guanine and Cytosine. Each nucleotide triplet, called a codon, can be "read" and translated into an amino acid to be incorporated into a protein being synthesized. In the cytoplasm, ribsomal RNA (rRNA) and protein combine to form a nucleoprotein called a ribosome. The ribosome serves as the site and carries the enzymes necessary for protein synthesis. There are about equal parts rRNA and protein. The ribosome attaches itself to m-RNA and provides the stabilizing structure to hold all substances in position as the protein is synthesized. Several ribosomes may be attached to a single RNA at any time. Transfer RNA (tRNA) contains about 75 nucleotides, three of which are called anticodons, and one amino acid. The tRNA reads the genetic code and carries the amino acid to be incorporated into the developing protein. There are at least 20 different tRNA's - one for each amino acid. The basic structure of a tRNA is shown in the left graphic. Part of the tRNA doubles back upon itself to form several double helical sections. On one end, the amino acid, phenylalanine, is attached. On the opposite end, a specific base triplet, called the anticodon, is used to actually "read" the codons on the mRNA.( C. E. Ophardt, 2003)
Figure 1-2: The structure of a strand of RNA
Astrobiology Magazine, 2008
In this experiment it was found that Glycogen and Protein were present in a sample of rat liver cells. Their presence can be detected by performing experiments with Iodine solution, Diphenylamine reagent, Biuret reagent and by applying heat through boiling. There was inaccurate pipetting during the quantative assay of protein, resulting in a difference in values between NP and CP results. It can be concluded that NP is more abundant in a rat liver cell than that of CP.