To be excreted from the cell, the insulin must go through the golgi apparatus to be packaged in a vesicle. The golgi apparatus is a stack of membrane-bound flattened sacks in the cytoplasm. The membrane surrounds an area of fluid where complex molecules (proteins, sugars, enzymes) are sorted and modified. The vesicle is like a 'bubble' of membrane, made from a phospholipid bilayer. It fuses with the bilayer of the cell membrane and the contents of the vesicle is excreted into the blood. This is called exocytosis.
The structure of the cell membrane enables the components needed for protein manufacture to be transported into the cell and for proteins/products to be transported out.
It is known as the fluid mosaic model.
It consists of a phospholipid bilayer: made of 2 layers of phospholipids. The hydrophilic phosphate heads face the extra-cellular fluid and cytoplasm. The hydrophobic tails are inside the membrane, protected from the water.
The bilayer allows diffusion (passive movement of substances from a high concentration to a low concentration) of non-polar, small molecules; for example, oxygen into the cell and carbon dioxide out of the cell.
Oxygen is required by the cell for respiration. Respiration occurs in the mitochondria, a double-membrane bound organelle. It has a folded inner membrane where respiration occurs. This generates the ATP needed to join the amino acids to the tRNA, and so to form the polypeptide chain that insulin is made from. The glucose molecules needed for respiration are transported into the cell from the blood by facilitated diffusion. This process uses the protein channels in the plasma-membrane. Facilitated diffusion moves substances from high to low concentration using protein channels. It is passive so uses no ATP.
Amino acids can be transported in to cells using this process, or using active transport. Active transport moves substances from a low concentration to a high concentration, using protein carriers and ATP.
Most amino acids are derived from digested proteins in the diet, which are transported in to cells and then assimilated into new proteins. However, 8 out of the 20 essential amino acids are manufactured by cells. The basic amino acid structure is made of carbon, hydrogen, oxygen and nitrogen. Other elements could be required to make the R-group.
If these elements are non-polar, they are transported by diffusion in to the cell. If they are large and/or polar, they are transported by facilitated diffusion, using protein channels or pumps in the case of sodium/potassium.
Insulin is made of 2 polypeptide chains joined by bonding between zinc ions in the R-groups of amino acids. To manufacture these in the cell, zinc ions would be transported in by protein channels, using facilitated diffusion, as they are polar.
Once in the blood the insulin is carried to all areas of the body where it binds to glycoprotein receptors in the cell membranes of its target cells.
Insulin stops the use of fat as an energy source by inhibiting the release of glucagon. Insulin is secreted in to the blood in a constant proportion to remove excess glucose from the blood, which otherwise would be toxic as it affects the osmotic potential of the blood.
Experiment on affect of temperature on enzyme activity.
Temperature (ºc)
Number of bubbles in 5 minutes
Test 1
Number of bubbles in 5 minutes
Test 2
Number of bubbles in 5 minutes
Test 3
Average number of bubbles in 5 minutes
0
34
51
66
50
20
72
78
85
78
30
224
184
211
206
40
90
101
98
96
60
40
57
57
39
30 - No potato
0
0
0
0
Catalase enzyme in potato breaks down hydrogen peroxide. Oxygen gas is given off. The rate at which oxygen is produced is the measure of the rate of reaction.
The catalase enzyme has an active site that is complementary to the substrate – hydrogen peroxide which is broken down into water and oxygen when it collides successfully with catalase. Temperature affects how quickly this happens. At low temperatures (below 30ºc) the enzyme and substrate have less kinetic energy, therefore move slowly. This means there are less successful collisions, so less enzyme-substrate complexes are formed and the rate of reaction is slow. Fewer bubbles of oxygen will be formed at these low temperatures.
Approximately 40ºc is the optimum temperature for enzyme activity. Molecules have sufficient kinetic energy to collide successfully therefore the rate of oxygen production is high. At 40ºc or above however, the protein that the enzyme is made from will begin to denature. The tertiary structure can not hold, so the shape of the active site is altered. This means that the substrate and active site are no longer complimentary and so fewer enzyme-substrate complexes form, slowing the rate of reaction. At 60ºc not all the enzymes will have denatured however, so there will still be some successful collisions and therefore, still some oxygen released.
In order to test the hypothesis that the temperature affects enzyme activity, many variables must be controlled. Firstly; the amount of catalase. The potato was cut to the same size and volume, ensuring the same surface area. All samples had their skin removed to prevent this affecting the enzyme action. Otherwise increasing the enzyme concentration would increase the rate of reaction.
Secondly; the volume and concentration of hydrogen peroxide had to be kept the same as increasing the substrate concentration or volume would have increased the rate of reaction.
Finally, each experiment was left for the same amount of time (5 minutes). Each temperature was also repeated 3 times to identify any anomalous results and to obtain an average.
A control experiment was also done in order to have evidence that it was the presence of catalase in the potato that caused the breakdown of hydrogen peroxide into oxygen bubbles. The control was set up in the same way as the other experiments, but without the potato. As expected there were no oxygen bubbles given off in this experiment as there was no catalase enzyme present to catalyse the breakdown reaction.
The results are as expected. At 40ºc the most oxygen were given off in 5 minutes in each repeat .
At 0ºc and 20ºc very few few bubbles were given off as the enzyme and substrate have little kinetic energy and, therefore, few successful collisions.
Although these results follow the expected pattern, the investigation method has many limitations, which makes the results less reliable. To improve this, it could be repeated with the following changes:
Firstly, different potatoes may have different amounts of catalase in them, especially if they are different ages. To avoid this affecting the investigation all the potato samples should be cut from the same potato, from the middle, and be freshly cut just before they are used. This would ensure that the amount of catalase available is constant for each test tube.
Secondly, the bubbles could be different sizes therefore counting bubbles in an imprecise way of measuring the rate of reaction. The oxygen could be collected in a measuring cylinder that is full of water as it would displace the water. This would give a quantitative value that is more accurate that counting bubbles.
Using bungs and delivery tubes also does not give the most precise results as oxygen could escape into the atmosphere and therefore not be measured. The apparatus should be sealed with petroleum jelly to reduce the loss of oxygen.
Enzyme action is greatly affected by changes in pH. Altering the pH of an enzymes environment (away from its optimum pH) will affect the bonding pattern of the tertiary structure of the enzyme, therefore changing the active site shape, temporarily denaturing the enzyme. As pH could change when hydrogen peroxide is broken down, a buffer solution should also be added to prevent this from happening. This would maintain the pH and therefore denaturing would not occur.
by
effie22 (student)
