The goal for this lab was to look for the conditions that show what chemicals in muscle fibers are essential for contraction and those prevent muscle contraction from developing in a simplified system in order to determine the minimum requirement for contraction.
In order to prepare muscle fibers, an individual thread was obtained from scores of glycerinated muscle materials that was about 0. 5 mm in diameter. The muscle was a rabbit psoas muscle in 50% glycerol (stored at -10 diplomas C) extracted from Carolina Biological. To be able to own an observation of what was taking place with the muscle, Nikon E400 was used. The fibers was placed over a glide in the existence of 0. 05 M KCl and 0. 005 M K phosphate buffer (pH 7). The first step was to detect the minimum requirement solution for muscle contraction to take place. This is done utilizing the following alternatives: 0. 001 M MgCl2, 0. 001 M CaCl2, and 0. 1 M ATP. The necessity was decided after observing the changes occurring when each of the solutions were added to the muscle individually, in combinations of two, and all three solutions together.
After the minimum amount requirement was motivated, chelators were used to see if they inhibited contraction in the presence of the perfect solution is that triggered contraction. The chelators were 0. 002 M EDTA and 0. 002M EGTA. The EDTA binds Ca ++ and Mg ++. EGTA binds only Ca++. The chelating brokers improve the solubility of magnesium and calcium and allow them to disseminate of the muscle fibers. This causes removing the ions from the actin and myosin environment. It was important that the chelators were added prior to the contracting brokers when inhibition had been tested. Otherwise, it might be impossible to find any changes after the contraction has occurred; contraction is not reversible once it occurs in a simplified system.
Then Solution A and Solution B were used to examine the localization of myosin and actin in the myofibrils. Solution As function was to solubilize and remove myosin in the form of monomers from its loci which is in the myofibril. Solution B experienced the same tendencies as the actin. The Solutions were located on the myofibril to see changes that took place and observations were made.
Discussion and Conclusion
During this laboratory, using the microscope we evaluated the changes that took place when certain solution were unveiled to the rabbit psoas muscle materials. The solutions brought on contraction, inhibited contraction that occurs, or possessed no influence on the sarcomeres whatsoever. We used glycerinated myofibrils from rabbit psoas muscle which really is a kind of striated muscle. Rabbit psoas muscle was a good model to utilize for this lab since the fibers are long and in a straight line. Also an added benefits was that there have been not a lot of connective tissues hooking up the muscle materials together. This is an in vitro model meaning that experiment was completed outside the living organism.
The Phase Distinction with magnification of 10X/ 40X was used during this lab to look at the slip because the cells are transparent and Phase Comparison is the best option to use in order to truly have a good resolution. Under a microscope the myofibers were striated and they acquired a repeating pattern of rings and lines. The pattern was brought on by parallel firm of necessary protein filaments within the myofibrils. Within the myofibril, there are two types of filament- the thick filaments which consist of the proteins myosin and slender filaments made up of the health proteins actin.
As shown on Stand 1, Mg2Cl2, Ca2Cl2, and ATP were the answer used to look for the changes occurring with the muscle. All of the three solutions were positioned on the slip which contains a thread of muscle dietary fiber and contraction of the muscle was witnessed. We could notify when contraction was taking place because the fibers was short long and it was easy to understand the changes like the shortening in length and the color change. When Mg2Cl2 and Ca2Cl2 were added independently to the fibre, nothing occurred. However, as soon as the ATP was positioned on changes were easily seen. ATP caused muscle contraction alone. The sarcomeres in the muscle fibre shortened long and the color transformed from light yellowish to darker yellow.
However, to be able to make certain this is the minimum requirement for muscle contraction, we added Mg2Cl2 with ATP and Ca2Cl2 with ATP.
With the MgCl2 and ATP, the contraction happened immediately as the solutions were added. The contraction was even faster than the ATP by itself. Then ATP and Ca2Cl2 solutions were introduced which also triggered contraction. Even though the combination of both solutions induced contraction to occur faster than ATP by itself, it was slower than the solutions of ATP and Mg2Cl2. Because of this, we figured ATP was the minimum amount requirement needed for the cells to contract. All of the solutions that brought on contraction weren't in one aspect because every component of a sarcomere was facing changes except the A music group which stayed the same. The I bands, the M lines, the Z lines, and the actin and mysosin- these were all decreasing long in order to cause contraction. By the end, it was decided that ATP was the necessity for glycerinated muscle contraction. When there is absolutely no ATP present, the myosin heads in the muscle will not be activated and it could not bind to the actin. In glycerinated tissues, the combination of KCl and MgCl2 with ATP increased the strength of muscle contraction. This is mainly due to myosins high affinity for these ions.
Table 2 shows whether contraction was inhibited in the occurrence of chelating realtors, EDTA and EGTA, when it was used in combination with the primary solution that induced contraction, ATP. EDTA and EGTA did not inhibit contraction from occurring, but the contraction was slower than when ATP was present. EDTA is a chelating agent that binds Ca++ and Mg++ and EGTA is a chelating agent that binds Ca++. The chelating realtors improve the solubility of Mg 2+ and Ca2+ so that they can leave the muscle fibres. With ATP and the chelating realtors, contraction took place and there was no inhibition occurring.
Table 3 shows two different solutions, Solution A and solution B, and their effect on actin and myosin. As shown in the stand, solution A had KCl, phosphate buffer, Na pyrophosphate, and MgCl2 while solution B had phosphate buffer. Both of these solutions did not cause any contraction in the muscle predicated on our observations. However, changes were observable because in both conditions the fibers improved color; they became lighter yellowish. This supposed that the muscles weren't contracting. Solution A made the mysosin more soluble and solution B acted very much the same as the actin.
When evaluating the muscle in living structure, the glycerinated muscle system differs. The glycerination strategy minimizes ions and ATP from the structure and disrupts the troponin/tropomyosin complex. When the complex is interrupted, the available binding sites on the actin materials are no longer clogged ( Cell and Molecular Biology). As a result, Ca2+ is not just one of certain requirements to cause contraction. Alternatively, since there is absolutely no ATP is in the glycerinated structure, the myosin heads cannot be turned on to cause contraction. Following the muscle contracted it didn't relax since there have been no opposing muscles to draw it. Also, muscle fibers do not long term contract when there are no stimulations or nerve indicators and this was one of the variations with glycerinated muscle and the living cell muscle.
Errors can have occurred in this laboratory if one used very thick muscle strands. Having thinner strands were better to have good results. Also, it is possible that a lot of the calcium was still in the sarcoplasmic reticulum of the glycerinated muscle, which could have lead into wrong results. Overall however, the lab was successful and we have obtained what we should were looking for- what alternatives cause contraction, which ones inhibit, and what is the minimum requirement for muscle contraction.
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