Neuronal and Cellular Signalling - Lab Report Example

Results Total Protein Calibration curve The concentrations of the proteins for each sample were obtained by substituting the absorbance in the equation of the standard curve. For example, for sample h the absorbance was 0.068, and the interpolated amount was 1.104 µg. Total protein content = 1.104 mg *100ml*10-3(mg)*1 800*10-3(ml) Total protein content=0.138mg The following table shows the total protein content for the samples Sample H P2 S m ppm pm Amount (mg) 1.104 1.059 1.178 6.823 3.970 19.872 Total protein content (mg) 0.138 0.132375 0.14725 0.852875 0.49625 2.484 % of P2 Pellet protein 3.246 3.114 3.464 20.064 11.674 58.437 Total protein in P2 Pellet=4.305375 mg Therefore, the % of P2 Pellet protein for h=0.138/4.305375*100 =3.205% 1. Choline Uptake Choline uptake average d.p.m/mg= average d.p.m Total protein synaptosomes *0.9/total fraction volume Protein content of 0.9ml synaptosomes= 0.14725µg*0.9/7ml =0.01893µg/ml = 0.00001893mg Therefore, protein content= 0.00001893mg/ml For sample 1 =9759.06/0.00001893mg/ml =5.1553×108 d.p.m/mg The choline uptakes of the other samples are summarized in the following table. Sample 1(Normal) 2(Normal) 3 (1mM HC-3) 4 (1mM HC-3) 5 (2-deoxyglucose & oligomycin) 6(2-deoxyglucose & oligomycin) Uptake (d.p.m/mg) 5.155×108 4.37 ×108 5.763×107 6.027×107 4.43×108 4.64×108 Theoretically, the values in test tubes 3 and 4 were included so that there could be uniform uptake of choline. In addition, the inclusion of these values was rather important to help in the calculation of the total and specific activities for AChE in the result as well as the total \protein content in the results. The addition of Oligomycin in 5-deoxyglucose medium induces the chemical anoxia in synaptosomes. In turn, oligomycin will inhibit the synthesis of ATP of mitochondria as well as glycogenolysis and glycolysis respectively. This resulted to the problems encountered during the rate of choline uptake by the synaptosomes. The composition of this mixture with the choline is identical to the Na+ ion medium, and there was the replacement of NaCl by the choline chloride respectively. The addition of Normal 1 and normal to the test-tubes will generally lower the inhibition rate as compared to 5, 6(deoxyglucose ) and oligomycin. With the lowered inhibition rate, the will be an increase in choline uptake by the synaptosomes. ATP synthesis will also be inhibited as well leading to the increase in choline uptake. Similarly, when 6(2-deosyglucose and olygomycin will also induce chemical anoxia in the synaptosomes as well as the mitochondria, however, there will be a slight increase synthesis of ATP inhibition as well as glycogenolysis and glycolysis that will eventually further increase the rate of choline uptake that will slightly increase. Total and specific activity of AChE in µmol substrate converted/min/mg protein Total activity=ΔA * 3* total volume *dilution factor (µmol/min) ɛ×I where ɛ=412nm:13.6µmol-1*cm-1 For the homogenate, ΔA=0.299-0.047 =0.084 3.5-0.5 (min) Total activity=0.084*3*0.1ml 412nm/13.6µmol-1*cm-1 =0.0252/30.294 =8.318×10-4 µmol substrate converted/min/mg protein Specific activity=Total activity µmol substrate converted/min/mg protein Total Protein =8.318×10-4 µmol substrate converted/min/mg protein 0.138 =0.006028 The total and specific activities for AChE in all the other fractions are indicated in the below table. Sample H P2 s m ppm Pm Total activity (µmol substrate converted/min/mg protein) 0.0008318 0.000475 0.000198 0.001753 0.002684 0.0000456 Specific activity (µmol substrate converted/min/mg protein) 0.006028 0.003591 0.001345 0.002055 0.654466 0.0000183 Total and specific activity of succinate DCPIP reductase in nMol substrate converted/min/mg protein Total activity=ΔA * 4.8* 3*total volume *dilution factor (nmol/min) I 0.1 0.1 For homogenate ΔA=0.147-0.183 *1= -0.036/6=-0.006 7-1 Total activity=-0.006*48*3*1 =-0.864 nMol substrate converted/min/mg protein Specific activity=Total activity Total protein =-0.864 nMol substrate converted/min/mg protein 0.138 =-6.26087 nMol substrate converted/min/mg protein The total activity and specific activities of succinate DCPIP reductase in the other fractions are indicated in the following table. Sample H P2 s m ppm pm Total activity (nmol substrate converted/min/mg protein) -8.64 -0.384 -0.168 0.47952 -1.296 -0.84 Specific activity (nmol substrate converted/min/mg protein) -62.6087 -2.90085 -1.14092 0.562239 -2.61159 -0.33816 Questions 1. The protein content was highest in the pure mitochondrial fraction and lowest in the p2 fraction. The order of the distribution of proteins from the highest to the lowest was pure mitochondria, myelin, partially pure mitochondria, synaptosomes, homogenate and P2 fraction. This distribution implies that there is little protein in the synaptosomes and that the higher quantity of protein in the pure mitochondrial fraction can be attributed to the presence of various respiratory enzymes. My recovery process was efficient because the sum of the percentages of the different fractions as proportions of P2 pellet protein gave 99.9%. 2. There exist mechanisms of choline uptake namely a saturable high-affinity and a high capacity low-affinity uptakes (Blusztajn and Wurtmman 617). Hemicholinium 3 inhibits the uptake of high-affinity choline uptake, but poor in hindering low-affinity uptake of choline. Conversely, Oligomycin interfere with the uptake of choline by inhibiting energy metabolism (Polak 144). Nevertheless, glucose enhances choline uptake by providing acetyl CoA that is vital for the synthesis of acetylcholine (Cooper, Bloom and Roth 152). Escalation in choline uptake in tubes 5 and 6 is due to the uptake mechanism, which has low-affinity uptake hence having not affecting by hemicholinium-3. 3. The results choline uptakes were unique due to the presence of hemicholinium-3 would inhibit the uptake of choline and lead to zero detection. However, small quantities of radioactivity were present in tubes 3 and 4 that contained HC3. These observations were due to inefficiencies in the washing process, which could have left traces of radioactive choline in tubes 3 and 4 hence the detected radioactivities. The pellets in tubes 1, 2, 5, and 6 were less than the pellets in tube 3 and 4 because little choline was used up from the contents of tube 3 and 4. 4. The fractions that would have the highest total activity of succinate DCPIP reductase and acetylcholine esterase in rat brain are the homogenate and partially pure mitochondrial fraction. The homogenate would have high total activities because it contains the actual fractions responsible for the activity of the enzymes. 5. The fractions ought to contain the highest specific activity of succinate DCPIP reductase of pure mitochondria and partially pure mitochondria because they are specific sites where oxidative phosphorylation and the tricarboxylic acid cycle take place (Aldridge and Johnson 275). In addition, the synthesis of acetyl-CoA occurs only in the mitochondria (Cooper, Bloom and Roth 152). The highest specific activities of cholinesterase are found in the synaptosomes because presynaptically localized vesicles that contain acetylcholine are found in the synaptosomes. Moreover, acetylcholine is the substrate for cholinesterase hence high activity of the enzyme should be where its substrate is in abundance (CIBA Foundation Symposium 54). The fractions that should contain the lowest specific activity of the enzymes are the mitochondria. However, the abundance of cholinesterase in the other fractions can also be because acetylcholine also acts as a hormone apart from a neurotransmitter (Proskocil et al. 2498). Therefore, the total and specific activity for each enzyme should follow a similar distribution because the total activity is an indicator of the specific activity. Works Cited Aldridge W. N. and Johnson, M. K. “Cholinesterase, Succinic Dehydrogenase, Nucleic Acids, Esterase and Glutathione Reductase in Sub-cellular Fractions from Rat Brain.” Biochemistry Journal, 73.2(1959):270-276. Print. Blusztajn, Jan K and Wurtman, Richard J. “Choline and Cholinergic Neurons.” Science 221(1983):614-620. Print. CIBA Foundation Symposium. Trends in Enzyme Histochemistry and Cytochemistry. Hoboken, NJ: John Wiley & Sons, 2009. Print. Cooper, Jack R. Floyd E. Bloom, Robert H. Roth. The Biochemical Basis of Neuropharmacology. New York: Oxford University Press, 2003. Print. Polak, R. L. “The influence of drugs on the uptake of acetylcholine by slices of rat cerebral cortex.” British Journal of Pharmacology 36. (1969): 144-152.Print. Proskocil BJ, Sekhon HS, Jia Y, Savchenko V, Blakely RD, Lindstrom J, Spindel ER. “Acetylcholine is an autocrine or paracrine hormone synthesized and secreted by airway bronchial epithelial cells.” Endocrinology 145.5(2004):2498-2506. Print. Read More