Isolation of Mitochondria

The role of an enzyme is to catalyse reactions within a cell. The enzyme present in a potato (Solanum Tuberosum) is catechol oxidase. In this experiment, the enzyme activity was tested under different temperature and pH conditions. The objective of this experiment was to determine the ideal conditions under which catechol oxidase catalyses reactions. In order to do this, catechol was catalyzed by catechol oxidase into benzoquinone at diverse temperatures and pH values. The enzyme was exposed to its new environment for 5 minutes before the absorbance of the catechol oxidase was measured at 420 nm using a spectrophotometer. The use of a spectrophotometer was crucial for the collection of data in this experiment. When exposed to hot and cold temperatures, some enzymes were found to denature causing the activity to decrease. Similarly, when the pH was too high or low, then the catechol oxidase enzyme experienced a significant decrease in activity. It can be concluded after completing this experiment that the optimal pH for catechol oxidase is 7 and that the prime temperature is 20º C. Due to the fact that the catechol oxidase was only tested under several different temperatures and pH values, it is always possible to get a more precise result by decreasing the increments between the test values. However, our experiment was able to produce accurate results as to the

vulgaris plants, via the formation of a standard curve prepared using varying concentrations of bovine serum albumin (BSA) solution. Following absorbance readings of the various BSA solutions, they were plotted against their concentrations providing an indirect measure for determining protein concentrations of the plant samples within the assay tubes, and through further calculations the sample protein concentration. The mean protein concentration for the control group was calculated to be 3.34 ± 1.30 mg/mL, while the mean treated group concentration was 2.01 ± 1.26 mg/mL. These results similarly like the chlorophyll results correlate with the literature articles, as a reduced protein content within the Paraquat treated plants can be expected to some extent (Chia et al., 1981). This reduction in protein concentration is the result of those superoxide anions produced by Paraquat, disrupting the chloroplast membranes and allowing for intracellular components including some proteins to leak out, hence the decrease in protein concentration in comparison to the non-treated plants (Qian et al., 2009). A slight outlier may exist within the treated groups protein concentrations as one of the groups provided a negative value for protein concentration which is not valid, but even after exclusion of that data value, results are still supportive of the expected outcome. Though these results support the claim of Paraquat toxicity causing membrane deterioration and leakiness, protein concentration values are rather more purposeful when used to analyze malondialdehyde (MDA) values on per mg of protein

The genes which encode for the mitochondria’s component proteins are in 2 separate genetic systems in 2 different locations. One of which is the cell nucleus, but the other is inside the organelle itself. There are relatively few genes inside the

Purpose: To determine the concentration of solute in the potato’s cytoplasm by measuring the change

It achieves this my measuring the mitochondrial enzyme activity and as such is used to determine the cytotoxicity of potential medical drugs. It measures the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT starts as yellow but as it enters the cells and passes into the mitochondria where it is reduced and changes in colour to a dark purple formazan product. The cells are then solubilised with an organic solvent and released, solubilised formazan reagent is measured using a spectrometer (Cree, 2011). Some errors that may occur while using this assay are that uneven evaporation of culture fluid in the wells may cause erroneous results. The MTT solution is stable for 6 months when stored at 0°C but at 2-8°C for more than 2 weeks may cause decomposition and erroneous results may occur. Microbial contamination will contribute to cleavage of MTT and the formation of MTT formazan, causing erroneous results (Sigma Aldrich,

Fig. 5 A. Mean number of mitochondria/µm2 ± SEM within 80 µm of the soma for wildtype mitochondria (WT - red) and Rett syndrome mitochondria (RTT – blue), both after 10 days in vitro. B. Mean number of mitochondria/µm2 ± SEM within 16-32, 32-48, 48-64 and 64-80 µm of the soma for wildtype

The crude protein extraction of E. huxleyi cells that were either grown in 23℃ or 18℃, was prepared by grinding the cells in liquid nitrogen until it was fine powder. The protein extract was denatured and solubilized by adding 800 μL of an extraction buffer (30mM Tris-HCl, 7 M urea, 2 M thriourea, and 4% CHAPS), 8 μL of a protease inhibitor cocktail, 16 μL of B-mercaptoethanol, and shaking it for half an hour at room temperature. This process was done in order for no contamination to occur because, this algae’s electrophoretic separation of proteins concentrations is low.   Then the extract was transferred to a 1.5 ml microfuge tube and it was centrifuged for 30 minutes at 15,000 xg at 4 °C. The liquid portion of the extract was transferred

The success of the macros was evident through their ability to conduct the entirety of the analysis for this research. This in turn, supports the second hypothesis. However, there is still room for optimization, as the macros have limitations. Firstly, they cannot differentiate between a cluster and a single mitochondrion. This is a problem, especially in the soma where there seemed to be large mitochondrial clusters. Therefore, the results from 0-16µm diameter from the soma were not used for statistical

Specific protein location and morphology of organelles can be found using DAPI stains. The DAPI stain is classified as a nuclear stain that binds A-T regions of DNA. DAPI significantly increases the fluorescent properties and can be identified as a blue fluorescence of DAPI when processed by UV light. The only implications include using fixed cells. Using a Mitotracker Red CMXRos, you can stain living cells mitochondria. Requiring an active mitochondrial membrane potential the mitochondria oxidizes the Mitotracker probe. Fluorescence property is maintained after fixation. An ER tracker viable stain uses the bodipy fluorophore. The glibenclamide probe selectively binds sulphonylurea receptors of ATP sensitive K+ channels that are localized in the ER. Another viable stain used was the Lysotracker. Its probes are very selective for acidic compartments. Its probe is retained in the lysosome via protonation of the probe. These different stains are identified as the mitotracker will be red, lysotracker is green and the Hoescht stain will be

C1.2.2. Optimization of analysis of movies of mitochondrial transport: In terms of acquiring the data for the large scale screen, we need to address important questions in terms of plate design and statistical analysis that build on our previous experience with such problems 18,44-47. In our initial studies described in C1.1.4, we assessed the effect of disrupting microtubules on mitochondrial transport by screening the plate before and after drug application. We did this because of the statistical power associated with paired measurements and because we were unsure how much well-to-well variation would impact the statistical analysis. Overall, we found that this provided an excellent means of measuring changes in mitochondrial transport. Having refined

To confirm, the P-values of the liver and the kidney is 0.003, this value is less than the null hypothesis of 0.05, as the value is greater than the null hypothesis it rejects the hypothesis depicting that the liver and kidney fractions are not the same. The comparison of the liver cytosol with the liver mitochondria enables us to determine the activity between the cell fractions, the calculated P-value, 0.0012, that disapproves the null hypothesis as 0.0012 ≤ 0.05 indicates that there is no relationship between the cytosol and mitochondrial fractions. Furthermore, the similarities in the Liver mitochondria and the kidney mitochondria activities of 0.109 ≥ 0.05 supports the null hypothesis, accordingly outlines a relationship between the liver and the kidney tissues in the mitochondrial fraction. Lastly, the kidney cytosol and the kidney present low levels of GST activity in the mitochondrial fraction, compared to the cytosolic, this indicates that the metabolic reactivity of the enzyme is more favorable in this specific location of the tissues. (evidence)

Previously mitochondria were considered to be static and isolated organelles. However, it has now been established that mitochondria form a complex, interconnected, and highly dynamic network. Mitochondria dynamics also involves changes in mitochondrial morphology, number in the cell and movement along the cytoskeleton. Mitochondrial dynamics is said to be tightly regulated by mitochondrial fusion and fission.

Mitochondria are double membrane organelles that are found in the cytosol in Eukaryotic cells. There are many within the cells, and their function is to produce ATP. The organelle originated from the endosymbiosis theory, which means the bacteria engulfed the cell and formed a mutualism relationship. Mitochondria can be used to measure age, health, and stress levels of individuals. Scientist use mitochondria as a measurement tool by using the reporter gene to produce a protein that when newly made appears green, and then when it ages, it appears red (UVA 2014). Furthermore, if mitochondria appear green they are producing a lot of ATP and functioning properly, if they appear red they are oxidized and are not performing correctly. (UVA 2014)

Mitochondria are double membraned cell organelle that plays central role in cellular energy provision . These organelles have their own genome which is small in size and are transmitted exclusively through female germ line. The human mitochondrial DNA (mtDNA) is a double-stranded, circular and has 16 569 bp which contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides which are required in oxidative phosphorylation.

Protein purification is a process that can be employed to separate a single protein from a larger starting material which may be anything from an organ to a cell. Isolating a purified protein from a larger fraction enables further analysis such as determination of amino acid sequence, potential biological function, and even evolutionary relationship. (Cuatrecasas 1970) In this experiment, the enzyme lactate dehydrogenase will be purified, this enzyme is found extensively in human cells and catalyzes the conversion of lactate to pyruvate, an essential part in energy production. LDH is a key part of anaerobic energy production especially within glycolysis in which LDH catalyzes the conversion of the reverse reaction, pyruvate to lactate, generating NAD+ from NADH, reproducing the oxidized form of the coenzyme which can be used for oxidative respiration. (Markert 1963) Due to the fact that number of purification steps correlates with the purity of the protein multiple purification techniques will be used to isolate a pure form of LDH. LDH will be isolated from a larger “cytosol” fraction collected from a homogenized rat liver in a previous fractionation exercise. Of the procedures that will be used to isolate and purify proteins from a larger fractionate are a set of techniques collectively known as chromatography. These techniques all have the same premise, in that they consist of a stationary phase, also known as the