Malate Dehydrogenase Isoenzymes - Lab Report Example

Summary This experiment examined the localisation of three enzymes within liver cells. The enzymes being examined were malate dehydrogenase (MDH), lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH). In addition, the concentration of protein within different cell fractions were determined. This was achieved through fractionating the liver cells into three different components: the supernatant, liver particulate and lysed liver particulate. MDH was localised primarily in the lysed liver particulate, while LDH was mainly localised within the supernatant and SDH was in all three fractions. The activity of protein also varied between the fractions, with the liver particulate and lysed liver particulate showing higher levels of protein than the supernatant. The results of the experiment indicate that the majority of energy converting reactions is centred within the mitochondria, with the MDH reaction causing the highest amount of absorption, suggesting a stronger reaction. The prevalence of protein within this portion of the cell supports this hypothesis. The use of electron acceptors to measure the strength of reactions was an effective approach to determining the prevalence of each reaction, as well as their localisation within the cell. Introduction Throughout the human body, different enzymes interact with one another to form comprehensive reactions and chains of reactions that play important roles in the functioning of the human body (Schilling et al., 1999). The exact combination of enzymes and reactions that occur differ depending on the part of the body that is being considered, and the function of the cell. For example, the cells in the brain may perform some of the same functions as cells in the liver, such as cell replication, while other functions are significantly different, resulting in the use of different enzymes. The liver has a range of roles, including the maintenance of blood glucose during starvation by the activation of glycogen. It is a critical organ for survival (Salway, 2012). As well as variation in function and enzyme composition across organs and areas of the body, this also occurs within organelles present within cells. Determining what enzymes are present in different cellular components and their level of activity can be achieved through the use of cell fractionation techniques (Scnaitman and Greenawalt, 1968). One cellular component of interest is the mitochondria, which are essential organelles, involved in cell death pathways as well as the production of metabolic energy (van Loo et al., 2002). Within cells, the enzyme malate dehydrogenase (MDH) is responsible for interconverting L-malate and oxaloacetate, making use of nicotinamide adenine dinucleotide (NAD) as a coenzyme. MDHs are some of the most active enzymes present in the mitochondria and cytosol (Gietl, 1992). As such, the reaction is dependent on the present of malate or oxaloacetate as a substrate, and the presence of NAD. NAD+ is used in the conversion of malate to oxaloacetate, and the reaction produces NADH (Worthington Biochemical Corporation, 2012). This reaction is important biologically, because NAD+ and NADH play critical roles in a range of cell functions and in apoptosis (programmed cell death) (Ying, 2008). Measuring the activity of MDH can be determined by using an electron acceptor to remove the H+, preventing the reverse reaction from occurring. Thus, the amount of electrons transferred to the acceptor can be used to measure the activity. One approach to this is using iodophenyl nitrophebyl tetrazolium chloride (INT) coupled with phenazine methosulphate (PMS), because this produces a red colour that can be directly measured (Moorjani and Lemonde, 1967). This approach can also be used to determine the activity of lactate dehydrogenase (LDH), which results in the production of pyruvate and NADH. Likewise, succinate dehydrogenase (SDH) acts as a catalysis for the conversion of succinate to fumarate can be measured using an electron activity. The aim of this experiment was to fractionate liver cells and determine the amount of lactate and malate dehydrogenase that were present in the cells, as well as the activity of succinate dehydrogenase. Finally, the experiment aimed to estimate the protein concentration in each of the cell fractions. The fractionation process resulted in the soluble components of the cell being left behind, which allowed for examination of the enzymes present within the mitochondria. Methods To create liver homogenate and cell fractions, fresh liver (30g) was homogenised in isotonic KCl (270ml). This homogenate was then strained and centrifuged (3,500rpm/10 minutes), the supernatant removed and the pellet resuspended in 10ml isotonic saline. Following this, 2ml of the mixture was transferred to a clean test tube and 2% Triton (0.1ml) was added and mixed. This created a fraction of lysed liver particulate. The next aspect of the experiment involved the determination of MDH and lactate. For each enzyme an assay was undertaken and optimised. This involved the addition of 0.1ml of enzyme to 2ml of an INT/PMS mixture. 25µl of the appropriate fraction was added to initialise the reaction, and this was stopped after 5 minutes by the addition of n-propanol/1M HCL (3ml). Finally, the absorbance was measured at 540nm for each incubation. Once the assay had been optimised, the procedure was ran three times for each enzyme. Following this, the experiment determined the activity of succinate dehydrogenase within the liver cells. This involved the addition of 4.75ml of a PMS/DCPIP/KCN cocktail into a test tube. 50µl of the appropriate fraction was then added, and the mixture preincubated for 5 minutes. After the 5 minutes, 0.2ml of de-ionised water was added to the tube for the control assay, and 0.2ml of succinate to the experimental tube. Both test tubes were then incubated in the dark for ten minutes, absorbance measured at 600nm. Following the optimisation of the assay, the control and experimental assays were run three times each. Finally, protein concentration was estimated. Five protein solutions were created with concentrations ranging from 0 to 10mg/ml. For each test solution and standard, 3ml of Biuret reagent was added, incubated for 30 minutes and then absorbance read at 60nm. Results The MDH assay detected an average of 0.012 absorbance/mg/ml. The majority of activity was recorded in the lysed liver particulate, while a small amount was observed in the supernatant and almost no activity was observed in the liver particulate. The LDH assay found a lower average level of activity (0.001 absorbance/mg/ml), and activity was primarily in the supernatant, with low levels of activity in the liver particulate and lysed liver particulate. Finally, the activity observed for SDH was relatively constant across the different factions, with slightly higher activity observed in the liver particulate and lysed liver particulate (Figure 1). Figure 1: Enzyme activity of lactate dehydrogenase, malate dehydrogenase and succinate dehydrogenase in each of the three cell fractions. To assay each fraction in relation to an unknown protein, volumes ranging from 6 to 7ml were taken, as these resided within the standard curve that had been established (Figure 2). The absorbance (AU/mg/ml) was lowest in the supernatant, and similar between the liver particulate and the lysed liver particulate, with the former being slightly higher. Figure 2: Standard curve for enzyme activity against the concentration of an unknown protein. Figure 3: Specific enzyme activity (AU/mg/min) for each of the three fractions. Discussion The assays showed that localisation of enzymes were different depending on the specific enzyme that was being examined. For LDH, the enzyme was mostly located in the supernatant, indicating that it was not present in any of the major organelles. The presence of a low amount of LDH within other fractions was likely to be due to ‘leaky’ membranes rather than the enzyme localising within the fraction. In contrast, MDH was predominantly in the lysed liver particulate, suggesting the enzyme was stored within the mitochondria. The fact that the enzyme was not observed in the non-lysed liver particulate strengthens this hypothesis; as it means that the mitochondria cells needed to be broken open before the enzyme could be detected. The prevalence of MDH in the mitochondria was expected, given that MDH is involved in an energy producing reaction, and the production of energy is one of the key functions of the mitochondria. However, it is interesting that LDH was not localised in the mitochondria, but instead in the supernatant. SDH behaved in a different manner, and was present in similar proportions in each of the fractions. The size of the activity and the similarity across fractions indicates that this was probably not an effect of the enzyme being ‘leaked’ from one fraction to the next. Instead, the distribution of the enzyme across fractions indicates that it is localised in multiple parts of the cell. The assay of activity for the three factions showed that the activity in both the liver particulate and the lysed liver particulate was similarly high, while activity in the supernatant was less than half the value. This suggests that enzyme activity is strong in and around the cell nucleus and mitochondria than it is in the surrounding liquid. Conclusion Malate dehydrogenase was localised primarily within the mitochondria Lactate dehydrogenase was localised primarily in the supernatant Succinate dehydrogenase was localised approximately equally across all three fractions Enzyme activity was lowest in the supernatant and higher in both the liver particulate and the lysed liver particulate References Gietl, C. 1992. Malate dehydrogenase isoenzymes: cellular locations and role in the flow of metabolites between the cytoplasm and cell organelles. Biochemica et Biophysica Acta, 1100, 217-234. Moorjani, S. & Lemonde, A. 1967. Dehydrogenases during postembryonic development of tribolium Confusum duval (Coleoptera): I. malate dehydrogenase and malic enzyme in the particulate and soluable fractions. Canadian Journal of Biochemistry, 45, 1393-1400. Salway, J. G. 2012. Medical biochemistry at a glance, West Sussex, John Wiley & Sons, Ltd. Schilling, C. H., Schuster, S., Palsson, B. O. & Heinrich, R. 1999. Metabolic pathway analysis: Basic concepts and scientific applications in the post-genomic era Biotechnological Progress, 15. Scnaitman, C. & Greenawalt, J. W. 1968. Enzymatic properties of the inner and outer membranes of rat liver mitochondria. The Journal of Cell Biology, 38. van Loo, G., Saelens, X., van Gurp, M., McacFarlane, M., Martin, S. J. & Vandenabelle, P. 2002. The role of mitochondrial factors in apoptosis: A Russiam roulette with more than one bullet. Cell Death and Differenciation, 9, 1031-1042. Worthington Biochemical Corporation. 2012. Malate Dehydrogenase [Online]. Available: http://www.worthington-biochem.com/mdh/default.html [Accessed August 7 2012]. Ying, W. 2008. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: Regulation and biological consequences. Antioxidants & Redox Signalling, 10, 179-206. Read More