This lab report discusses the Cellular Location of Metabolic Enzymes. Regarding methodology, the experiment was conducted in three sessions during the first session liver homogenate and cell functions were prepared. The first session was carried out by homogenizing the liver in an isotonic media. The Maltase and lactate dehydrogenase were determined in the second session, and protein concentrations were estimated in the third session. The Maltase dehydrogenase (MDH) and Lactase dehydrogenase (LDH) were used to catalyze the reactions in session two. The biuret reaction was used to estimate the protein in the last session. The relationship between absorbance at 600 nm and protein standard was established using the equation Y=0.3424+ 0.022.The concentrations were 1.554 mg/ml, 0.572 mg/ml and 0.534 mg/ml for fractions A, B and C respectively. The MDH/LDH activity was 0.6936 (Au/min/ml), 0.064 (Au/min/ml) and 0.072 (Au/min/mg0 for fractions A, B and C respectively. The specific activity was 0.446 (AU/min/mg protein), 0.112 (AU/min/mg protein) and 0.135 (AU/min/mg protein for fractions A, B and C respectively.
The multienzyme and enzyme systems possess characteristic intracellular locations within the cell. For instance glycolysis in the eukaryotic cells takes place in the cytoplasm whereas the citric acid cycle takes place in the mitochondrial membrane's matrix. Furthermore, oxidative phosphorylation and electron transport take place in the inner mitochondrial membrane. Nevertheless, the metabolic compartmentalization does not imply that the systems are independent of one another but rather they are transport mechanisms that facilitate the transportation of metabolic intermediates in various compartments. Through the addition of a detergent such as Triton, the mitochondrial membranes lyse completely, and it liberates the proteins that may be present in the mitochondria's matrix (Raimundo 2014). It is imperative to note that the mitochondria prepared through the centrifugation procedure may not exhibit properties that can be demonstrated in isolated preparations, as such the membrane become “leaky” and subsequently small molecules permeate the membranes of the mitochondria. This experiment aims at fractionating liver cells with an objective of determining the cellular location of crucial metabolic enzymes. The experiment was divided into three sessions, and the first session focused on preparing the cell fractions and liver homogenate. Session two focused on the determination of the Maltase and Lactate Dehydrogenase whereas Session three dealt with the estimating Protein concentration.
2.1 Session 1: Preparation of the Cell fractions and Liver Homogenate
2.2 Session 2: Determination of the Maltase and Lactate Dehydrogenase
First and foremost 9 test tubes were labeled in the following sequence Lactate/Fraction A; Lactate/Fraction B; Lactate/Fraction C; Maltase/Fraction A; Maltase/Fraction B; Maltase/Fraction C; Water/Fraction A; Water/Fraction B; Water/Fraction C). After labeling, 2ml of INT/PMS were pipette into every test tube then 0.1 ml of the lactate were added into the first, second and third test tube to assay for LDH. Also, 0.1 ml of maltase was added into the fourth, fifth and sixth test tubes to assay MDH and all the contents were mixed well. The enzyme reaction was started by adding 250ml* of Fraction C was added to the third, sixth and eight tubes then mixed thoroughly. The volumes of the fractions that were used were altered, and the assays were re-run by assessing the enzyme activities. In cases where the absorbance was high, small fractions were used whereas in those tubes where absorbance was low, big fractions were used. The reactions were stopped by adding 3 ml of 1 N HCL in all the test tubes and after the addition, the contents were nixed well. The absorbance was determined for all tubes at 540nm after each incubation.
2.3 Session 3: Estimating Protein Concentration
10 mg/ml of a protein solution were used to prepare five standards of solutions with concentrations ranging from 0 to 10 mg/ml. The three fractions (A, B and C) were mixed with a Biuret Reagent of 3 ml thus; the total number of tubes was eight. The tubes were incubated for thirty minutes, and the absorbance was read at 600 nm. The concentration of the protein in each fraction was calculated, and it was measured in the form of mg/ml.
A graph that depicts the correlation between the standard protein absorbance at 600nm is provided in the figure below.
Figure 1: Protein assay
According to the graph 1, y = 0.3424x + 0.022, hence to the concentration of protein in mg/ml of each fraction can be computed the in the following steps
x= Fraction A/B/C-0.0220.3424
The MDH/ LDH activity is provided in Table 2 below, and it is used to compute the change in the absorbance/min/ml for every fraction in the MDH/LDH activity.
The absorbance of the Fractions A, B and C is divided by five because the assay is five minutes and this yields the per minutes. Accordingly,
250μl of Fraction A is used, so it is multiplied by 4 to obtain per ml
25μl of Fraction B and C are used; they are multiplied by 40 to yield per ml.
The specific activity is computed by using the protein concentration data and the data on the LDH and MDH activity. The data from Table 3 is divided by the respective protein concentrations for fractions A. B and C contained in Table 1 b.
0.6936÷1.554=0.446 (AU/min/mg protein)
0.064÷0.572=0.112 (AU/min/mg protein)
0.072÷0.534=0.135 (AU/min/mg protein)
The specific activity data is summarized in Table 4
Additionally, the specific data activity is plotted in figure 2 below
Figure 2: Specific activity data
4.1 Session 1
After the homogenization of liver in an isotonic media through mild procedures, the mitochondria, and cell nuclei stay intact. Centrifugation o the sub cellular structure out of the homogenate leaves behind the liver’s soluble components in the supernatant and the soluble components include cytoplasmic enzymes (Raimundo 2014).
4.2 Session 2
The reaction is catalyzed by Maltase dehydrogenase (MDH) as represented by the equation below.
Maltase + NAD+.NADH + H+
The activity of MDH is obtained by measuring the Production of NADH. The role of NDAH is to reduce the dye. After the reduction of the dye, it becomes red colored due to the formation of NADH. Accordingly, a spectrophotometer can be used to determine the MDH activity and the formation of NADH. There are two electrons that are employed in the process, and they include phenazine methosulphate (PMS) and iodophenyl nitrophenyl tetrazolium chloride (INT) (Suzuki et al.2013).The role of Phenazine methosulphate is to transfer electrons from the NADH to the electron acceptor that is iodophenyl nitrophenyl tetrazolium chloride (INT) (Trabbic et al. 2013). A summary of NADH formation is provided below.
In the same way, Lactate dehydrogenase plays an instrumental role in catalyzing the reaction and the reaction is summarized in the following equation.
Lactate + NAD+ +
The LDH activity leads to the formation of NADH, and this can be established spectrophotometrically which entails using an INT cocktail or PMS to monitor the generation of the red color (Wang et al. 2012).
4.3 Session 3
The color of chelate that is formed at the room temperature between the nitrogen atoms in the peptide bonds and the copper alkaline solutions can be used to estimate the concentration of polypeptide. The estimation is referred to as Biuret reaction. The color reaction is standardized by bovine serum albumin. Since proteins have unique combinations of amino acids, they yield varying colors in every unit mass of the polypeptide. In the case of assays of unknown proteins, the method yields results that are expressed in the form of equivalent concentration of the BSA (Gatica‐Sosa et al. 2012).
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