Succinate Dehydrogenase Activity of Mitochondria

Mitochondria 1

Succinate Dehydrogenase Activity of Mitochondria

Albana Gashi

BIO 355

April 8, 2010

Introduction

Mitochondria are important organelles found in most eukaryotic cells. They are usually referred to as “cellular power plants” because they are responsible for energy conversion that produces ATP. Mitochondria are highly mobile because they use cytoskeletal proteins as tracks for their movement (Anesti and Scorrano 2006). These cytoskeletal proteins have the ability to influence shape and function of the organelle. They contain the enzymes of the citrate cycle in the matrix space and have relatively permeable outer membrane (Sherratt 1991). In aerobic cells, mitochondria are the main site of ATP production (Sherratt 1991).

Adenosine triphosphate (ATP) is produced through cellular respiration when sugars, fatty acids, and amino acids are broken down to carbon dioxide and water. The chemical energy produced during this process is ATP. A reaction that takes place is known as the Krebs cycle, which is complex pathway that involves metabolic intermediates and many enzymes, such as succinate dehydrogenase (SDH). SDH is part of a multiprotein complex of the electron transport chain and is the only enzyme of the Kreb’s cycle bound to the inner mitochondrial membrane (Burke et al. 1982).

In this experiment, we wanted to measure the initial velocity of the succinate using a variety of concentrations of the enzyme by adding different volumes of the re-suspended mitochondrial fraction. The velocity of the reactions will be determined by measuring the change in absorbance with time. We expected the velocity of the enzyme reactions to be altered by the presence or lack of an inhibitor, such as malonate or succinate.

Methods

The mitochondrial fraction was isolated from cauliflower cells by removing the outer layer of the cauliflower surface with a single-edge razor blade. The tissue was placed in chilled mortar with 40ml of ice-cold mannitol grinding medium and 5g of cold purified sand. The tissue was grinded with a chilled pestle for four minutes. The suspension was filtered through four layers of cheesecloth into a chilled 50-ml centrifuge tube while ringing out the juice into the tube. The filtrate was centrifuged at 600g for 10 minutes at 0*-4*C while making sure the centrifuge tubes were balanced. The postnuclear supernatant was decanted into a clean, chilled centrifuge tube and spun again at 10,000g at 0*-4*C. The postmitochondrial supernatant was decanted and discarded. 7.0ml of ice-cold mannitol assay medium was added to the mitochondrial pellet. The mitochondrial pellet was scraped from the wall of the centrifuge tube and then the sediment was re-suspended in the assay medium using a Pasteur pipet. The mitochondrial suspension was transferred to a test tube and placed in an ice-water bath where it was kept during the entire experiment.

The next series of steps were performed to measure the succinate dehydrogenase activity. The spectrophotometer was turned on and the wavelength was set at 600nm. Ten cuvettes were labeled 1-7 and blank 1, 2, and 3. All solutions were kept at room temperature except the ice-cold mitochondrial suspension. The mitochondrial suspension for tube 7 was prepared by heating a 0.6-ml aliquot in a boiling-water bath for 5 minutes and then cooled in an ice-water bath. Various solutions were added to all cuvettes except for the mitochondrial suspension (Table 1). The correct volumes of assay medium, azide, DCIP, malonate, and succinate were added to all tubes as indicated in table 1. After the correct solutions were added to each tube each cuvette was covered with Parafilm and inverted twice to mix the contents. The mitochondrial suspension was thoroughly re-suspended with a Pasteur pipet and the correct volume was added to each cuvette while recording the time in table 2 and 3. As soon as the mitochondrial suspension was added, the cuvette was covered with Parafilm and inverted twice to mix contents. The Parafilm was removed and the tubes were placed in the test tube rack. As soon as the mitochondrial suspension was added to tube 1, the spectrophotometer was adjusted for blank 1 the absorbance reading for tube 1 was taken. Then it was adjusted for blank 2 and tube 2 absorbance reading was taken. Then blank 3 and tube 3-7 absorbance readings were taken. This process was repeated every 5 minutes for 35 minutes. All of the absorbance readings were recorded in figure 2.

Table 1. Volumes of each solution to be added to each tube.

Tube / Assay Medium / Azide (0.04M) / DCIP (5x10-4M) / Malonate (0.2M) / Succinate (0.2 M) / Mitochondrial suspension
Blank 1 / 3.7ml / 0.5ml / ----- / ----- / 0.5ml / 0.3ml
1 / 3.2ml / 0.5ml / 0.5ml / ----- / 0.5ml / 0.3ml
Blank 2 / 3.1ml / 0.5ml / ----- / ----- / 0.5ml / 0.9ml
2 / 2.6ml / 0.5ml / 0.5ml / ----- / 0.5ml / 0.9ml
Blank 3 / 3.4ml / 0.5ml / ----- / ----- / 0.5ml / 0.6ml
3 / 2.9ml / 0.5ml / 0.5ml / ----- / 0.5ml / 0.6ml
4 / 2.7ml / 0.5ml / 0.5ml / 0.2ml / 0.5ml / 0.6ml
5 / 3.4ml / ------/ 0.5ml / ----- / ----- / 0.6ml
6 / 3.4ml / 0.5ml / 0.5ml / ----- / 0.5ml / 0.6ml
7 / 2.9ml / 0.5ml / 0.5ml / ----- / 0.5ml / 0.6ml

Results and Discussion

The total change in absorbance at each time interval is the difference between the 5-minute reading for tube 7 and the reading for each tube at the specified time (Fig 1). The 5-minute absorbance reading for tube 7 was taken as the 0-minute reading for all tubes because it’s a control tube. Tube 7 was heat shocked, which denatures the protein. This would result in the graph line being horizontal.

The 0.9ml of mitochondrial suspension had the highest initial velocity because tube 2 reacted the quickest (Fig 2). When the enzyme concentration is doubled or tripled, the initial velocity will also increase as well because they are proportional to each other. The initial velocity in tube 4 was different from tube 3 because tube 4 contained malonate and was inactive, while tube 3 did not contain malonate and had a quicker initial velocity. Tube 4 was inactive because malonate is an inhibitor that causes mitochondrial collapse and swelling which induces cell death (Fernandez-Gomez et al. 2005).

It was evident the electron transport system was functioning in the isolated mitochondria due to the reduction in DCIP. DCIP is used because it can perform rapid analyses as a redox color indicator (Yoshida et al. 2002). This was visible through the color changes taking place. The solution went from blue to colorless. There is evidence there was succinate in the mitochondrial fraction due to the inactivity in tube 6. It did not undergo a reaction. This indicates the mitochondria could not produce its own succinate when cellular respiration is not occurring due to the lack of glucose, which caused tube 6 to be inactive. However, when succinate was added, the reactions were not inactive.

Figure 1. Total change in absorbance between the 5-minute reading for tube 7 and the reading for each tube at the specified time.

Figure 2. Absorbance readings of tubes 1-7 showing the initial velocity of each reaction.

References

Anesti, V., L. Scorrano. 2006. The relationship between mitochondrial shape and function and the cytoskeleton. Bioenergetics 1757: 692-699.

Burke, J.J, J.N. Siedow, D.E. Moreland. 1982. Succinate dehydrogenase: a partial purification from mung bean hypocotyls and soybean cotyledons. Plant Physiology 70: 1577-1581.

Fernandez-Gomez, F.J., M.F. Galindo, M. Gomez-Lazaro, V.J. Yuste, J.X. Comella, et al. 2005. Malonate induces cell death via mitochondria potential collapse and delayed swelling through an ROS-dependent pathway. British Journal of Pharmacology 144: 528-537.

Sherratt, HS. 1991. Mitochondria: structure and function. Rev Nuerol (Paris) 147: 417-430.

Yoshida, N., S.J. McNiven, T. Morita, H. Nakamura, I. Karube. 2002. A simple, multiple simultaneous spectrophotometric method for bod determination using DCIP as the redox color indicator. Analytical Letters 35: 1541-1549.