A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Summary

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Abstract

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

Introduction

Mitochondria are best known as the power-producing organelles in most eukaryotic organisms, which produce the energy currency, ATP, through the tricarboxylic acid cycle (i.e., Krebs cycle) and oxidative phosphorylation. Mitochondria are also found to play important roles in a lot of other physiological processes, such as regulation of apoptosis¹, Ca²⁺ homeostasis^2,3, reactive oxidation species (ROS) generation⁴, and endoplasmic reticulum (ER)-stress response⁵. Mitochondrial dysfunction can affect any organ in the body at any age and is a primary cause of metabolic, aging-related⁶, and neurodegenerative diseases⁷. Intact mitochondria are mechanistically related to mitochondrial function. Thus, proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function⁸. Citrate synthase, a rate-limiting enzyme in the first step of the tricarboxylic acid cycle⁹, is localized in the mitochondrial matrix within eukaryotic cells, and thus can be used as a quantitative marker for the presence of intact mitochondrial mass^9,10. Citrate synthase activity can also be used as a normalization factor for intact mitochondrial proteins^11,12.

The fruit fly, Drosophila melanogaster, is an excellent model system for studying mitochondrial function, as many molecules and pathways that play pivotal roles in mitochondria are evolutionarily conserved from Drosophila to humans^13,14,15. Here, we present a protocol for a fast and simple method for measurement of citrate synthase activity by a colorimetric assay in Drosophila tissue homogenates¹⁶ in a 96 well plate format. In the citrate synthase activity assay, citrate synthase in Drosophila tissue homogenate catalyzes the reaction of oxaloacetate with acetyl coenzyme A (acetyl CoA) to form the citrate CoA-SH and H⁺. CoA-SH subsequently reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to generate a colored product, 2-nitro-5-thiobenzoate (TNB), which can be easily measured spectrophotometrically at 412 nm. Citrate synthase activity can be reflected by the rate of color production.

Protocol

1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster¹⁶

1. Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype.
2. Prepare 500 µL of ice-cold extraction buffer containing 20 mM HEPES (pH = 7.2), 1 mM EDTA, and 0.1% triton X-100 in a 1.5 mL test tube for each sample.
3. Anesthetize adult flies with CO₂ on an anesthesia pad and isolate the desired tissues. To isolate the adult fly thoraxes, for example, fix the fly thoraxes by a pair of forceps, and then isolate the fly abdomens by cutting along the border of the thorax and abdomen using a pair of scissors. Collect the fly thoraxes for the muscle citrate synthase activity assay.
   NOTE: Determine the fresh weight of tissue at this step if using it to normalize citrate synthase activity.
4. Transfer 10 adult fly thoraxes to 100 µL of ice-cold extraction buffer immediately and homogenize with a pestle on ice. To keep the samples ice-cold, homogenize the samples for 5-10 s on ice, then have the samples sit on ice for 5 s, and repeat until all the tissues in the tube are homogenized completely.
   NOTE: Tape the tube, check the homogenates to make sure that there are no clots in the homogenates and that the tissues in the tube are homogenized completely. All sample treatments should be performed on ice.
5. Take 10 µL of each homogenized sample in a new tube for protein content measurement. Keep the samples for protein measurement on ice.
   NOTE: The protein samples can be frozen and stored at -80 °C for later analysis. The protein concentrations serve as an internal parameter for normalization of citrate synthase activities.
6. Add 400 µL of ice-cold extraction buffer to each remaining sample to make a total volume of 500 µL. Mix well by gently pipetting up and down at least 5x, avoiding bubble formation. For each reaction, add 1 µL of diluted cell lysate to 150 µL of the freshly prepared reaction solution (20 mM Tris-HCl (pH 8.0), 0.1 mM DTNB, 0.3 mM acetyl CoA, 1 mM oxaloacetic acid). Mix thoroughly and immediately by gently pipetting, avoiding bubble formation. Measure the absorbance at 412 nm every 10-30 s for 4 min at 25 °C using a plate reader capable of measuring absorbance at 412 nm at minimum intervals of 10 s.
   NOTE: Change the tip before pipetting a different reagent and avoid forming bubbles in the wells. Incomplete mixing of the reagents may cause variations in the measurements. The citrate synthase activity assay should be done at room temperature immediately after the samples are collected, as this assay is used to quantify intact mitochondrial mass. The reaction buffer should be prepared immediately before use and should not be stored.
7. Plot data as optical density (OD) absorbance (abs) (Y-axis) versus time (in minutes) (X-axis). Then calculate the slope for the linear portion of the curve, where the reaction rate is
   
   
   
   Divide the value by the sample protein concentration to normalize the citrate synthase activity. The citrate synthase activity is calculated as
   
   
   
   NOTE: The sample protein concentration can be measured by a protein concentration assay kit.

Results

Figure 1 presents an example of the kinetic curves for the OD absorbance at 412 nm over time obtained using the citrate synthase activity colorimetric assay to measure the Drosophila thorax tissue homogenates of different genotypes. It is well known that PGC-1α is a master regulator of mitochondrial biogenesis. PGC-1α is functionally conserved between Drosophila and humans. Drosophila RNF34 (dRNF34) is an E3 ubiquitin ligase for

Discussion

Metabolic studies using Drosophila as a model must take into consideration the genetic background, diet, and stock maintenance of the flies¹⁸. To avoid the effects of different genetic backgrounds on the measurement of citrate synthase activity, different strains of Drosophila should be backcrossed to the control strain for 10 generations. The genetic background of all Drosophila strains used in our experiments is w¹¹¹⁸, so we used w¹¹¹⁸

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)	Sigma-Aldrich	V900477
2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA Base)	Sigma-Aldrich	V900483
Acetyl-CoA	Sigma-Aldrich	A2181
Dithio-bis-nitrobenzoic acid (DTNB)	Sigma-Aldrich	D8130
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	V900106
Oxaloacetate	Sigma-Aldrich	O4126
Pellet pestle	Sangon	F619072
Pellet pestle motor	Tiangen	OSE-Y10
Plate reader	BioTek	Eon
Protein BCA Assay kit	Beyotime	P0010S
Scissors	WPI	14124
Triton X-100	Sangon	A110694-0100