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....

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 ....

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