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1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster
<ol>
	<li>Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype.</li>
	<li>Prepare 500 &#181;L 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.</li>
	<li>Anesthetize adult flies with CO2&#160;on an anesthesia pad and isolate the desired tissues. To isolate the adult fly thoraxes, for example, fix the fly t...

citrate synthase activity assay: a colorimetric assay to measure mitochondrial citrate synthase activity in drosophila tissue homogenate

Source: Wei, P., et al., <a href="https://www.jove.com/v/59454">A Colorimetric Assay of Citrate Synthase Activity in&#160;Drosophila Melanogaster.</a> J. Vis. Exp. (2020)
This video describes a colorimetric assay to measure the mitochondrial citrate synthase activity assay present in the tissue homogenate of Drosophila.

Citrate Synthase Activity Assay: A Colorimetric Assay to Measure Mitochondrial Citrate Synthase Activity in Drosophila Tissue Homogenate

Source: Wei, P., et al., A Colorimetric Assay of Citrate Synthase Activity in&#160;Drosophila Melanogaster. J. Vis. Exp. (2020)
This video describes a ...

Within the mitochondria, citrate synthase, localized in the matrix of the inner mitochondrial membrane, is the rate-limiting enzyme that catalyzes the reaction between acetyl coenzyme A and oxaloacetic acid to form citrate, the first step of the citric acid cycle.&#160;
To analyze citrate synthase activity, transfer adult Drosophila fly thoraxes abundant in mitochondria into a tube filled with a chilled extraction buffer containing ethylenediaminetetraacetic acid, EDTA, and a non-ionic detergent.
Homogenize the thorax tissues on ice to prevent heat-mediated protein denaturation. The applied shear force, in conjunction with the detergents, lyses the tissue and releases intracellular components, including mitochondria.
Further, the rupture of the inner mitochondrial membrane leads to the release of mitochondrial matrix proteins, including citrate synthase. EDTA binds to divalent cations and inhibits protease activity, thereby preventing protein degradation.
Dilute the homogenate using extraction buffer and add an appropriate volume into the reaction mixture containing wells of the multi-well plate. The reaction mixture comprises acetyl coenzyme A, oxaloacetic acid, and dithionitrobenzoic acid.
Citrate synthase in the homogenate catalyzes the reaction between oxaloacetic acid and acetyl coenzyme A, forming citrate and coenzyme A with a thiol group. The thiol group reacts with the colorless dithionitrobenzoic acid, forming a yellow product, thionitrobenzoic acid.
Using a microplate reader, measure the absorbance of the yellow-colored solution. The absorbance could be related to citrate synthase activity in the tissue homogenate.

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Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

JoVE Journal

Biochemistry

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Citrate Synthase Activity Assay: A Colorimetric Assay to Measure Mitochondrial Citrate Synthase Activity in Drosophila Tissue Homogenate

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