This study investigates the activity of citrate synthase, a key enzyme in the citric acid cycle, using adult Drosophila fly thoraxes. The methodology involves homogenizing the thorax tissues and measuring the absorbance of the resulting reaction mixture to determine enzyme activity.
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.
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.
Transfer 10 adult fly thoraxes to 100 microliters of ice-cold extraction buffer immediately, and homogenize them with a pestle on ice. Keep the samples cold by homogenizing for five to 10 seconds on ice, letting them sit on ice for five seconds, then, repeating until all tissues are completely homogenized.
Aliquot 10 microliters of each homogenized sample into a new tube for protein content measurement, keeping these tubes on ice as well. Add 400 microliters of ice-cold extraction buffer to each remaining sample for a total volume of 500 microliters, and pipette up and down gently to mix.
For each reaction, add 1 microliter of diluted cell lysate to 150 microliters of the freshly prepared reaction solution. Mix thoroughly by gently pipetting, making sure to avoid bubble formation. Then, use a plate reader to measure the absorbance at 412 nanometers every 10 to 30 seconds for four minutes at 25 degrees Celsius.
Plot the data as absorbance over time, then, calculate the slope for the linear portion of the curve. Finally, divide the reaction rate or slope by the sample protein concentration to normalize the citrate synthase activity.
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