How do you measure aconitase activity?
How do you measure aconitase activity?
In the aconitase activity assay protocol, aconitase catalyzes an equilibrium between aconitate, cis-aconitate and iso-citrate. These reactions are monitored by measuring the increase in absorbance at 240 nm associated with the formation of the cis-aconitate which has an extinction coefficient of 2.2 OD/mM per well.
What is aconitase Characterised by?
For the determination of kinetic constants, aconitase was heterologously overproduced, purified, reactivated and biochemically characterised. Aconitase was active over a wide pH and temperature range with maximal activity between pH 7.5 and 7.75 and at about 50 °C.
What does aconitase do in the citric acid cycle?
The enzyme aconitase is a key player in the central pathway of energy production. As part of the citric acid cycle, it converts citrate into isocitrate.
What type s of reaction does aconitase catalyze?
Aconitase from pig (PDB 7acn) is a single polypeptide (Mr 83kD) that catalyzes the reversible isomerization of citrate and isocitrate. It is the second enzyme in the Citric acid cycle, which is a series of enzyme-catalysed chemical reactions that is crucial to aerobic cellular respiration and the production of ATP.
What is mitochondrial Aconitase?
Mitochondrial aconitase is a reversible enzyme that catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid cycle. Mitochondrial aconitase is very sensitive to oxidative inactivation and can aggregate and accumulate in the mitochondrial matrix causing mitochondrial dysfunction.
What happens if aconitase is inhibited?
The inhibition of aconitase can lead to accumulation of citrate because aconitase mediates its conversion to isocitrate.
Is aconitase reversible?
Aconitase (aconitate hydratase; citrate hydrolyase; EC 4.2. 1.3) catalyzes the reversible stereo-specific isomerization of citrate to isocitrate via cis-aconitate as an intermediate in a non-redox-active manner (Fig. 1). Aconitase belongs to the family of iron–sulfur-containing dehydratases.
What is the function of cytosolic aconitase?
Cytosolic aconitase links iron metabolism, unfolding and proteolytic degradation of proteins with ROS metabolism. Oxidation of aconitase by ROS/RNS results in loss of labile iron, inactivating the enzyme and switching the function to regulation of iron metabolism as iron regulatory protein 1 (IRP1).
Is aconitase a isomerase?
The iron-responsive element-binding protein (IRE-BP) and 3-isopropylmalate dehydratase (α-isopropylmalate isomerase; EC 4.2. 1.33), an enzyme catalysing the second step in the biosynthesis of leucine, are known aconitase homologues.
Which is the best aconitase activity assay kit?
Guaranteed product quality, expert customer support. Find out more. Aconitase Activity Assay Kit ab109712 is a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates. Unlike other aconitase assays this is not a coupled reaction and therefore only aconitase activity is required and measured.
How is aconitase activity measured in a spectrophotometer?
Using a UV–vis spectrophotometer, aconitase activity can be quantified according to the formation of NADPH (Kennedy et al., 1983; Rose & O’Connell, 1967 ). The reaction is best monitored in an instrument that can perform kinetic measurements, as the reaction is typically complete within 10 min.
What is the extinction coefficient for the aconitase assay?
In the aconitase activity assay protocol, aconitase catalyzes an equilibrium between aconitate, cis-aconitate and iso-citrate. These reactions are monitored by measuring the increase in absorbance at 240 nm associated with the formation of the cis-aconitate which has an extinction coefficient of 2.2 OD/mM per well.
What is the role of aconitase in the tricarboxylic cycle?
Aconitases are enzymes that catalyze the stereospecific isomerization of citrate to isocitrate in the tricarboxylic cycle [1 ]. They are iron–sulfur proteins that contain [4Fe—4S] clusters.