Aconitase Activity Assay Kit ab109712 is a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates.
Colorimetric
Cell culture extracts, Tissue
Enzyme activity
2h
Select an associated product type
Catalyzes the isomerization of citrate to isocitrate via cis-aconitate.
ACO1
Aconitase, Citrate hydro-lyase, ACO2
Aconitase Activity Assay Kit ab109712 is a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates.
Colorimetric
Cell culture extracts, Tissue
Enzyme activity
2h
Microplate reader
Blue Ice
Multi
Multi
Please refer to protocols
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.
In the aconitase activity assay protocol, aconitase catalyzes an equilibrium between citrate, 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. Therefore the rate of cis-aconitate production is proportional to aconitase activity.
Aconitase preservation solution, assay buffer, reagents and an essential UV microplate are provided for this measurement. The entire assay can be completed within 2 hours.
Note – mitochondrial and cytoplasmic aconitase activities are indistinguishable. Therefore, to measure the mitochondrial activity only, first isolate mitochondria, or for both activities fractionate the cells into cytoplasmic and mitochondrial.
Previously called Aconitase Enzyme Activity Microplate Assay Kit.
Aconitase (aconitate hydratase; EC 4.2.1.3) is an iron-sulfur protein that catalyzes the reversible inter-conversion of citrate and isocitrate, via a cis-aconitate intermediate, in both the TCA and glyoxylate cycles. The enzyme contains a [4Fe-4S] cluster which interacts directly with the substrates. In eukaryotes there are both mitochondrial and cytosolic forms of the enzyme. The mitochondrial form functions not only in the TCA cycle, but also to stabilize mtDNA thereby influencing mitochondrial gene expression. The cytosolic form can function as an aconitase as well as an iron regulatory protein.
The active form of the enzyme is inhibited by citrate analogs, and fluoracetate. Other inhibitors include oxidative stress agents such as peroxynitrite, hydrogen peroxide and superoxide, which inactivate the enzyme by changing the [4Fe-4S] to a [3Fe-4S] cluster. Aconitase is considered a good marker of mitochondrial and cellular oxidative stress. This change in mitochondrial aconitase can lead to a decreased energy production, whereas in cytosolic aconitase it triggers binding of the enzyme to mRNA iron response elements resulting in increased expression of iron uptake proteins and decreased transcription of iron sequestering protein.
A hydroxyl scavenging solution (Aconitase preservation solution) is supplied with this aconitase assay kit to maintain aconitase activity during sample preparation. An inactivated [3Fe-4FS] aconitase may be activated in vitro by the addition of iron and cysteine.
This supplementary information is collated from multiple sources and compiled automatically.
Aconitase also known as aconitate hydratase is an enzyme with a mass of approximately 82 kDa. It catalyzes the reversible isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid (TCA) cycle. Aconitase is expressed in the mitochondria where it functions as mitochondrial aconitase and in the cytoplasm in a slightly different form. The enzyme contains an iron-sulfur cluster that is important for its activity. Scientists often use aconitase kits to measure its activity in different samples.
Mitochondrial aconitase plays an important role in energy production by facilitating the TCA cycle which is essential for ATP generation. The enzyme can also serve as an iron regulatory protein in the cytoplasm contributing to the regulation of iron metabolism. Aconitase exists as part of a larger complex with its iron-sulfur cluster playing a critical role in both its enzymatic and regulatory functions. By shifting between its enzymatic and regulatory roles aconitase helps in maintaining homeostasis within cells.
Aconitase operates in the TCA cycle an important metabolic pathway for energy production. It participates in the metabolism of acetyl-CoA converting it into isocitrate through interactions with citrate. This key pathway integrates with glycolysis and aids in the oxidative phosphorylation process. Aconitase's function is related to proteins such as isocitrate dehydrogenase which further processes isocitrate in the TCA cycle.
Mutations and defects in aconitase can contribute to neurodegenerative diseases and iron-sulfur cluster deficiencies. A connection exists between aconitase activity and disorders like Friedreich’s ataxia a condition featuring impaired iron-sulfur cluster biosynthesis. Ferritin a protein associated with iron storage links with aconitase through pathways involved in iron regulation suggesting potential therapeutic targets in disorders of iron metabolism.
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Mitochondria were isolated from HL-1 cells following starvation protocol and approximately 50μg of mitochondrial preparation were placed in each microplate well. Equal amounts of the substrate isocitrate were added to all wells and the absorbance at 240nm was recorded for 30 minutes. The catalytic activity was measured by the rate of formation of cis-aconitate as detected by the increase in absorbance.
Figure 1. Bovine heart mitochondria were treated with increasing concentrations of hydrogen peroxide and peroxynitrite to generate aconitase activity IC50's for these oxidative stress agents.
Figure 2. Using cellular fractionation kit Cell Fractionation Kit - Standard ab109719, whole cells were separated into cytoplasmic, mitochondrial and nuclear fractions.
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