Description
Key facts
Detection method:Colorimetric,
Sample types:Cell culture extracts, Tissue,
Assay type:Enzyme activity,
Assay time:2h,
Assay Platform:Microplate reader
Product details:
Aconitase Assay Kit ab109712 is a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates.
Aconitase assay principle
In the aconitase 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. The rate of cis-aconitate production is proportional to aconitase activity.
The active form of aconitase 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.
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 also be activated in vitro by the addition of iron and cysteine (not provided in this kit).
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.
Aconitase assay protocol summary
- prepare samples and used aconitase preservation solution to stabilize aconitase activity
- add samples and reagents to plate
- incubate at room temperature
- readout on a colorimetric plate reader at 0-min and 30-min
Related Aconitase assay kits
< br/> For an alternative aconitase assay method, based on an enzyme-coupled reaction with isocitrate dehydrogenase, use Aconitase assay kit
ab83459
ab83459
is our most popular Aconitase assay kit.
Other notes
Previously called Aconitase Enzyme Activity Microplate Assay Kit and Aconitase Activity 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.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-Multi, Appropriate long-term storage conditions-Multi, Storage information-Please refer to protocols
Supplementary Information:
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.
Biological function summary
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.
Pathways
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.
