Aconitase Activity Assay Kit (ab109712)
Key features and details
- Assay type: Enzyme activity
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 2 hr
- Sample type: Cell culture extracts, Tissue
Overview
-
Product name
Aconitase Activity Assay Kit
See all Aconitase kits -
Detection method
Colorimetric -
Sample type
Cell culture extracts, Tissue -
Assay type
Enzyme activity -
Assay time
2h 00m -
Product overview
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 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. 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.
-
Notes
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.
-
Platform
Microplate reader
Properties
-
Storage instructions
Store at +4°C. Please refer to protocols. -
Components 96 tests Detergent 1 x 1ml 96-well UV microplate 1 unit Aconitase Preservation Solution 1 x 20ml Buffer 1 x 50ml Isocitrate (25X) 1 x 800µl Manganese (100X) 1 x 200µl -
Research areas
-
Relevance
Aconitase (aconitate hydratase; EC 4.2.1.3) is an iron-sulfur protein containing an [Fe4S4]2+ cluster that catalyzes the stereospecific isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle, a non-redox-active process. Tissue contains two aconitases, a mitochondrial (m-) and a cytosolic (c-) aconitase. They are related, but distinctly different enzymes and are coded for on different chromosomes. Loss of aconitase activity in cells or other biological samples treated with prooxidants has been interpreted as a measure of oxidative damage. -
Cellular localization
ACO1: Cytoplasmic ACO2: Mitochondrial -
Alternative names
- ACO 1
- ACO 2
- ACO1
see all
Images
-
Mitochondrial aconitase enzymatic activities were measured using ab109712 Haitham E et al., PLoS One, 11(8). Fig 2d. doi: 10.1371/journal.pone.0160380 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
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.
-
ELISA - Aconitase Enzyme Activity Microplate Assay Kit (ab109712)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. -
ELISA - Aconitase Enzyme Activity Microplate Assay Kit (ab109712)Figure 2. Using cellular fractionation kit ab109719, whole cells were separated into cytoplasmic, mitochondrial and nuclear fractions.
