Aconitase Assay Kit

Cayman Chemical Item Number 705502

Aconitase Assay Kit

Description

Aconitase is an iron-sulfur protein containing a [Fe₄S₄]²⁺ cluster that catalyzes the stereospecific isomerization of citrate to isocitrate via cis-aconitate. Whereas exposure of aconitase to oxidants renders the enzyme inactive, loss of aconitase activity in cells or in biological samples treated with pro-oxidants has been interpreted as a measure of oxidative damage. Cayman’s Aconitase Assay Kit provides a simple, reproducible, and sensitive tool for assaying aconitase from tissue homogenates or cell lysates. In this assay, citrate is isomerized by aconitase into isocitrate, which is then converted to α-ketoglutarate in a reaction catalyzed by isocitric dehydrogenase. These reactions are monitored by measuring the increase in absorbance at 340 nm associated with the formation of NADPH. The rate of NADPH production is proportional to aconitase activity.

Stability

6 months

Storage

-20°C

Shipping

Wet ice in continental US; may vary elsewhere

Background Reading

Emptage, M.H., Dreyer, J., Kennedy, M.C., et al. Optical and EPR characterization of different species of active and inactive aconitase. J Biol Chem 258(18) 11106-11111 (1983).

Bulteau, A., Lundberg, K.C., Ikeda-Saito, M., et al. Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/reperfusion. Proc Natl Acad Sci USA 102(17) 5987-5991 (2005).

Delaval, E., Perichon, M., and Friguet, B. Age-related impairment of mitochondrial matrix aconitase and ATP-stimulated protease in rat liver and heart. Eur J Biochem 271 4559-4564 (2004).

Gardner, P.R., Nguyen, D.D.H., and White, C.W. Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and rat lungs. Proc Natl Acad Sci USA 91 12248-12252 (1994).

Villafranca, J.J., and Mildvan, A.S. The mechanism of aconitase action. J Biol Chem 246(18) 5791-5798 (1971).

Kennedy, M.C., Mende-Mueller, L., Blondin, G.A., et al. Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci USA 89 11730-11734 (1992).

Beinert, H., Kennedy, M.C., and Stout, C.D. Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein. Chem Rev 96 2335-2373 (1996).

Sipos, I., Tretter, L., and Adam-Vizi, V. Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals. J Neurochem 84 112-118 (2003).

Yan, L., Levine, R.L., and Sohal, R.S. Oxidative damage during aging targets mitochondrial aconitase. Proc Natl Acad Sci USA 94 11168-11172 (1997).

Kennedy, M.C., Antholine, W.E., and Beinert, H. An EPR investigation of the products of the reaction of cytosolic and mitochondrial aconitases with nitric oxide. J Biol Chem 272(33) 20340-20347 (1997).

Rose, I.A., and O'Connell, E.L. Mechanism of aconitase action. The hydrogen transfer reaction. J Biol Chem 242(8) 1870-1879 (1967).

Hausladen, A., and Fridovich, I. Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. J Biol Chem 269(47) 29405-29408 (1994).

Bulteau, A., Ikeda-Saito, M., and Szweda, L.I. Redox-dependent modulation of aconitase activity in intact mitochondria. Biochemistry 42 14846-14855 (2003).

Tsui, K., Chang, P., and Juang, H. Manganese antagonizes iron blocking mitochondrial aconitase expression in human prostate carcinoma cells. Asian J Androl 8(3) 307-315 (2006).

Gruer, M.J., and Guest, J.R. Two genetically-distinct and differently-regulated aconitases (AcnA and AcnB) in Escherichia coli. Microbiology 140 2531-2541 (1994).

Gardner, P.R. Aconitase: Sensitive target and measure of superoxide. Methods Enzymol 349 9-23 (2002).

Schapira, A.H.V. Mitochondrial involvement in Parkinson’s disease, Huntington’s disease, hereditary spastic paraplegia and Friedreich’s ataxia. Biochim Biophys Acta 1410 159-170 (1999).

Sadek, H.A., Humphries, K.M., Szweda, P.A., et al. Selective inactivation of redox-sensitive mitochondrial enzymes during cardiac reperfusion. Arch Biochem Biophys 406 222-228 (2002).

Tretter, L., and Adam-Vizi, V. Inhibition of Krebs cycle enzymes by hydrogen peroxide: A key role of a-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress. J Neurosci 20(24) 8972-8979 (2000).

Kennedy, M.C., Emptage, M.H., Dreyer, J., et al. The role of iron in the activation-inactivation of aconitase. J Biol Chem 258(18) 11098-11105 (1983).

Lin, K., Pasinelli, P., Brown, R.H., et al. Decreased intracellular superoxide levels activate sindbis virus-induced apoptosis. J Biol Chem 274(19) 13650-13655 (1999).

Show all 21 Hide all but first 3

705502-96well

96-Well Solid Plate (Colorimetric Assay)