Arachidonate → Arachidonic Acid

Participants

Reaction Steps

The enzyme activity was measured according to the modified method of Ochi et al. [K. Ochi, T. Yoshimoto, S. Yamamoto, K. Taniguchi, T. Miyamoto: Arachidonate 5-lipoxygenase of guinea pig peritoneal polymorphonuclear leukocytes, J. Biol. Chem. 258, 5754-5758 (1983)]. The principle of the method is as follows: in in vitro systems, during the reaction catalysed by 5-LO 5-hydroxyperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) is formed from arachidonic acid, then it is transformed to 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE). Both 5-HPETE and 5-HETE have absorption maximum at 236 nm, therefore it is possible to determine the activity of 5-LO enzyme by measuring the change of absorption at 236 nm. 5-LO was prepared from human polymorphonuclear leukocytes by a simple purification procedure. The cells were washed and lysed by a sonic disintegrator. The lysate was centrifuged and the enzyme in the cytosol was partially purified by ammonium sulphate fractionation (the enzyme precipitates at 50% saturation). The reaction mixture (50 mM tris-HCl pH=7.5, 2mM CaCl2, 0.1 mM ATP, 0.02 mM arachidonic acid) was incubated at 37° C. for 5 minutes. The reaction was started with the addition of arachidonic acid and stopped by the addition of 10 mM of citric acid. The thus-obtained 5-HPETE and 5-HETE were extracted with a 6:4 mixture of diethyl ether and hexane and the absorption of the organic phase was measured by spectrophotometry at 238 nm.

Provenance & Evidence