Product identification and determination of optimal condition of 8, 11, 14-eicosatrienoic acid desaturation in rat liver microsomes.

delta 5-Desaturation of 8, 11, 14-eicosatrienoic acid in washed rat liver microsomes was studied in the presence of NADH, ATP, and coenzyme A. The product was identified as arachidonic (5, 8, 11, 14-eicosatetraenoic) acid by radiogaschromatography. Cytosol fraction stimulated delta 5-desaturation but did not participate in 8, 11, 14-eicosatrienoyl-CoA synthesis. The pH optimal to the desaturation was 6.8. The minimal concentrations of ATP and pyridine nucleotides which gave the maximal desaturation activity were 2 mM and 0.75 mM, respectively. Both NADH and NADPH were able to be electron donors for delta 5-desaturase, but cytochrome P-450 did not take part in the enzyme reaction because CO gas did not inhibit its activity. Apparent Km and Vmax for 8, 11, 14-eicosatrienoic acid were 18 microM and 154 pmol/min/mg, respectively.